Detection of HPV DNA in cervical specimens collected in cytologic solution by ligation-dependent PCR

OBJECTIVE: To determine the feasibility and sensitivity of detecting human papillomavirus (HPV) in specimens collected in Cytyc PreservCyt fluid (Boxborough, Massachusetts, U.S.A.) using ligation-dependent polymerase chain reaction (LD-PCR) and to demonstrate the diagnostic value of HPV DNA testing as an adjunct to cytology in the detection of cervical squamous intraepithelial lesions (SIL), especially in cases of atypical squamous cells of undetermined significance (ASCUS). STUDY DESIGN: LD-PCR is a recently invented DNA amplification technology that utilizes a capture probe for target isolation and 2 hemiprobes for target detection. The hemiprobes are designed in such a way that when they hybridize to their target, the 5' end of one probe and the 3' end of the other probe are brought together. Two hemiprobes can then be ligated into a full probe that can serve as a template for PCR amplification. A total of 94 cervical specimens were collected in cytologic fluid and tested with LD-PCR. The results were compared with those of the Digene Hybrid Capture II assay (HC II) (Beltville, Maryland, U.S.A.) and consensus PCR. RESULTS: The overall sensitivity for detecting HPV was 41.5% (39/94) by LD-PCR, 50% (47/94) by consensus PCR and 37.2% (35/94) by HC II. The prevalence of HPV by HC II, consensus PCR and LD-PCR were 87.5%, 100% and 87.5% in the high grade SIL group; 100%, 90.9% and 90.9% in the low grade SIL group; 30%, 52.5% and 40% in the ASCUS group; and 14.2%, 22.8% and 17.1% in women with normal cytology. These results indicate that all 3 methods have similar sensitivity in patients with SIL. However, there is greater variation in detection rates in the ASCUS and normal cytology groups. CONCLUSION: LD-PCR is a useful method of detecting HPV in liquid-based gynecologic cytologic preservatives, and HPV testing as a method adjunct to the liquid-based Pap test could be useful in detecting SILs, especially for the management of patients with ASCUS.     

Novel multiplexed genotyping of human papillomavirus using a VeraCode-allele-specific primer extension method

A VeraCode‐allele‐specific primer extension (ASPE) method was applied to the detection and genotyping of human papillomavirus (HPV)‐DNA. Oligonucleotide primers containing HPV‐type‐specific L1 sequences were annealed to HPV‐DNA amplified by PGMY‐PCR, followed by ASPE to label the DNA with biotinylated nucleotides. The labeled DNA was captured by VeraCode beads through hybridization, stained with a streptavidin‐conjugated fluorophore, and detected by an Illumina BeadXpress® reader. By using this system, 16 clinically important HPV types (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were correctly genotyped in a multiplex format. The VeraCode‐ASPE genotyping of clinical DNA samples yielded identical results with those obtained by validated PGMY‐reverse blot hybridization assay, providing a new platform for high‐throughput genotyping required for HPV epidemiological surveys.     

Can human papillomavirus DNA testing of self-collected vaginal samples compare with physician-collected cervical samples and cytology for cervical cancer screening in developing countries?

Background: To determine human papillomavirus (HPV) types by polymerase chain reaction (PCR)-reverse line blot assay and examine the concordance between HPV by Hybrid Capture 2 (HC2) and PCR on self-collected vaginal and physician-collected cervical samples and cytology. Methods: This was a cross-sectional study of 546 sexually active women aged ≥30 years with persistent vaginal discharge, intermenstrual or postcoital bleeding or an unhealthy cervix. Participants self-collected vaginal samples (HPV-S) and physicians collected cervical samples for conventional Pap smear and HPV DNA (HPV-P) testing and performed colposcopy, with directed biopsy, if indicated. HPV testing and genotyping was done by HC2 and PCR reverse line blot assay. Concordance between HC2 and PCR results of self- and physician-collected samples was determined using a Kappa statistic (κ) and Chi-square test. Results: Complete data were available for 512 sets with 98% of women providing a satisfactory self-sample. PCR detected oncogenic HPV in 12.3% of self- and 13.0% of physician-collected samples. Overall, there was 93.8% agreement between physician-collected and self-samples (κ = 76.31%, 95% confidence interval [CI]: 64.97–82.29%, p = 0.04)—complete concordance in 473 cases (57 positive, 416 negative), partial concordance in seven pairs and discordance in 32 pairs. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of self-sampling for detection of cervical intraepithelial neoplasia (CIN)2+ disease were 82.5%, 93.6%, 52.4% and 98.4%, respectively; for physician-sampling they were 87.5%, 93.2%, 52.2% and 98.9%, respectively; and for cytology they were 77.5%, 87.3%, 34.1% and 97.9%, respectively. Concordance between HC2 and PCR was 90.9% for self-samples (κ = 63.7%, 95% CI: 55.2–72.2%) and 95.3% for physician-collected samples (κ = 80.4%, 95% CI: 71.8–89.0%). Conclusions: Self-HPV sampling compares favourably with physician-sampling and cytology. A rapid, affordable, HPV self-test kit can be used as the primary method of cervical cancer screening in low-resource situations.     

