The SureX HPV genotyping test (SureX HPV test), which targets the human papillomavirus (HPV) E6/E7 genes was compared with the Cobas 4800 and Venus HPV tests for detecting 14 high-risk HPV (HR-HPV) types in clinical referral and follow-up patients to evaluate its value for cervical cancer screening.Two different populations were enrolled in the study. The first population comprised 185 cases and was used for comparing the SureX HPV test  with the Cobas 4800 test . The second population comprised 290 cases and was used for comparing the SureX HPV test  with the Venus HPV test . Polymerase chain reaction (PCR) sequencing was performed for further confirmation of discordant results.P < 0.01). In the second population, the overall agreement rate was 95.5%. Thirteen discordant cases were confirmed by PCR sequencing, which showed that the agreement rates were 76.9% between the SureX HPV test and PCR sequencing and 23.1% between the Venus HPV test and PCR sequencing (P < 0.01). With cervical intraepithelial neoplasia grade 2+ (CIN2+) as the reference standard, the sensitivity values of the SureX HPV test and the Venus HPV test were 93.5% and 92.0%, (P > 0.05), while the specificity values were 43.3% and 46.7%, respectively (P > 0.05).In the first population, the overall agreement rate was 95.6% for 14 high-risk HPV types. Eight discordant cases were confirmed by PCR sequencing, which showed that the agreement rates were 75.0% between the SureX HPV test and PCR sequencing and 25.0% between the Cobas 4800 test and PCR sequencing (The SureX HPV test had good consistency with both the Cobas 4800 and Venus HPV tests for 14 HR-HPV types. In addition, it avoided some false negatives and false positives. Therefore, the SureX HPV test can be used for cervical cancer screening. Cervical cancer is one of the most common malignant tumors of the reproductive system in women. There were approximately 570,000 new cases of cervical cancer and 311,000 deaths worldwide in 2018 . LargelyTwo different populations were enrolled in this study. The first population comprised 185 cases with cervical lesions, which contained detectable HPV DNA and underwent cervical cytopathological evaluation in the Department of Pathology of Guangdong Provincial People’s Hospital between November 2017 and March 2018. The second population comprised 290 cases with cervical lesions, which contained detectable HPV DNA and underwent cervical histopathological evaluation in the Department of Clinical Laboratory of the National Cancer Center/Cancer Hospital between March 2018 and October 2018. The inclusion criteria included an age between 21 and 80 years, the absence of pregnancy, an intact cervix, no history of cervical lesions, and no history of chemotherapy, radiotherapy or surgical treatment.In this study, for each patient, flushed the female genital tract with normal saline, probed a specimen collection brush into the cervix, turned the brush to take the cervical secretions after 5 s, and placed the brush in a 2 ml volume collection tube. The cervical specimens collection were performed by an experienced doctor as detailed for each test. And the cervical specimens were stored in a refrigerator (4 °C) and analyzed within 14 days.For the SureX HPV test, total cellular DNA was extracted from cervical specimens using the extraction work station Smart LabAssist-16/32  according to the manufacturer's instructions. For Cobas 4800 HPV test, HPV DNA was extracted from cervical specimens using the automatic nucleic acid extractor cobas × 480 DNA extractor . For the Venus HPV test, HPV DNA was extracted from cervical specimens using the automatic nucleic acid extractor Autrax workstation . For the detection of 14 HR-HPV types, the three different method were used in accordance with the manufacturer's instructions. Negative and positive controls provided in the kits were included in each PCR test.The SureX HPV test  utilized amplification of target HPV DNA by multiplex polymerase chain reaction (PCR) and capillary electrophoresis to detect and genotype 25 HPV types according to the length of specific amplification fragments. The HPV types were identified by the test including HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 81, 82 and 83. For the SureX HPV test, specifically designed primers were targeted on early genes E6, E7 and E1 of HPV types, plasmid pcDNA 3.1(+) (pcDNA for short) and human β-globin locused to make sure the length of the amplified PCR products were at least 3 nucleotides difference. So, via PCR amplification of target DNA, 27 targets could be identified by capillary electrophoresis in a single analysis according to the length of PCR products. The measure of β-globin served as a quality control mechanism to confirm that a negative result was not due to inappropriate sample collection or failure of DNA extraction. The internal control pcDNA could monitor the PCR process and ensured that the testing procedure had been properly performed. Therefore, a validated specimen should show the specific peaks of pcDNA and β-globin. The peak height of pcDNA ought to be equal to or greater than 500 RFU (≥ 500). When the peak of β-globin was absent indicating insufficient cervical cells, we suggested resampling should be conducted. For PCR amplification products (1 µl) subjected to capillary electrophoresis in an ABI 3500 Dx/3500xL Dx genetic analyzer, the cutoff value for determining specimens of HPV positive was:i) Signal of a HPV type ≥ 300 RFU; or ii) Peak area ratio (the ratio of the peak area of a HPV type to the peak area of pcDNA) ≥ 0.2.The cobas4800 HPV test  used primers to define a sequence of approximately 200 nucleotides within the L1 region of the HPV genome, which specifically detected 14 high-risk types . The cobas4800 test mainly included two processes. Firstly, HPV DNA was extracted through automated sample preparation, and then the HPV and β-globulin target DNA sequences were amplified by PCR primers. The amplified target DNA sequences were combined with their corresponding fluorescent probes. There were 4 types of fluorescent probes for detection, namely HPV16, HPV18, β-globulin and 12 other high-risk HPVs. The probes were labeled with different fluorescent dyes, and the subtype of HPV in the sample were determined by real-time monitoring of fluorescent signals. In addition, β-globin was used as an internal control (IC) to ensure a sufficient sample quantity for HPV DNA detection. If the cycle threshold (Ct) cutoff value for HPV16 was ≤ 40.5, a sample was considered HPV-positive; if the Ct cutoff value was > 40.5 and β-globin was effective, it was considered negative; otherwise, it was considered invalid. If the Ct cutoff value for HPV18 or any of the 12 other high-risk types was ≤ 40, a positive result was determined; and if the Ct cutoff value was > 40 and β-globin was effective, a negative result was determined; otherwise, the result was considered invalid.The Venus HPV genotyping test  was based on real-time fluorescence PCR technology and contained a specific ready-to-use system for the detection of 15 15 types of HR- HPV genotypes, including HPV 16, 18, 31,33,35,39,45,51,52,56,58,59,66, 68, 82. Detection of amplified HPV DNA fragments was performed in the fluorimetric channels FAM, HEX/VIC/JOE, TEXAS RED/Cal Red 610 and CY5 with the fluorescent quencher BHQ1. Human minibrain homolog (MNBH) was amplified as an internal control (IC) to indicate the presence of sufficient