Comparison of human papillomavirus genotyping using commercial assays based on PCR and reverse hybridization methods

Different tests for human papillomavirus (HPV) screening are commercially available, detecting high‐risk oncogenic HPV types with a pool of genotype‐specific probes. However, it is necessary to establish reliable methods for the identification of individual genotypes. The purpose of this study was to compare three different commercial methods for HPV genotyping: INNO‐LiPA HPV Genotyping v2 (LiPA), Linear Arrays HPV Genotyping Test (LA) and Clinical Arrays Human Papillomavirus (CA). A total of 83 HPV DNA‐positive samples by hybrid capture method were genotyped (82, 78 and 81 by LiPA, LA and CA, respectively). Comparison analysis was limited to the HPV genotypes common to the three assays. There were concordant results (absolute agreement between assays) in 31 samples (39.7%) and compatible results (correspondence for some but not all genotypes) were found in 44 samples (56.4%). Only three samples (3.8%) were considered as discordant (did not show any similarity between the tests). Analyzing kappa values we have a very good agreement (>0.8) for HPV16 and HPV31 and good agreement (0.6–0.8) for HPV types 6, 18, 53 and 66 when all methods are compared. We conclude that all genotyping methods tested are highly comparable and suitable for clinical and epidemiological studies.     

Universal human papillomavirus genotyping by the digene HPV Genotyping RH and LQ Tests

BackgroundHigh-risk (hr)HPV testing plays an important role in primary cervical cancer screening. Subsequent hrHPV genotyping might contribute to better risk stratification. The majority of hrHPV tests do not include identification of individual hrHPV genotypes.ObjectivesThe digene HPV Genotyping RH Test (strip-based) and LQ Test (xMAP-based) allow genotyping of GP5+/6+ amplimers, but their probes target a region in the L1 ORF, which is also amplified by other broad-spectrum hrHPV assays, e.g., the Roche Amplicor HPV Test (Amplicor) and the Roche Linear Array. The goal was to test whether the RH Test and LQ Test can be used as an universal hrHPV genotyping test.Study designSelf-collected cervico-vaginal specimens (n = 416) from an epidemiologic study were analyzed with Amplicor. The amplimers obtained were also tested with the RH Test and LQ Test for identification of 18 HPV types, including the 13 hrHPVs targeted by Amplicor.Results197 specimens were positive by Amplicor, in which the RH Test and LQ Test identified one of the 13 hrHPVs in 94.4% and 98.0%, respectively. In 219 specimens remaining negative by Amplicor, the RH Test and LQ Test, performed on the Amplicor amplification products, still detected one of the 13 hrHPVs in 3.7% and 5.5%, respectively, and include identification of HPV53, 66, and 82. Overall, the RH and LQ Tests demonstrated high concordance with Amplicor for hrHPV detection (κ = 0.908 and κ = 0.923, respectively).ConclusionsThe digene HPV Genotyping RH and LQ Tests can be directly used for amplimers generated by the Amplicor HPV Test.     

Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results

Galan‐Sanchez F, Rodriguez‐Iglesias MA. Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results. APMIS 2010; 118: 681–4. We have evaluated Cervista HPV HR for papillomavirus detection in 65 samples previously borderline negative by the hybrid capture method (Digene), using InnoLipa and sequencing as confirmatory techniques. Nine samples were found to be positive by Cervista HPV HR, of which five (7.6%) were confirmed by InnoLipa. Four samples (6.1%) were false positive, of which three samples were reactive for A9 probes. The Cervista HPV HR assay can detect HPV‐positive samples from those with hybrid capture borderline results but can produce false‐positive results when tested for reactivity with A9 probes.     

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.     

子宫颈刮片中人乳头瘤病毒的基因分型

目的 确定不同型人乳头瘤病毒（ＨＰＶ）感染的自然历程以及其持续感染在子宫颈癌发展过程中的作用。方法 应用聚合酶链反应检测荷兰１５５名妇女子宫刮颈片中的ＨＰＶ ＤＮＡ，应用线样探针分析法（ＬｉＰＡ）进行，包括ＨＰＶ６，１１，１６，１８，３１，３３，３５，４０，４２，４３，４４，４５，５１，５２，５６和５８的基因分型。结果 １５５例妇女子宫颈片中ＨＰＶ ＤＮＡ检出率为６０％，其中在宫颈细胞学检查正常或     