The evaluation of human papillomavirus genotyping in cervical liquid-based cytology specimens; using the Roche Linear Array HPV genotyping assay

Objective:  To ascertain the usefulness of the Roche Linear Array human papillomavirus (HPV) genotyping assay for assessing HPV genotypes in liquid-based cytology (LBC) samples and to evaluate this methodology within a cytopathology laboratory. These tests are of importance as persistent infection with high-risk HPV genotypes is considered a causal factor in the development of cervical cancer.
Methods:  A total of 175 cervical LBC samples were tested using the Roche Linear Array HPV genotyping test. The suitability of the assay use in routine cytopathology laboratory was considered. HPV genotypes were matched to the cervical cytology results, which included negative, borderline nuclear abnormalities, mild, moderate and severe dyskaryosis.
Results:  The assay could be applied to screening samples with the combined result available at the reporting stage. There were no test failures. All samples used after cytological analysis had sufficient DNA for testing. The results were reproducible and easily read and there was concordance of results between biomedical scientists. The results of the assay showed co-infection with multiple HPV genotypes was common in both high-grade and low-grade cytology samples. The percentage of HPV+ samples in the normal cytology samples (although in this grouping the number of samples was low) was 37%. In the cytology samples reported as severe dyskaryosis the HPV genotypes most commonly found were HPV16 and HPV51.
Conclusion:  The assay was able to detect multiple HPV infection with a wide range of genotypes in LBC samples sent for routine cytological analysis. It would be suitable for use in a cytopathology laboratory. The results of the assay show that the genotype profile has some variation from other geographical regions, and more work is needed to determine population prevalence, to ascertain the impact of the HPV vaccine, to evaluate test for cure and HPV triage management.     

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India

Background

Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc.

Methods

A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR).

Results

The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit.

Conclusions

Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.     

Chlamydia trachomatis detection in cervical PreservCyt specimens from an Irish urban female population

Objective:  The aim of this study was to determine the prevalence of cervical Chlamydia trachomatis infection by polymerase chain reaction (PCR) in urban women undergoing routine cervical cytological screening and to investigate the relationship with age, cytology, smoking status and concurrent human papillomavirus (HPV) infection.
Methods:  A total of 996 women (age range 16–69 years) attending general practitioners for routine liquid-based cervical smear screening in the Dublin area were recruited in the study of prevalence of C. trachomatis . Informed consent was obtained and liquid-based cytology (LBC) specimens were sent for cytological screening. DNA was extracted from residual LBC and tested for C. trachomatis by PCR using the highly sensitive C. trachomatis plasmid (CTP) primers and for HPV infection using the MY09/11 primers directed to the HPV L1 gene in a multiplex format.
Results:  The overall prevalence of C. trachomatis was 5.4%. Prevalence was highest in the <25 years age group (10%). Coinfection with HPV and C. trachomatis occurred in 1% of the screening population. A higher rate of smoking was observed in women positive for C. trachomatis , HPV infections or those with abnormal cervical cytology. Chlamydia trachomatis infection was not associated with abnormal cytology.
Conclusions:  Women (5.4%) presenting for routine cervical screening are infected with C. trachomatis . Opportunistic screening for C. trachomatis from PreservCyt sample taken at the time of cervical cytological screening may be a possible strategy to screen for C. trachomatis in the Irish female population.     