nucleic acid from the human MNBH gene. The Ct value was calculated. If the Ct value was ≤ 38.0, a sample was considered HPV-positive. If the Ct value of the IC was ≤ 32.0, and "undetermined" or "no CT" was displayed in the other channels, the sample was determined to be HPV-negative. If the Ct value was 38.0–40.0, the reaction was repeated. If the Ct value remained in this range and the amplification curve was a typical S-shape, the sample was considered HPV-positive; if the amplification curve was not a typical S-type, as the sample was considered HPV-negative.https://www.ncbi.nlm.nih.gov/BLAST).PCR sequencing was performed for further confirmation of discordant results. Sequencing reactions was targeted on type-specific E6/E7 gene and performed using the ABI PRISM BigDye Terminator V3.0 kit (Applied Biosystems) and analyzed in an ABI 3730 genetic analyzer (Applied Biosystems) at Sangon Biotech Co. (Shanghai). DNA sequences were then compared with the sequences of known HPV types using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information website  and higher (CIN2+) considered positive. Cytological diagnosis was performed by the Department of Pathology, Guangdong Provincial People’s Hospital; pathological diagnosis, by the Cancer Hospital, Chinese Academy of Medical Sciences.P values of < 0.05 (two-tailed) were considered statistically significant.All statistical analyses were conducted using SPSS 23.0. The consistency checks were evaluated by the Kappa (k) values. Using CIN2+ as a reference, the sensitivity, specificity, and area under the receiver operating characteristic (ROC) curve (AUC) were calculated. All differences with P > 0.05)  and 70.3% (130/185), respectively. Among the 185 cases, there were 12 NILM, 15 LSIL, 75 ASCUS, 43 HSIL+. In the different cervical cytopathological categories, no significant difference was observed in the positive rates of the 14 HR-HPV types between the SureX HPV test and the Cobas 4800 test (The results of the SureX HPV test and the Cobas 4800 test were shown in Table P < 0.01).There were 8 discordant results between the SureX HPV test and the Cobas 4800 tests; these results were confirmed by PCR sequencing, as shown in Table P > 0.05).Among the 290 cases, there were 33 diagnosed with normal cervix 11.4%), 56 CIN1(19.3%), 28 CIN2 (9.7%), 35 CIN3 (12.1%) and 138 cancer (47.6%), and the percentage of CIN2 was 30.7% and CIN 2+ was 69.3%.The overall positive rates of the 290 cases for the SureX HPV test and Venus HPV test were the same , as shown in Table %, 56 CINP < 0.01).The results of the SureX HPV test and the Venus HPV test were shown in Table P < 0.01).Thirteen results were discordant between the SureX HPV test and Venus HPV test; these results were confirmed by PCR sequencing, as shown in Table P > 0.05), and the specificity values were 43.3% and 46.7%, respectively (P > 0.05). The AUCs for the SureX HPV test and Venus HPV test were 0.751  and 0.727 , respectively  curve analysis was used to calculate the sensitivity and specificity. The sensitivity values of the SureX HPV test and Venus HPV test were 93.5% and 92.0%,  between the SureX HPV and the Cobas 4800 tests, and were 94.5%  between the SureX HPV and the Venus HPV tests. Therefore, good concordance was shown for detecting the 14 HR-HPV types between the SureX HPV and the Cobas 4800 tests and between the SureX HPV and the Venus HPV tests.Persistent HR-HPV infection is the key factor for cervical cancer. The most common HPV genotypes causing cervical cancer are HPV 16 and 18 , 22. TheTo determine the actual HPV infection and show the accuracy of the SureX HPV test, PCR sequencing targeted on type-specific E6/E7 gene was performed to further confirm discordant results. In our study, 6 cases were positive by the SureX HPV test and PCR sequencing but negative by the Cobas 4800 test, and 7 were positive by the SureX HPV test and PCR sequencing but negative by the Venus HPV test. As both the Cobas 4800 and the Venus HPV tests target HPV L1 DNA, these 13 results may be false negatives due to missed detection of HPV L1 DNA. In addition, 2 cases were positive by the Cobas 4800 test and PCR sequencing, while 3 cases were negative by the SureX HPV test, and 3 cases were positive by the Venus HPV test and PCR sequencing but negative by the SureX HPV test. The reason for this discrepancy may be that the results of capillary electrophoresis in the SureX HPV test showed a peak height of pcDNA in the five samples of approximately 500 RFU, which may have resulted in low amplification efficiency because of low levels of HPV DNA in the cervical cell specimens. Although HPV DNA was extracted by three different automatic nucleic acid extractor in our study, the three automatic nucleic acid extractor were all magnetic bead methods and the three test had an internal control (IC) to indicate the presence of sufficient nucleic acid.Additionally, studies had shown that although the HR-HPV DNA test had high sensitivity, the potential hazard of this method was that it could detect a large number of women with false-positive results, who were likely to have transient infections. After a few months, HPV is cleared naturally by the body without causing cervical cancer or precancerous lesions . Three pP < 0.01) and the Venus HPV test were 0.727 . Therefore, there was reasonable consistency between the SureX HPV test and Venus HPV test.The method of screening cervical cancer and precancerous lesions by detecting HPV DNA was characterized by high sensitivity, while specificity was related to the positive rate of HPV. The specificity differed greatly among different populations . With CIIn the study, there were 8 cases was negative to three different HR-HPV test and PCR sequence in HSIL+ or CIN2+ population. The possible reasons may be inappropriate sample collection, or the low copies of HPV DNA in the sample, or the patient factors, such as the patient had been treated but not informed.Compared with previously developed and wisely used HPV detection methods such as real time PCR, the SureX HPV test described PCR-capillary electrophoresis method could achieve detection and identification of 25 HPV genotypes in one tube with targeting on specific oncogenic E6/E7. Normally it took around 6 h to have 96 specimens detected and reported if the capillary electrophoresis platform was 24-channel equipped. The testing cost is competitive with regular test methods.In summary, in this study, we compared a novel HPV genotyping test, the SureX HPV test, with the Cobas 4800 and the Venus HPV tests. The SureX HPV test had good consistency with the Cobas 4800 and the Venus HPV tests for detecting 14 HR-HPV types. In addition, the SureX HPV test could avoid some false-negative and false-positive results, and its sensitivity and specificity for pathological grade CIN2+ lesions was equivalent to that of the Venus HPV test. Therefore, the SureX HPV genotyping test is a novel method detecting HPV DNA, which utilizes PCR amplification and capillary electrophoresis to identify 25 HPV types in a single analysis, and it is an accurate, safe, and inexpensive HPV detection method.However, as the population selected for this study was the primary screening-positive population rather than the general population, further comparative analysis of these three methods through large-sample studies in the general population to provide a basis for the development of a large-scale cervical cancer screening strategy.


