Clinical Evaluation Of HPV DNA Test Combined With Liquid-based Cytology In The Diagnosis Of Cervical Disease

Human papillomavirus (HPV) is responsible for99.7%of cervical cancer and arethe most common sexually transmitted infection world widely. Several methods ofHPV detection have been proposed and still studies are going on regarding thesensitivity of the tests as screening method. HPV lesions are thought to arise from theproliferation of infected basal keratinocytes. It is limited to basal cells of stratifiedepithelium, which could be exposes to infectious virus through disturbed epithelialbarrier as might occur after minor skin abrasions or sexual intercourse. Most of HPVinfections in young females are only temporary and are gone in1-2year duration. Bursome “high risk” viruses may take10-15years to progress into invasive cervicalcancer. Genital HPV infections cause significant health burden, because cervicalcancer is the second most common and lethal cancer detected in women worldwide.Progression to cervical cancer can be almost always prevented when standardprevention strategies are applied. More than40types of HPV are identified which cantransmit through sexual contact. These are DNA viruses and targets stratifiedsquamous epithelia of the body. A subset is also able to infect the grandular epitheliaof the cervix. Human papillomavirus is primarily transmitted through penetrativesexual intercourse. Most of HPV infections are cleared up without medical attentionor consequences, and the re-infection seems to be less, probably due to developmentof some level of natural immunity. Several months to years may elapse beforesquamous intraepithelial lesion (SIL) develop and be clinically detected. Humanpapillmavirus (HPV) has potential to turn the normal cells into abnormal after theinfection. Normally, the body fights off HPV naturally and the infected cells, but attimes when body fails to fight HPV, the visible changes could turn into the form ofgenital warts or cervical intraepithelial lesions. Human papillomavirus (HPV) beingthe most common sexually transmitted infection in the world, only handful ofpreventable methods have been identified and proposed. With proper preventionmethod used, the chance of getting HPV-related infections are less. Recently,vaccines are made available and along with it, condoms and microbicides are thoughtto help in prevention in transmission of infection. Two vaccines are available,Gardasil and Cervarix marketed by GlaxoSmithKine, which prevents from HPV-16 and HPV-18. These vaccines are most effective when given before a person’s firstsexual contact. These are given in three doses over six months to get the bestprotection. But it provides little benefit to woman who has already been exposed tothe infection. These vaccines are also recommended to men who have sex with menand immunocompromised. Though human papillomavirus (HPV) infection is the mostcommon worldwide, but with proper and regular screening method cervical dysplasiacould be diagnosed early and cancer could be prevented. Various precautions shouldbe obtained for the best sample of cervical cytology. The collection should be avoidedduring menstruation, and at least for5days after the period has stopped. And after thesample has acquired, the patient should be advice to avoid vaginal intercourse,douching, use of tampons and also placement of intravaginal devices, medications andcontraceptives. There are basically two main conventional methods of collectingsample for cervical cytology. In conventional method, the use of a special woodenspatula or Ayre spatula is properly positioned in the external cervical os and rotatedtwice. The excessive mucosal bleeding should be avoided. The endocervix sample istaken by cervical brush rotating it to180degree. The collected material is added tothe slide and treated with cell fixative. Other method is the liquid-based cytologyapproach. The cervical broom is placed in the cervix and rotated two to four times andthe sample is transferred into a liquid transport medium. There are two commercialsystems available, Thin-Prep (Hologic Corp) and Sure-Path (BD-TriPath Imagning).The main advantage of liquid base methods is that the transport media containing theremaining cells is stored and these cells could be used for HPV DNA testing withHybrid Capture II assay.Bethesda system also provides the detailed guidelines on how to manage thepatients with cervical cytologic