改进探针标记法斑点杂交在轮状病毒VP7分型中的应用

目的建立一种更加简便的A组人轮状病毒（HRV）核酸斑点杂交VP7分型方法,以便用于对HRV流行情况进行调查。方法在HRVVP7编码基因各G基因型问高度变异而型内高度保守区域设计分型探针,在该区域的两侧相对保守区域设计一对通用引物,利用PCR分别将地高辛标记HRV5种常见型别（G1～4,G9型）的DNA探针,建立基于VP7的斑点杂交方法;选取经抗原检测和聚丙烯酰胺凝胶电泳（PAGE）检测均为HRV阳性的2006至2008年住院腹泻患儿粪便标本200份,RT—PCR扩增VP7全基因,并对扩增阳性产物应用斑点杂交方法进行G型别分析。结果建立的斑点杂交方法在5种型别探针间无交叉反应,各型探针的检测灵敏度可达到10Pg。200份PAGE阳性标本中162份RT—RCR扩增VP7基因阳性,斑点杂交显示G1型41例（25．3％）,G2型2例（1．2％）,G3型63例（38．9％）,G9型35例（21．6％）,混合感染19例（11．7％）,杂交未分出型2例（1．2％）,未检测到G4型HRV。结论本研究所建立的斑点杂交方法敏感度和特异度强,适合在HRV大规模分子流行病学调查时应用。通过该方法的初步应用,发现北京地区婴幼儿HRV除了常见的G1、G2和G3型外,还有G9型感染。     

微量酶标夹心杂交法定量检测人iNOS-mRNA

目的：建立一种高灵敏度、高特异性、精确、简便的微孔板酶联夹心杂交技术，对人iNOS-mRNA进行定量检测。方法：设计两条特异探针，其中一条为捕获探针，5'端连接氨基，能与微孔板表面的NOS基团共价结合，“竖直”地包被在微孔中，另一个为检测探针，3'端带有生物素标记。用脂多糖（LPS）刺激人外周血单个核细胞24 h后，提取巨噬细胞总RNA，进行RT-PCR扩增，PCR产物热变性后加入到已包被捕获探针的微孔板内，并加入检测探针进行夹心杂交，最后加入亲和素-辣根过氧化物酶（AV-HRP）及底物，在450 nm波长检测杂交信号。结果：该方法检测iNOS-mRNA的灵敏度明显高于RT-PCR-琼脂糖凝胶电泳；特异性高，RT-PCR和酶联夹心杂交均无非特异阳性信号出现；精密度良好。结论：本方法操作简单、灵敏度高、特异性强、结果数据化，适合于iNOS-mRNA的定量检测。     

Nucleic acid spot hybridization with nonradioactive labeled probes in screening for human papillomavirus DNA sequences

We examined 161 human tissue samples using the spot hybridization technique with nonradioactive labeled DNA probes of human papillomavirus (HPV). Whole cells were spotted on nitrocellulose filters; DNA of the cells was denatured and fixed to the filter. Then the DNA spots were hybridized to nonradioactive labeled DNA and monitored by a sandwich immunoenzymatic reaction. This technique is simple, sensitive, specific, requires no special equipment, and can be used in clinical settings. HPV DNA was found in 92% of samples in which, on the basis of histologic and colposcopic criteria, its presence was suspected, as well as in 31 samples where it was not suspected.     

Quality assurance of genotyping array for detection and typing of human papillomavirus

The aim of this study was to evaluate the sensitivity, specificity, reliability and reproducibility of the EasyChip^® HPV blot for human papillomavirus (HPV) genotyping. Type-specific sensitivity and specificity for 39 types of HPV (HPV 6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7 and MM8) were examined. The operating environment, reliability, reproducibility and blot interpretation were assessed by a quality assurance system. Each batch experiment contained samples from 89 cervical specimens and 7 extrinsic controls. Caski, HeLa and Jurkat cells, male human blood cell DNA and sterile water were used to assess reliability. Furthermore, pairs of sibling controls were used to assess reproducibility. The overall sensitivity of HPV detection was 1–50 copies of HPV genome equivalent. There was no cross-reactivity with amplicons of other HPV genotypes. One hundred batch experiments demonstrated that the reliability was excellent. The intra-batch and inter-batch reproducibility was 98 and 97%, respectively. It was concluded that the EasyChip^® HPV blot is a highly sensitive, reliable and reproducible tool for detection and identification of HPV genotypes.     

Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women

Cervical cancer is a leading cause of cancer‐related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV‐positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%). J. Med. Virol. 81:678–684, 2009 © 2009 Wiley‐Liss, Inc.     