Human papillomavirus testing as an established screenings test allow for the early detection and treatment of cervical cancer. Testing positive for HPV may have adverse consequences for women. This study aimed to explore the psychological impacts of testing positive for HPV on women in a developing country with a distinct cultural and religious background.Qualitative face-to-face semi-structured interviews were conducted with 40 Iranian women who received a positive high-risk HPV result. Content analysis approach was used to data analysis through MAXQDA10.Three main categories were emerged: initial confrontation; STD-related psychological burden; and rebuilding health. Initial reactions to positive HPV results were shock, unrealistic fear, confusion, distress, and financial concerns. Stigma was manifested in form of self-blame, fear of HPV-disclosure, negative body image, being stigmatized by healthcare providers, and receiving health care anonymously. Refusal to use insurance services showed how evident and powerful the stigma was. Most women reported lifestyles and sexual behaviors modifications to help their immune system to clear HPV; indicating that the screening can work as a valuable opportunity to improve women's physical and sexual health. Regular follow-up, safe sex and a focus on spirituality enable women infected with HPV to take control of the situation. Worrying about other HPV-linked cancers  and fears of partner infection indicated that women consider HPV to be more than just a cause of cervical cancer.The findings implied to the HPV-positive women's need to support and factual information. Designing and implementing interventions that mitigate the psychological effect of positive HPV test results can highlight the potential benefits of screening for women's health. HPV is a globally common virus in the sexually active population that can lead to certain types of cancers, including cervix, vagina, and vulva in women, penis in men, and anus, and back of throat in both women and men . The onlThere are several limitations in the available literature. To date, although extensive qualitative research has explored women's adverse psychological responses to HPV positive results, most of studies were conducted in the countries where HPV is common and HPV vaccination programmes have been introduced .Despite the availability of the vaccine, national HPV vaccination programmes are very rare in the Middle East: only 1 country (the United Arab Emirates) has effectively introduced the vaccine through a national programme and very few others have intended to implement it in the near future . In IranIn Iran, according to Globocan (2020), age-standardized incidence rate for cervical cancer and mortality is 2.3 and 1.5 in 100,000 individuals, respectively . It has A qualitative study was conducted based on the conventional content analysis approach to understand the psychological responses of women to positive result of HPV test , 21.This study was carried out at a referral gynecology-oncology outpatient clinic (equipped with colposcopy and directed by oncologist-gynecologist SSH and her colleagues), which serves a large population of women from all around the country with diverse backgrounds, and socio-economic status. The services in this clinic (including colposcopy) are very low-priced and offered to all patients, regardless of their insurance coverage.) The clinic is located in a large, busy, university-based, and geographically accessible complex, at the center of Tehran, the capital of Iran.There is no unified HPV-related educational content (or leaflet) for Iranian women undergoing HPV testing. Health care providers who perform sampling or interpret the result for women will explain HPV to them. As a result, what women learn from their provider about HPV may vary in quality and quantity in different settings. All women referred to clinic with their Pap smear and hrHPV-positive results . Moreover, the authorities of hospital agreed to cooperate in the study. Direct quotes that are representative of the participants have been presented. The data (audio files) will be destroyed 18 months from the ending of this qualitative study (June 1st). All methods were carried out in accordance with relevant guidelines and regulations.Participants' characteristics shown in Table The results are presented in terms of three main categories: initial confrontation, STD-related psychological burden, and rebuilding health. Examples of codes and sub-categories are shown in Table All the participants in the study were asked about the initial experience of HPV positivity. Initial confrontation reflects their feelings and thoughts firstly after receiving a positive HPV result, consist of four categories.As evidenced by the various respondents, the typical reactions to results were shock, fear, and distress. A 45-year-old widow recently tested positive for HPV stated: “My daughter said: 'why you are so scared?' I said: 'We are subconsciously afraid of everything we don't know about. To me, HPV is like jinn; unknown and scary!” .A subdivision of participants has used coping mechanisms, such as denial. They generally rationalized their results with the probability of human errors of laboratory technicians. Some women did not expect their positive results due to being monogamous and having rigid hygienic habits.Distress had led to losing appetite and sleep problems in several women. Some reported feeling overwhelmed by irrational fears and obsessive concerns. Several interviewees reported a disrupting of their roles (as an employee or student) due to distress and sadness. A single woman mentioned that severe anxiety and fear of death caused her neurotic back pain which led her to job loss. Few women, who all have received positive HPV results within the last month, had catastrophizing psychological conditions such as thinking of death and suicide.Almost all participants reported that they had little or no knowledge of HPV on the day of the test. After receiving positive results, they had tried to find out more about HPV on the Internet. They described the Internet as a private accessible source of comprehensive knowledge, but confusing and stressful as well. Confusion is well described as: “I was frantically searching. My mind was like a busy intersection, and I felt directionless.” .Women commonly mentioned feeling uncertainty and confusion about the source and the time of contracting the virus. In most cases, the patient did not know whom to dump her misdirected anger. Most participants held their last partner accountable for infection (partner-directed guilt and anger). Women with mixed-status types stated that the partner having genital warts first was almost considered the source of infection.Only two married women stated they had been cheating on their husband and accepted being accountable for the contamination (self-blame and self-directed guilt).Acquiring virus from non-sexual sources , as a defense mechanism or myth, mentioned only by married women with a long-term relationship that their husband claims being life-long monogamous.Few stated that others  were accountable for infection (deflected guilt and anger).Some women revealed being vigilant about other STIs after getting information on the Internet. They worried that they might be infected with other STIs too.Some women assumed that a positive hrHPV result means they definitely develop cervical cancer in the future. Women with more hrHPV genotypes, including 16/18 and abnormal cytology CIN expresseIn our study, the main concerns of women with normal cervical cytology were other HPV-related cancers . There is no approved test to determine HPV in the mouth, throat, and anus. Women revealed concerns over where