采用PCR-测序法对北京地区325例妇女宫颈脱落细胞样品中人乳头瘤病毒进行检测和基因分型

为了探讨PCR-测序法在宫颈脱落细胞样品中人乳头瘤病毒 (Human papillomavirus, HPV) 临床检测中的应用价值,采用HPV通用引物PGMY09/11针对HPV L1区基因序列进行PCR扩增,并通过DNA测序法对HPV进行基因分型。对于混合感染样品,利用HPV型别特异性引物PCR的方法进行基因分型。325例临床样品中,228例为HPV阳性,其中66例为混合感染。共发现27种不同的HPV型别,其中HPV 16比例最多,其次是HPV 58和52。高危型HPV检出率随病变程度加重显著性增加     

多重PCR同时检测人乳头瘤病毒、巨细胞病毒和沙眼衣原体

为了应用聚合酶链反应同时检测人巨细胞病毒（CMV）、人乳头瘤病毒（HPV）、沙眼衣原体（CT）,参照文献报道的基因序列,设计合成了三对能扩增370bp、450bp、510bp基因片段的引物,并对PCR扩增条件进行了优化。0.1fgHPV－DNA、CMV－DNA、CT－DNA即可被检出,得到了与设计片段相同的产物,且不扩增大肠杆菌、白色念球菌、解尿支原体等病原的核酸。对395例标本进行检查,各病原体的检出率分别是：HPV19.2%、CMV14.9%、CT5.1%。其中混合感染42例。PCR同时检测CMV、HPV、CT经济、快速、敏感、特异,可用于临床诊断和实验研究。     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC‐US)

M. Waldstrom and D. Ornskov
Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC‐US) Objective: The effect of triaging women with atypical squamous cells of undetermined significance (ASC‐US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high‐grade disease. We evaluated the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non‐oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types. Methods: We identified 369 consecutive PreservCyt samples diagnosed with ASC‐US tested for HPV DNA using the LA test. The Aptima HPV test was performed on residual material in the same vial. Follow‐up of 325 women was available. The gold standard used was histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2+ or 3+. Results: LA and Aptima HPV assays were positive in 44.3% and 31.7% of the cases, respectively. The concordance was 81.2%. The two tests had identical sensitivity for detecting CIN3+ [92.6% (95% CI, 75.7–99.1)] but the Aptima HPV assay showed a significantly better specificity of 73.8% (95% CI, 68.5–78.7) versus 60.1% (95% CI, 54.3–65.7) for LA for detecting CIN3+. When using CIN2+ as the gold standard the sensitivity for LA was higher than for the Aptima HPV assay [93.8% (95% CI, 82.8–98.7) versus 87.5% (95% CI, 74.8–95.3)], but the specificity was higher for the Aptima HPV assay: 78.0% (95% CI, 72.6–82.7) versus 64.3% (95% CI, 58.3–69.9). Conclusions: Both tests showed good and equal clinical sensitivities for detecting CIN3+, but the Aptima HPV assay had significantly higher specificity for detecting CIN2+ and CIN3+ in women aged 30 years or older with ASC‐US.     