abnormalities. Usually about30%patient diagnosedas ASC-US on cervical cytology could be subsequently identified as CIN. And hence,it is recommended to undergo reflex HPV DNA testing and determine the presence ofhigh-risk HPV types. In colposcopic examination, LSIL could be diagnosed which iscorrelalted with mild dysplasia or CIN I and may progress to more severe dysplasia. Itis recommended to continue surveillence with cytology and colposcopy examinationif the disease persists or worsens. HSIL found on colposcopic biopsy correlates frommoderate to severe dysplasia and could lead into carcinoma in situ and/or invasivecarcinoma. It is recommended to undergo cervical conization to obtain a larger specimen and to assess for the presence of invasive disease. HPV DNA testing can beperformed using the same media from concomitant liquid-based cytologic screeningof cervical cytology. It is based on the detection of HPV DNA by Hybrid Capture IItest (Digene Corporation). In this assay, the mixture of single-stranded RNA probebinds with the complementary single-stranded HPV DNA. The DNA-RNA complexgives a semi-quantitative reading of the viral load of the sample. The benefit ofscreening with HPV DNA testing in conjunction with cervical cytology is consideredbeneficial for women over30years. Among women who were positive,90%werepositive in the vulvovaginal region,46%in the cervix. Studies have concluded thatHPV test reults are more sensitive, although less specific than Pap smears in detectinghigh-grade dysplase in older women. Currently, there is no treatment to eradicatehuman papillomavirus infections. And hence, the main goal of the treatment is toeliminate or control the disease by destruction of the lesion by physico-chemicalmeans, or by induction of an inflammatory or immune response.Objectives: To evaluate the sensitivity and accuracy of the HPV DNA test inconjunction with Thinprep cytology test as a screening method of cervical disease. Tostudy weather the cervical erosion is related to high risk HPV infection. To determinethe mean age distribution who are more prone to HPV infection at Changchun.Material and Method: The study is a retrospective cohort implemented to determinethe real performance of liquid based medium and HPV DNA testing combined inNo.1Hospital, Gynecology Department, and Changchun, Jilin University. The studygroup included total150patients from1stJanuary2011to30thDecember2012. Acomputerized search identified patients with Thinprep test results and high risk HPVDNA testing during a2years period was recruited. The patients were chosen afterproper speculum examination followed by Thinprep cytology (TCT) and HPV DNAtest. Cytological specimens were obtained with endocervical brush, which was rinsedinto the vial of Cytyc. The manufactures protocol was followed for HPV DNA testingusing Hybrid Capture II. Colposcopic biopsy was performed in patients who hadatypical squamous cells of undetermined significance (AUS-US), low-intraepitheliallesion (LSIL) or high-grade intraepithelial lesion (HSIL) in cytology and withpositive results of high-risk HPV DNA. The diagnostic criteria were based on theBethesda System (TBS). Participants: The study group included150women who were randomly selected,with the inclusion criteria that had proper medical history, age group more than20years, who were going through the examination for the first time and those who hadabnormal either in the speculum examination or in liquid based medium (TCT).Results: The high risk HPV–positive women with abnormal cytology had a CIN Irisk of73(86%), whereas35(23.3%) high-risk HPV positive women out of109(72.7%) normal cytology who underwent histological biopsy had CIN I16(10.7%).The risk for cervical intraepithelial neoplasia (CIN) in women with high-risk HPVpositive with normal cytology was higher among women invited for the first time31-40years of age12(8%) than among older women1(0.7%). Out of44(29.3%) womenwho had I degree erosion with38(86%) had normal histological biopsy showing nostatically significant between them. Thin-cytology test reviewed,109(72.7%) hadnormal results,13(8.7%) had atypical squamous cells of undetermined origin (ASC-US),14(9.3%) had low squamous intraepithelial lesion (LSIL) and14(9.3%) had highsquamous intraepithelial lesion (HSIL). The total number of patients who had, Idegree erosion in speculum examination were44(29.3%), II degree erosions were40(26.7%) and III degree erosions were (2.7%). With HPV DNA load threshold of1pg/ml considering to be positive in29.3%I degree erosion38(86%) had normal resultshowing there is no statistically significant relation between HPV infection and degreeof erosions (P>0.05). atypical squamous cells of undetermined origin was diagnosedin liquid based cytology.13(8.7%) were diagnosed as ASC-US in thin-cytology test.Of these patients,1(0.7%) had high-risk HPV DNA identified by Hybrid Capture IIand all went through histological examination. Overall,13(8.7%) of patient withASC-US interpretation underwent histologic follow-up and8(5.3%) has cervicalintraepithelial neoplasia (CINI) positive whereas,14.2(94.7%) had CIN negativeresults. It shows the relationship between ASC-US and HPV DNA load is non-significant (P>0.05). the variation in the liquid based cytology and HPV DNA testing.Of total patients,109(72.7%) patients had a normal thin-cytology test (TCT) results.72.7%underwent HPV DNA testing who had some degree of erosions in speculumexamination to rule out the HPV infection and further histological examination ifHPV DNA load was higher than the average. Out of109(72.7%) normal TCT results,HPV DNA test positive were35(23.3%) and cervical intraepithelial neoplasia (CIN I)was found to be in16(10.7%). The significance of the combined test of TCT and HPV DNA test were specifically high (P<0.05) than alone. The mean age distributions whowere more prone to HPV infection were37.8±10.44. At age20-30years40(26.7%),5(3.3%) had cervicitis and4(2.7%) had CIN I. Age31-40years50(33.3%),5(3.3%)had cervicitis and12(8%) had CIN I and1(0.7%) had cervical carcinoma. At age41-50years46(30.7%),2(1.3%) had cervicitis,8(5.3%) had CIN I and1(0.7%) hadcervical carcinoma. Following age51-60years10(6.7%),1(0.7%) had cervicitis,2(1.3%) had CIN I. And at age61-70years,0(0%) had cervicitis,1(0.7%) had CIN Iand1(0.7%) had cervical carcinoma. These age variation shows decline in cervicitisand inclined in cervical carcinoma. The risk for CIN is higher among women31-40years of age12(8%) than the older women1(0.7%).Conclusion: The data from this study confirms that HR-HPV DNA testing is muchmore sensitive than cytology alone and that HPV DNA testing helps in identifyingwomen with high risk of serious cervical disease in an efficient and medicallyacceptable manner. Other most significant advantage of this cervical cancer screeningmethod is that women who are HPV DNA positive can easily and quickly referred forColposcopic examination within one year, which could identify the precancer andcancer stage. And those who are HPV DNA negative can safely have much longerscreening intervals saving considerable costs. With mean age being38±10years, ageolder than30years should undergo HPV DNA testing with cytology triage in primaryscreening. But in woman younger than30years using HPV DNA assay, as an initialscreening step can lead into unnecessary treatment and due to strong body’s immunesystem their could be change in HPV related infection. However, close follow-up isessential if the initial biopsy is negative because considerable number of women mayhave HPV infection positive in subsequent studies. In the present study population,there were a substantial number of atypical squamous cells of undeterminedsignificance (ASC-US) cases with a positive HPV DNA test result and a negativebiopsy result, supporting the validity of the negative biopsy diagnosis. The studieshave proved that the HPV infections can be transient and episodic, especially if thewoman is young. If the HPV infection is newly acquired the chance to resolve ishigher than the longer persistent infection. The study shows that, the probability ofresolving the newly acquired infection is31%in the first6-months period and39%during second period. However, with the time period the resolution of infection drops11%in the third3-months period after infection.Even in our studies, it is reasonable to assume that some newly acquired HPV infections might have resolved even beforethey underwent colposcopic examination and biopsy resulting in false-positive HPVDNA testing. In the study, if only the cases of ‘negative for dysplasia’ to ‘high-gradedysplasia’ were considered,10.7%of the patients had an upgrade in their diagnosisafter the additional biopsy examination. This significant number is enough to believethat recommending additional biopsy examination in HPV positive patients withpositive cytologic findings for intraepithelial lesion.