exactly the virus exists. Women reporting participation in oro-genital practices were anxious about HPV potentials for causing oropharynx cancers. For instance, one woman reported she had mixed her salivary glands up with oral cancer. In response to these concerns, few said some laboratories had taken oral samples for HPV testing without a doctor's request, which is unnecessary and costly. It seemed women did not know what specialist they should see for sore in the mouth or anal canal. Questions about anal cancer mentioned by a woman complained of severe anal itching: “I think my anus is infected with the virus too. Is there a test like Pap smear for anal cells?” .The majority of women mentioned that HPV testing, performing further procedures such as freezing or colposcopy, and following tests for other STIs  were expensive. “In the first weeks, it cost me three to four million Toman. Except for HIV test, other tests and procedures were costly since they're not covered by insurance.” .Much of the adverse emotional reactions related to the sexually transmitted nature of HPV were escalated by religious values, cultural norms, and social rules. This category was strongly linked to the context of the study.Responses surrounding stigma were identified in different forms. A few participants, mostly single women, had assumed acquiring the virus as a punishment or repaying for individual mistakes. Three single women mentioned intense self-blame and guilt exacerbated by the self-perceived risk of cancer. They described it a devastating combination of shame and fear: “Dying of genital cancer due to a sexual disease is horribly embarrassing.” .I didn't want to use my private insurance for colposcopy so that my family wouldn't know I have HPV. I think this story holds for so many girls because when I was in the doctor's office, I saw patients anxiously calling outside and borrowing money for colposcopy. I was recently hired at a health center. I'm afraid they will fire me if they find out I have HPV. A form of stigma experienced in this context was related to receiving health care anonymously. However, there are governmental-funded colposcopy clinics in almost every province; some women interviewed pointed out that they prefer not to use governmental services.When I took my mother to the gynecologist for HPV screening, Doctor said: 'Hajieh,The majority of women reported that healthcare providers treat them with respect. Few women felt being stigmatized by healthcare providers and perceived as promiscuous and likely infected with other STIs. One of them reported:Another woman stated that her husband had been rejected by his urologist who was supposed to examine his warts: “as soon as he find out I have genital warts, he said without even looking at us, 'Get out of my office. I am not examining this disease!' And we got distraught.” .As interviews showed, three-quarters of participants had revealed their results to at least one person . Experiencing rejection and isolation, several women had regretted communicating their test results with others. They explained how misinformation about passing the virus through casual contacts would cause a social denial.HPV positivity, whether with or without genital warts, had dramatically changed women’s feelings about their body image. Some women described feeling “disgusting”, “contaminated”, and “sexually unattractive”. Others reported a sense of “ugly” or “leper”.Touching my warts makes me sick. I don't want my partner to even look at them. I feel inadequate. I feel losing a part of my womanhood. I believe all the beauty of a woman is in her lips, breasts, and genitals. If one of these three has a problem, a woman gets devastated. Given the growing attention women have about their genitals' appearance, they suffer more in the presence of warts.Few women had concealed their HPV from their partner. Concealment had led to receiving less financial support and going for appointments alone. These women were more likely to feel overwhelmed and helpless.The consequences of disclosing the results with a partner were different. Separation due to the HPV was common among singles because it was considered as a sign of infidelity and being unhealthy. One woman stated that her divorce was mostly because of HPV. “He was not only a disgusting sex partner but also a traitor. Divorcing him, I protected myself from further infections.” .A newlywed woman expressed disappointment as: “It just felt unfair! I feel I don't deserve to get an STD! I am so unlucky that after years of virginity, my first sexual relationship ended up like this!” .Very few women said they had been unfaithful. Considering themselves as the source of the infection, women often hid their HPV from their husband. Only a woman whose husband had noticed her infidelity, treated her with contempt and rejection.A divorcee woman stated that her ex-husband accused her of deceiving him by hiding her incurable contaminating disease and divorced her.Some couples tried to accept HPV experiencing painful emotional challenges and conflict. Women described it as an emotional dilemma. The women were finally able to forgive their husbands who attributed the virus to their ex- partners. HPV-positive women with normal cervical cytology, whose husband was responsible for the infection and took good care of them, had a milder psychological response to HPV.Worries about auto-inoculation were raised by some women. They thought they might spread the virus from one region of their body to another, . Few women reported obsessive hygienic behaviors such as not using toilet paper and avoiding touching their genitals for fear of topical spreading virus to nearby tissues. “My doctor said that if I touch my genitals and then my mouth, it's like I had oral sex, and I have to avoid it, so I wear disposable gloves in the bathroom.” .Some women had stopped having sex; Most of them for the protection of themselves (self-protection) and a small number for the protection of their partners (partner-protection). Although some women reported using condoms usually, there was also worry and confusion about its protective role, which caused some of them not to have sex at all. Abstaining from sex was associated with concurrent "abnormal" cytology and hrHPV. These women maintain the physical act of sex would deteriorate their condition. “Realizing that sex is a risk factor of cervical cancer, I was more afraid of sex. My health is more important than sex.” .Women with mixed HPV genotypes and perineum warts were worried about internalizing more virus load from genital skin warts to their cervix during penetrative sex even with condoms.A subset of women appeared to be more affected by concerns about risks associated with anal or oral sex or even kissing. It sometimes resulted in abstention from these practices.Women who were sure they had lately gotten the virus (recently losing virginity or newly married women) were more likely to stop having sex for a while to clear the virus up. A woman stated that her decision was influenced by what they have learned about the virus: “HPV goes away on its own within six months to a year” and “re‐infection not allowing the virus to be cleared.” .Some patients considered non-sexual modes of HPV contagion. They were nervous about passing the virus to others through casual contacts, surfaces, towels, clothes, and common utensils . Participants who were mother noted that they were more concerned about infecting their children than serious complications of the virus, such as CC. Excessive hand washing has been reported by several women to protect families from contracting the virus. “Look at all dry cracks in my hands. I use bleach every day. My fingers are chapped and wounded. I was even thinking