Evaluation of a reverse line blot assay for genotyping common human rotaviruses

To allow high-throughput genotyping of group A rotaviruses (RVA) in a routine surveillance setting, we developed a reverse line blotting method for the determination of the most common human RVA G- and P-genotypes: G1–G4, G9, G12, P[4], P[6] and P[8]. Using the reverse line blotting method on 951 clinical RVA positive feces samples, in 905 (95%) of the samples the G-genotyping yielded a result while in 945 (99%) of the samples the P-genotyping was successful. Comparison of the reverse line blotting-method as it is used currently to a sequence based method for genotyping RVAs showed an agreement of 96% for single strain infections (75 out of 78) but only 48% for mixed infections (10 out of 21).     

Genomic characterization of a novel human papillomavirus (HPV-117) with a high viral load in a persisting wart

Warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. Human papillomaviruses (HPV) are considered the causal agents for the development of such warts. We isolated the novel type HPV-117 from a persisting wart by rolling circle amplification. One hundred eighteen warts from immunocompetent patients (IC) and 49 warts from OTR were analyzed by HPV-117 E6 type-specific PCR. As inferred from a phylogenetic analysis, the new type HPV-117 belonged to alpha-PV species 2, including the most similar types HPV-10 and HPV-94. The general prevalence of HPV-117 in warts was 2% in IC (2/118), and 12% in OTR (6/49). The high viral load in dysplastic cells of a Verruca vulgaris was shown by in situ hybridization. Our results suggest an active role of the novel type in the development of cutaneous warts of OTR.     

Analytical performance of a low-cost multiplex polymerase chain reaction human papillomavirus genotyping assay for use in Sub-Saharan Africa

We have tested a multiplex polymerase chain reaction (PCR) human papillomavirus (HPV) genotyping assay to fill the need for rapid and low-cost HPV detection in Sub-Saharan Africa. This method allows high throughput genotyping and simultaneous detection of 14 high-risk and two low-risk HPV types, by PCR amplification of HPV DNAs in a single reaction tube. In this study, we describe stepwise experiments to validate the multiplex HPV PCR assay for determination of HPV genotypes from 104 cervical brush samples from Tanzanian women. Assay performance was evaluated by determination of intra-laboratory reproducibility, sensitivity, and specificity. Further performance was assessed by comparison with the widely accepted and validated HPV My09/My11 amplification and hybridization assay. Statistics; the Cohen kappa (κ) and McNemar P values were used to analyze interobserver and intermethod agreement. Overall concordance between the multiplex and line blot hybridization assays was 99% (per sample) with a κ value equal to 0.95; and 96.49% (per detection event) with a κ value of 0.92. Interobserver reproducibility of the assay per sample was 95.76% with κ of 0.91. These results demonstrate that the multiplex HPV PCR assay has high analytical sensitivity and specificity in detecting as many as 16 different HPV genotypes and that its simplicity and low cost makes it well suited for sub-Saharan Africa.     

Evaluation of human papillomavirus DNA detection in samples obtained for routine Chlamydia trachomatis screening

BackgroundThe costs and logistics involved in obtaining samples is a bottleneck in large-scale studies of the circulation of human papillomavirus (HPV), which are useful for monitoring and optimisation of HPV-vaccination programs. Residual samples obtained after screening for Chlamydia trachomatis could constitute a convenient, low-cost solution.ObjectivesWe evaluated HPV DNA detection and typing using (i) the residual samples routinely taken for C. trachomatis screening or (ii) the sample types used in large-scale phase III HPV vaccination trials (cervical, vulvar, labial, perineal, perianal, scrotal and penile shaft samples).Study designSamples from 127 men and 110 women attending two sexual health clinics were analysed using PCR for HPV DNA, with typing using mass spectrometry.ResultsThe HPV DNA prevalence was 7.1% in male urine samples, but 57.3% in female urine/vaginal samples, which was even higher than the HPV prevalence found in cervical samples (54.1%). The sensitivity for HPV DNA detection in the urine/vaginal samples was 7.9% (95% CI 3.0–16.4) for men and 78.9% (95% CI 67.6–87.7) for women, using detection in any one of the reference samples as reference. With cervical samples as reference, the sensitivity was 89.3 % (95% CI 78.1–95.9).ConclusionsAmong men, low sensitivity of urine for HPV detection suggests limited usefulness. Among women, the high sensitivity of urine/vaginal samples for HPV detection suggests a useful low-cost solution for the study of HPV epidemiology.     