Cervical cytology/histology discrepancy: a 4‐year review of patient outcome

E. L. Moss, A. Moran, G. Douce, J. Parkes, R. W. Todd and C. E. W. Redman Cervical cytology/histology discrepancy: a 4‐year review of patient outcome Objective: To investigate the diagnosis, review and management of women identified as having a cytology/histology discrepancy. Methods: A review of all patients diagnosed with a discrepancy between referral smear and cervical histology was performed between January 2003 and December 2004. Cases were followed for a minimum of 4 years and patient management and outcome reviewed. Results: A significant discrepancy was identified in 79 cases, 0.1% of all smears (n = 80 926) analysed during the study period. A discrepancy between cytology and histology, obtained from large loop excision of the transformation zone (LLETZ), was confirmed by multidisciplinary review in 42 cases (53.2%). In 37 cases (46.8%) the cytological and/or histological diagnosis was revised; the cytology was significantly more likely than the histology to be amended (chi square P = 0.005), most often because cytology had been overcalled. Of the confirmed discrepancy cases, 33 (78.6%) were due to high‐grade squamous cell or glandular abnormalities on cytology with a negative, inflammatory or human papillomavirus (HPV) infection on histology (HGC/NH). HGC/NH cases were managed by cytological follow‐up in 29 (87.9%), of which 72.4% of the smears were negative when performed at least 6 months post‐excision. During the 4‐year follow‐up period six women with a confirmed HGC/NH underwent a repeat cervical excision (hysterectomy or LLETZ), and of these, HPV effect was seen in two cases but no cervical intraepithelial neoplasia was detected in any of the histological specimens. Conclusion: Cytology overcall was responsible for the majority of cytology/histology discrepancies. A confirmed discrepancy is not an indication for a further excisional biopsy but follow‐up is essential because a small percentage of patients may have disease that has been missed.     

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

Aim: The aim of this study was to develop a multiplex real‐time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. Methods and Results: A primer set and three species‐specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia‐associated non‐Erysipelothrix spp. bacterial isolates. Cross‐reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. Conclusion: The multiplex real‐time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. Significance and Impact of the Study: The developed real‐time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.     

Improved rate of high-grade cervical intraepithelial neoplasia detection in human papillomavirus DNA hybrid capture testing

BACKGROUND: Numerous studies have established a link between human papillomavirus (HPV), squamous intraepithelial lesions (SIL) and carcinoma of the cervix. Testing for HPV DNA in addition to cytology in screening programs for cervical cancer has been suggested to increase detection rates. STUDY DESIGN: HPV DNA testing (performed by hybridization antibody capture assay I or II), cytology and biopsy (performed within 1 month of each other) were retrospectively reviewed for a series of 155 women. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HPV testing vs. cytology were calculated using biopsy as the gold standard. These values were also calculated in a subgroup of 37 individuals older than 35 years. RESULTS: The sensitivity, specificity, PPV and NPV of DNA hybrid capture HPV testing for detecting high-grade cervical intraepithelial neoplasia (CIN) were 86%, 44%, 26% and 93%, respectively. The respective values for cytology detection of high-grade CIN were 17%, 97%, 56% and 82%. CONCLUSION: HPV testing was significantly more sensitive for detecting high-grade CIN than cytology (86% vs. 17%). Our data support immediate colposcopy and biopsy, rather than follow-up Papanicolaou testing, if the test for HPV DNA is positive for an intermediate- to high-risk type.     

Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses

Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negaive with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued.     

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Aims: To develop a multiplex real‐time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Methods and Results: Real‐time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real‐time PCR assay. The multiplex real‐time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C_t values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. Conclusions: This multiplex real‐time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Significance and Impact of the Study: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.     

Chlamydia trachomatis infection and human papillomavirus in women with cervical neoplasia in Pernambuco-Brazil

Chlamydia trachomatis (CT) is the most common bacterial cause of sexually transmitted disease. High-risk human papillomavirus (HR-HPV) is considered the main etiological agent for cervical neoplasia. Evidences showed that the presence of co-infection of CT and HR-HPV plays a central role in the etiology of cervical intraepithelial neoplasia (CIN) and cervical cancer. The goals of this study were: evaluate the human papillomavirus (HPV) and CT prevalence among Brazilian women with abnormal cytology and provide the effect of this association on the severity of cervical neoplasia. The population of this study was composed by 142 women with incident histological incidence of CIN grades I, II, III or cervical cancer from Recife, Northeast of Brazil. The polymerase chain reaction method on a cervical brush specimen was used to detect both agents and the automatic sequencing method was used for HPV genotyping assay. The prevalence of HPV and CT was 100 and 24.65 %, respectively. Thirteen types of HPV were detected; HPV 16, 18, 31 and 33 were the most common. The most prevalent HPV types were HPV 16 and 18. A significant association between CT positive and HPV 16 infection was found (p < 0.0106; OR = 5.31; 95 % IC 1.59–17.67). In the study population, there was diversity of HPV infections, with high-risk types being the most common. Also, the data collected suggest that CT infection may play an important role in the natural history of HPV infection.     