about living apart from my family so that my children wouldn't be infected.” .Despite a positive HPV result being distressing news for many women, it also has the potential to improve health outcomes. Interviews demonstrated that women had more focus on their physical, sexual, and spiritual health after receiving positive HPV results.I will accept anything if I can stay safe from cancer. I have bought everything anyone recommended to the extent that I need a daily plan for taking those. Although HPV has no cure and it goes away by the person's immune system, interviews verified that some women consider strengthening their immune system to be a treatment for HPV. To increase the likelihood of virus clearance, they adopt lifestyles changes. Almost all participants reported arbitrary use of vitamins, minerals, mushrooms, etc.Almost all women interviewed stated they want to continue regular CC follow-ups but few women believed identifying hrHPV means that they will not get CC. These women were less upset and had milder negative psychological response to HPV positivity.Most women received positive HPV results 3–6 months before the interview reported lifestyle modifications to boost their immune system such as regular exercise, smoking cessation, reducing drinking habits, vegetarian diet, fasting, sugar cessation, healthy tea drinking, and adjusting sleep routines. These women were more likely to repeat the HPV test earlier than scheduled (12 months later) to check the effectiveness of the assumed treatment.The majority of participants and their partners had taken one dose of the HPV vaccine and planned to complete their vaccination.Some participants reported that after receiving their positive HPV results, they had adopted a positive attitude towards safe sex and practiced sexual behaviors like abstinence, limiting the number of sexual partners, using condoms, and avoiding oral and anal sex.Women revealed a strong need for talking and receiving emotional supports from their significant others. “I needed to talk it out instead of keeping it bottled up.” .Several women felt less stigma and gained emotional support from being in online groups with other patients. Most participants felt better after the interview because they could ask their questions and talk to somebody listening to them carefully.It may sound odd, but knowing I have HPV gave me a new idea or angle on things. I made some positive changes in my life. I think HPV may very well keep my family and me healthier and prevent other potential health issues or other STDs like HIV down the road. I am glad that I'm being vigilant about controlling what I can… A subset of participants expressed some positive reactions such as cherishing the results to prevent worse outcomes like cervical cancer. Regular check-ups after receiving positive HPV test can be viewed as a blessing in disguise.Thank God, I know it. My friend got ovarian cancer, and within three months, she went through a total hysterectomy. I'll never get cervical cancer because doctors can handle it at the precancerous stage. It's a bit of good luck. Women mentioned their religious beliefs, as a source of support that had helped them manage their stress. They hoped for healing and were grateful.Two single women who felt deeply guilty talked about repentance. One stated: “I always say: God forgive me! I feel very helpless and I ask God to give me another chance! I pray that it will go away.” .This study provides an insight into the psychosocial responses of testing positive for HPV. In line with other studies, a positive HPV result was associated with stigma, distress, unrealistic fear of cancer, and concerns over deteriorating of emotional relationships , 26–29. Excessive fear of cancer was seen in women who besides HPV (type 16/18), also had abnormal cytological results. Concern about other HPV-related cancers  and fear of partner infection show that women consider HPV more than cervical cancer agent.Similar to another study , InterneIt has been suggested that Iranian health insurance companies should be committed to including CC screening (Pap smear and HPV-testing) in their basic service packages . It needSome of the findings are specific to religious, social, and cultural norms and cannot be generalized to other communities. According to McCaffery, HPV testing does represent a conflict for women from specific cultural and religious backgrounds . It seemIn societies that HPV is common normalization can help de-stigmatization  but in oThe studies, though limited, verified low health literacy among Iranians related to HPV . PromotiThe most concern of participants who were mothers was related to contaminating their children. Since Sabeena publicized “oral HPV infection appears to be significant in the transfer of HPV among family members” , the worInterviews showed women take boosting their immune system as a treatment. Also, despite FDASince no cure for HPV is presently available, the recommendations toward the treatment of HPV-positive patients are often conservative management with a close follow-up. Although WHO has mentioned that CC is mostly preventable , a limitInterviews were carried out over a fifteen-month period, due to researcher availability. However, we do not think this would have affected our findings as there was no major news or online media coverage of HPV during this time period, or change in health professional communication guidance.Results indicate that healthcare providers need to know women's negative psychological responses to positive HPV result and address their concerns by providing more attention and support.Some considerations are needed to how HPV-information is conveyed since misinformation received from invalid sources amplified women's fear of cancer. Attention should be paid to questions and concerns of women with mixed-status since they may experience more rejection and fear of spreading the virus. Asking about concurrent genital warts in hrHPV screening is suggested. The participants who were mothers feared of contaminating their children, so they should be educated in preventive behaviors and encouraged to vaccinate their teens. Being in online groups with other HPV-positive patients can lessen stigma and provide emotional support from women. Lifestyle modification to boost immunity to clear HPV indicated that the positive results of screening may have the potential to enhance women's physical and sexual health. Health authorities can provide multidisciplinary self-care guidance, including healthy eating, regular sleep, safe sex, and regular follow-up to promote women empowerment and shared decision making in the process of cervical cancer screening. More research is suggested to determine factors associated with adverse psychological responses to the positive result of HPV.This study provides an overview of negative psychological responses to hrHPV positivity. Women with HPV are in a difficult situation and need support and factual information. Providing deep understanding and wider resonance, these findings can help health authorities and policymakers of countries that are in the process of adopting HPV testing for cervical screening. In addition to cervical cancer, other HPV-associated cancers need to be taken into account when medical professionals are counseling HPV-positive women. Therefore, clinicians and other medical professionals involved in the HPV testing, need to be prepared to provide HPV-positive women with update comprehensive information, and emotional support. In conclusion, findings may help clinicians and educators to create an evidence-based educational programme or psychological-based intervention to improve women's physical, mental and sexual health.Additional file 1: Interview guide.