安阳市宫颈病变患者人乳头瘤病毒感染现状及基因分型分析

目的:分析安阳市宫颈病变患者人乳头瘤病毒（human papillomavirus,HPV）感染现状,并分析其基因型。方法:选取2018年6月至2020年12月在本院门诊就诊或住院治疗的宫颈病变患者372例,参照宫颈组织病理活检结果分为宫颈炎、宫颈上皮内瘤变（cervical intraepithelial neopl...     

Clinical significance of signal pattern of high-risk human papillomavirus using a novel fluorescence in situ hybridization assay in cervical cytology

The present study aimed to evaluate a novel fluorescence in situ hybridization (FISH) assay for detecting the high-risk human papillomavirus (HR-HPV) DNA and signal pattern in cervical cytology specimens and for identifying cervical intraepithelial neoplasia (CIN) lesions. One hundred and ninety-six liquid-based cytology specimens with CIN were recruited. The signal pattern (punctate, mixed punctate and diffuse, and diffuse) detected by FISH was compared with E6 mRNA and correlated with histological classification. FISH and E6-type specific polymerase chain reaction (PCR) had fair to good agreement for detecting HPV DNA across all grades of CIN (kappa coefficient, 0.37–0.73). Among 44 samples of negative FISH and positive E6 type-specific PCR in HPV 16, 18, 31, 33, 52 and 58, 82% (36/44) of E6 mRNA were not detected, in contrast to 41% (48/118) of positive FISH and positive E6 type-specific PCR (p <0.0001). Among HR-HPV DNA positive cases tested by the FISH assay, the specificity of predicting CIN3 using the punctuate pattern is higher than that using E6 mRNA (96.3% vs. 44.8%). The punctate pattern was 0% in patients with <CIN1 lesions, 8.7% for CIN1 lesions, 6.1% for CIN2 lesions, and 34.0% for CIN3 lesions (p 0.001). The odds ratios were 8.7-fold higher (2.7–27.8, p <0.0001) for the punctate pattern versus the mixed punctate and diffuse pattern, and the diffuse pattern, for predicting CIN3 lesions. The novel FISH assay is comparable to PCR for detecting HPV DNA in cervical cytology with CIN lesions. The punctate signal pattern detected by the FISH assay can be more biologically and clinically relevant for clinically detecting CIN3 lesions.     

Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds

A rapid, sensitive and automated assay procedure was developed for the in vitro evaluation of anti-HIV agents. An HTLV-I transformed T4-cell line, MT-4, which was previously shown by Koyanagi et al. (1985) to be highly susceptible to, and permissive for, HIV infection, served as the target cell line. Inhibition of the HIV-induced cytopathic effect was used as the end point. The viability of both HIV-and mock-infected cells was assessed spectrophotometrically via the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The procedure was optimized as to make optimal use of multichannel pipettes, microprocessor-controlled dispensing and optical density reading. The absorbance ratio of the mock-infected control to the HIV-infected samples was about 20. This allowed an accurate determination of the 50% effective doses, as demonstrated for 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddCyd), dextran sulfate and heparin. The technique significantly reduced labor time as compared to the trypan blue exclusion method, and permits the evaluation of large numbers of compounds for their anti-HIV activity.     

Evaluation of a novel multiplex human papillomavirus (HPV) genotyping assay for HPV types in skin warts

Human papillomaviruses (HPV) of the genera alpha, mu, and nu induce benign tumors of the cutaneous epithelia that constitute a significant burden for immunocompromised adults. Currently, no gold standard for genotyping of these HPV types exists. In this study, we describe the prevalence of genus alpha, mu, and nu HPV types in cutaneous warts. We developed a novel multiplex HPV genotyping assay, BSwart-PCR/MPG (BSwart), to type sensitively and specifically 19 cutaneous HPV types frequently found in warts. BSwart-PCR/MPG is based on a multiplex PCR using broad-spectrum primers and subsequent multiplex hybridization to type-specific probes coupled to Luminex beads. In a first application comprising 100 cutaneous warts, the assay was compared to another, recently described genotyping assay, the HSL-PCR/MPG. When a 10-fold dilution series was used, the detection limit was between 10 and 100 HPV genomes per PCR. When comparing the two assays, there was an excellent agreement in detecting dominant HPV types; however, we also obtained evidence for a higher sensitivity of the BSwart assay for multiple infections in these cutaneous warts. Using BSwart, HPV was found in 95% of wart preparations, with HPV1 being most prevalent, followed by types 27, 57, and 2. Both novel BSwart and HSL-PCR/MPG HPV genotyping assays are powerful high-throughput tools that could be used to learn more about the natural history of cutaneous HPV. They would be advantageous to monitor the efficacy of future skin HPV vaccines and to identify novel HPV vaccine candidates.