Multiplex detection and SNP genotyping in a single fluorescence channel

Probe-based PCR is widely used for SNP (single nucleotide polymorphism) genotyping and pathogen nucleic acid detection due to its simplicity, sensitivity and cost-effectiveness. However, the multiplex capability of hydrolysis probe-based PCR is normally limited to one target (pathogen or allele) per fluorescence channel. Current fluorescence PCR machines typically have 4–6 channels. We present a strategy permitting the multiplex detection of multiple targets in a single detection channel. The technique is named Multiplex Probe Amplification (MPA). Polymorphisms of the CYP2C9 gene (cytochrome P450, family 2, subfamily C, polypeptide 9, CYP2C9*2) and human papillomavirus sequences HPV16, 18, 31, 52 and 59 were chosen as model targets for testing MPA. The allele status of the CYP2C9*2 determined by MPA was entirely concordant with the reference TaqMan® SNP Genotyping Assays. The four HPV strain sequences could be independently detected in a single fluorescence detection channel. The results validate the multiplex capacity, the simplicity and accuracy of MPA for SNP genotyping and multiplex detection using different probes labeled with the same fluorophore. The technique offers a new way to multiplex in a single detection channel of a closed-tube PCR.     

HPV testing in liquid cytology specimens: comparison of analytic sensitivty and specificity for in situ hybridization and chemiluminescent nucleic acid testing

OBJECTIVE: To compare the analytic sensitivity and specificity of Hybrid Capture 2 (HC2) (Digene Corporation, Gaithersburg, Maryland, U.S.A.) with in situ hybridization (ISH) in detecting high-risk types of HPV (HR-HPV). STUDY DESIGN: Performance characteristics of ISH and HC2 were compared in 99 consecutive cervical cytology samples diagnosed as low grade squamous intraepithelial lesion and processed by either the ThinPrep (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) or SurePath (TriPath Imaging Systems, Research Triangle Park, North Carolina, U.S.A.) method at 2 geographically different centers. Polymerase chain reaction (PCR) was used as the gold standard. RESULTS: Of the samples, 67% were positive for HR-HPV viral types by PCR. ISH had a sensitivity of 0.87 as compared to 0.95 (p = 0.11) for HC2. The specificity of ISH was significantly different from that of HC2 (0.57 vs. 0.13, respectively; p = 0.0004). The performance characteristics of ISH were not affected by the processing method or population tested. CONCLUSION: The sensitivity of ISH is comparable to that of HC2, with significantly superior specificity, and is therefore an efficacious alternative to HC2 for triaging patients with abnormal cervical cytology results.     

宫颈癌患者人乳头瘤病毒(HPV)主要型别及其感染研究

本文探讨了江西省和广东省宫颈癌患者人乳头瘤病毒(Human papillomavirus,HPV)感染及其型别分布,分析了高危型HPV对各种宫颈病变的感染情况,为宫颈癌的早期发现和临床诊治提供科学依据。首先采用细胞学、HPV DNA检测(第二代杂交捕获法,HC2)、电子阴道镜和宫颈化学着色方法筛查宫颈癌患者,经病理镜检确诊,然后用GP PCR-SBT法对宫颈癌患者进行HPV基因分型。江西省溪口镇、古市镇及修水县城宫颈癌癌前病变发生率为5．7‰。HC2方法发现宫颈癌患者13种高危型HPV DNA阳性率为89．9％,宫颈上皮内瘤样病变的为84．8％,对照组为24．5％。采用GP PCR-SBT方法进行基因分型发现,江西省宫颈癌患者存在HPV16、58、31、33、18、66、6、11、56和81十种型别,其中HPV81型在国内外鲜有报道。据此提出生殖道高危型HPV感染是妇女宫颈癌发病的重要因素。并发现江西省宫颈癌高发区妇女高危型HPV感染率为24．5％。建立了HPV基因分型的方法,对HPV致宫颈病变的分子机制进行了分析。     

Sensitivity of HPV tests on stained vs. unstained cervical smears

OBJECTIVE: To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests. STUDY DESIGN: Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis. RESULTS: After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found. CONCLUSION: The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.