Several determining factors are involved in HPV infection outcomes; human leukocyte antigen (HLA) polymorphisms have been described as related factors. This study has ascertained the effect of genetic variation on HLA-DRB1 and DQB1 genes on HPV-16/-18/-31/-33/-45 and -58 clearance and redetection in Colombian women. PCR and qPCR were used for viral identification and the Illumina MiSeq system was used for HLA-typing of cervical samples (n = 276). Survival models were adjusted for identifying alleles/haplotypes related to HPV clearance/redetection; L1/L2 protein-epitope binding to MHC-II molecules was also predicted. Significant associations suggested effects favouring or hampering clearance/redetection events depending on the viral type involved in infection, e.g. just DRB1*12:01:01G favoured HPV-16 (coeff: 4.8) and HPV-45 clearance (coeff: 12.65) whilst HPV-18 (coeff: 2E-15), HPV-31 (coeff: 8E-17) and HPV-58 hindered elimination (coeff: 1E-14). An effect was only observed for some alelles when configured as haplotypes, e.g. DRB1*04:07:01G (having the greatest frequency in the target population) was associated with DQB1*02:01:1G or *03:02:03. Epitope prediction identified 23 clearance-related peptides and 29 were redetection-related; eight might have been related to HPV-16/-18 and -58 persistence and one to HPV-18 elimination. HLA allele/haplotype relationship with the course of HPV infection (clearance/redetection) depended on the infecting HPV type, in line with the specific viral epitopes displayed. The causal relationship between persistent high-risk types of human papillomavirus (HR-HPV) infections and the development of cervical lesions progressing to cervical cancer (CC) has been extensively demonstrated5. Many infections (around 90%) are eliminated during an average period of 2 years; however, some of them are latent as non-productive infection is limited to the epithelium’s base layer and they do not become detected7. A complex interaction between viral and host factors is responsible for CC’s clinical course and development9. Host immunological and genetic factors play an essential role in HPV infection outcome11. Human leukocyte antigen (HLA) system alleles and haplotypes have been reported as being responsible for antigen presentation, recognition of infected cells and HPV elimination13.Human papilloma virus (HPV) is the most common sexually-transmitted viral infection, having around 291 million infections annuallyDRB1*07:02, DRB1*13:01, DQA1*01:03 and DQB1*06:0316. However, most reports contain inconsistencies given the differences in the populations being analysed, the typing techniques, the outcomes considered and the methodological design19. Few longitudinal studies have evaluated these molecules’ association with HPV’s natural history of infection (considering them as being generic infections) and just HPV-16 and -18 have been considered at type-specific level22.Some HLA alleles and haplotypes have been described as being associated with CC, such as DRB1 and DQB1 alleles related to the clearance and redetection of the 6 HPV types having the greatest distribution in Colombia  in a cohort of Colombian women using next generation sequencing (NGS) for HLA typing and quantitative PCR assay for viral detection. L1 and L2 protein peptides fitting into alleles were analysed for predicting which of them might have been related to infection events regarding each viral type. Such information is relevant to understanding specific infections’ natural history and the genetic factors modulating them. The results should prove useful in identifying inmunological biomarkers enabling establishing HPV infection suceptibility and its clinical course.This study was aimed at identifying HLA-Two hundred and seventy-six women were considered eligible for this study; 12 were excluded as complete information was missing (lack of information regarding HPV or HLA typing). Cohort follow-up lasted 32.3 months; 206 women were visited four times and 59 women a fifth time during the follow-up period. Table DRB1 and DQB1 allele and haplotype frequencies. The results regarding type-specific infection clearance for 219 women out of the 276 included in this study have already been reported23; Table Supplementary Tables Evaluating time-related redetection percentage revealed that most viral types  became positive again after 5 months’ non-detection Fig. .Figure 1Calculating multicollinearity between the variables  and tolerance) revealed no collinearity between the variables included in the model. Cox multivariate and log-normal models were thus adjusted for identifying alleles and haplotypes related to infection clearance/persistence (Supplementary Tables Alleles/haplotypes having associations favouring clearance/redetection were found (considered in the model as having greater and earlier probability of occurrence) as well as hindering associations (considered as lower probability and later occurrence).DRB1 and nine DQB1 allele associations with HR-HPV infection clearance were found  and 6 for DQB1  continued being significant after p values (p ≤ 0.001) had been corrected  and five for DQB1  after the p values had been corrected (p ≤ 0.001), 17 of these associations favouring infection redetection and 33 hindering it  events found for this viral type in the study population . Twenty-four of these were positively associated with the probability of infection clearance whilst 63 had a negative effect, i.e. favouring HPV infection persistence   Fig. , 64 favoDRB1* 04:11:01 favouring HPV-31, -45 and -58 redetection. Different associations were found depending on HR-HPV type (favouring and hindering a particular event), e.g. DRB1*03:02:01 was associated with favouring HPV-31 and -33 clearance, whilst HPV-16, -45 and -58 hindered clearance , e.g. DRB1*15:02:01G-DQB1*06:01:01G)  as was modification of the effect when alleles were associated and not associated independently (e.g. DRB1*11:04:01G-DQB1*02:01:01G for redetection)  Figs. . HaplotyDRB1*11:02:01 was associated with a lower probability of HPV-31 clearance and earlier redetection of the same viral type. DRB1*03:02:01-DQB1*04:02:01 was associated with earlier HPV-31 clearance and its later redetection  for the same HPV type, i.e. an allele/haplotype was associated with hindering clearance of an HPV type and also associated with a greater probability of redetection of the same viral type. For example, DRB1*01:03, DRB1*07:11 (HPV-16 and -45) and DRB1*13:04 ). Others could have explained the differences regarding an event due to being specific for each virus   Table  to S19.TDRB1*04:08 and DRB1*15:03, HPV-18 L1-GHYIILFLRNVNVFPIFLQM presented by DRB1*04:08, DRB1*13:03 and DRB1*15:03 and HPV-18 L2-TTSFAFFKYSPTISSASSYS presented by DRB1*04:08, DRB1*07:11 and DRB1*15:03.Interestingly, some peptides could have favoured or hindered a clearance event depending on the allele presenting it, e.g. HPV-16 L1-QFPLGRKFLLQAGLKAKPKF presented by DRB1*01:03 (HPV-18 and HPV-45), DRB1*13:04, DRB1*15:02 (HPV-31 and HPV-58), whilst peptides presented by the other alleles could explain the differences found regarding events .Differences were seen throughout follow-up regarding infection patterns for all 6 HR-HPV types, this being consistent with previous studies23. Differences concerning redetection rates were seen to depend on the HR-HPV type; the percentages and rates reported for every viral type were similar to those reported previously28. However, redetection events could not be differentiated between new infection and/or latent infection due to study design29.This study’s findings regarding infection clearance correlated with those of a prior cohort study carried out with part of the population in this study30. Cumulative post-clearance redetection in this study, including all types of HR-HPV analysed, was 7.42% and cumulative redetection following non-detection was 22.10%  were found to be positive (greater probability or earlier occurrence of an event) or negative (lower probability or later occurrence of an event) Figs.  and 3. T33. Immunoinformatics led to identifying viral peptides which could be considered factors favouring viral persistence in a host since they were related to a lower probability/later occurrence of a clearance event and the greater probability/earlier occurrence of redetection , could have favoured HPV replication due to lower antigen presentation capability favouring the replication of such latent infections in a host, so that only when the amount of copies exceeded the detection threshold could they have been identified and diagnosedl Tables  and 4.DRB1 and DQB1 alleles and haplotypes identified in the study population, highlighting differences in the relationship with the clinical course of such infection in a host  and haplotypes (DRB1*03:01:01G-DQB1*02:01:01G and DRB1*13:01 01G-DQB1*06:03:01G) previously reported as associated were not significant for this study’s population36.Specific associations with HPV type were found for most HLA-st Figs.  and 3. Sst Figs.  and S14.DRB1*01:03, DRB1*04:02, DRB1*04:08, DRB1*04:10, DRB1*04:11 DRB1*08:04, DRB1*11:14, DRB1*13:03, DRB1*13:04 and DRB1*13:05 alleles and DRB1*03:02:01-DQB1*04:02:01G and DRB1*04:02:01-DQB1*03:02:01G haplotypes) were not consistent with that reported previously or constituted new findings. This was especially true regarding redetection, this being the first study to consider this outcome 19.Other associations  and demonstrating their potential usefulness as therapeutic anti-HPV peptide vaccines.This study has thus reported, for the first time, alleles and haplotypes (typed by NGS) associated with clearance/persistence and redetection events, as well as L1 and L2 epitopes from the six most frequently occurring HPV types which are responsible for around 85% of CC cases. The information obtained through this study provides relevant knowledge for understanding the genetic component in the immune response against HR-HPV types and the natural history of CC. The associations described here enable constructing the bases for future studies aimed at evaluating the impact and effectiveness of anti-HPV vaccines and treatments (current and future), considering each population’s genetic particularities.23. The women had been attending hospitals in the Colombian cities of Bogotá, Girardot and Chaparral between January 2007 and March 2010; they voluntarily participated in the study and declared that they had not changed their place of residence in at least two years after the start of the study. Detailed information regarding the study population and the procedures related to the 6 HR-HPV types’ detection and quantification have been described in previous publications23.The study’s target population consisted of a cohort of women who had been participating in a Fundación Instituto de Inmunología de Colombia (FIDIC) multicentre study evaluating 6 HR-HPV types’ persistence, clearance and reinfectionDRB1 and DQB typing were excluded from the analysis  and real-time PCR viral identification results were included in the study. Women whose cervical samples did not have the minimum amount of DNA and/or required quality for HLA-A QIAamp 96 DNA Blood Kit  was used for extracting genomic DNA (gDNA) from the 276 aliquots containing the cervical samples (stored in FIDIC’s DNA bank), following the manufacturer’s recommendations and specifications. The cervical samples’ gDNA quality for HLA typing was determined as >10 ng/μL concentration and ≥1.8/2.0 absorbance ratio at 260/280 nm.38. The human β-globin gene was amplified by PCR for evaluating DNA integrity, using GH20/PC04 primers. This was followed by using two sets of generic primers targeting a region of the L1 gene for the generic detection of HPV (GP5 +/GP6 + and MY09/MY11)  and qPCR was used for quantifying the viral load, following previously described proceduresE5, E6 and E7 genes; these were specific for HR-HPV-16, -18, - 31, - 33, -45 or -58 (Supplementary Table 38.Samples proving positive by at least one generic primer set were typed using primers targeting a region located on the 11–106) serial dilutions were obtained from plasmid DNA (known concentration) from each viral type and the HMBS gene for making the calibration curve. The human HMBS gene was used for validating DNA integrity and determining the amount of viral copies per cell. The samples were analysed for the aforementioned six HR-HPV types, involving absolute  and relative quantification (HPV copies /cell = HPV copies /(HMBS/2 copies)) of each type’s load. Six dilutions of plasmid DNA were included for each type and included as controls for HMBS in each analysis along with a negative NTC control (no template control)23.Real-time PCR detection was used for determining the amount of viral copies and the CFX96 Touch qPCR detection system was used for analysis; the primers and TaqMan probes used here are described in Supplementary Table loci (3x resolution) from good quality gDNA samples40. The IPD-IMGT/HLA database (https://www.ebi.ac.uk/ipd/imgt/hla), published in January 2018 (3.31.0), was used for assigning alleles. WHO Nomenclature Committee for Factors of the HLA System guidelines were followed when reporting alleles, using National Marrow Donor Program (NMDP) codes and G codes for ambiguous alleles 41.Illumina MiSeq  was used for typing HLA-DRB1 and DQB1 l molecules from exons 2 and 3 from every Frequencies and percentages were used for qualitative variables and measures of central tendency (median and mean) for quantitative ones, along with their measures of dispersion (interquartile ranges (IQR) and standard deviation (SD).DRB1 and DQB1 allele and haplotype frequencies. Persistence was considered as being the detection of the same type of HPV in at least two consecutive visits, whilst clearance was determined as no viral detection in two consecutive samples after a positive detection when analysing infection by each of the 6 HPV types . Redetection was taken as being positive for the same viral type following its prior non-detection (regardless of infection clearance) .An expectation-maximisation (EM) algorithm was used for obtaining HLA- DRB1 and DQB1 relationship with type-specific infection (clearance/redetection) outcome44. Multicollinearity between the variables included in the models was also evaluated by VIF and tolerance.Cox-proportional hazard and accelerated failure time (AFT) models were adjusted when the assumption of proportionality was not met to evaluate allele and HLA-45. Hazard ratios (HR) and time ratios (TR) were reported, depending on the model used47. Independent models were run for each HPV type. TR values less than 1 denoted an earlier occurrence of an event, whilst values greater than 1 indicated a later occurrence of such event47.Every AFT models’ goodness of fit was evaluated in line with Akaike information criterion (AIC) and Bayesian information criterion (BIC), selecting the model having the best fit p < 0.200 value in univariate analysis was taken when selecting the independent variables for adjusting the models and the change in the crude estimator when added to the models. Origin, age, the amount of sexual partners, a background of abortion, coinfection (i.e. infection by 2 or more HR-HPV types) and absolute viral load  were taken as independent variables.A p values, considering the multiple alleles and haplotypes identified in the study population and that there was no a priori hypothesis concerning the associated alleles/haplotypes48. Two-tailed tests were used for hypotheses testing (0.05 significance) and STATA14 software was used for the aforementioned analysis.The Bonferroni method was used for correcting each model’s raw 49, using the Technical University of Denmark’s Systems Biology Department’s Center for Biological Sequence Analysis’ NetMHCIIpan 3.2 server prediction method50. Peptides predicted to have a strong binding threshold (<5% Rank) were sought manually by aligning each HR-HPV type’s complete L1 or L2 capsid protein reference sequences . This was aimed at finding similar or different regions and determining possible explanations for the associations found between HLA-II and HR-HPV. Peptide sequences coinciding with the secretion signal sequence were not considered for analysis.The Immune Epitope Database (IEDB) was used for predicting binding peptides (20 aa long) for every HLA-II alleleSupplementary information.


Objectives: This study aimed to describe the study design, and to analyze the type-specific distribution of cervical high-risk human papillomavirus (hrHPV) infection and its association with cytological and histological results in a large population-based screening program in Buji Street, Shenzhen, China.Methods: A total of 10,186 women aged 21-70 years were co-tested by Cobas4800 HPV assay and liquid-based cytology. Women were referred to colposcopy by virtue of being HPV16/18-positive, Other hrHPV-positive/ cytology ≥ASCUS, or HPV-negative/ cytology ≥LSIL. Three-year histological follow-up data were recorded.Results: The overall prevalence of hrHPV infection was 11.1%; among them, the highest type was Other hrHPV (8.9%), followed by HPV16 (1.6%) and HPV18 (0.6%). Moreover, the prevalence of hrHPV and that of HPV16 increased with cytological severity (trendP <0.001). In the baseline phase, 106 women had cervical intraepithelial neoplasia 2/3 (CIN2/3) and six had cervical cancers. During 3-year follow-up, 12 cases of CIN2/3 and no cancers were identified. For HPV16-positive women with normal cytology, the baseline risks of CIN2/3 or worse (CIN2+/CIN3+) were 15.5% (7.0-23.9%) and 4.2% (1.4-8.5%) respectively. For Other hrHPV-positive women with normal cytology, the cumulative 3-year risks of CIN2+/CIN3+ were 3.1% (1.0-5.2%) and 0.7% (0.3-2.1%) respectively. Strikingly, 75.8% (322/425) of abnormal cytology and 50.9% (29/57) HSIL cytology were attributed to Other hrHPV infection in HPV-positive women. Similarly, Other hrHPV infection led to large proportions of CIN2 (62.7%) and CIN3+ (43.9%) over 3-year follow-up.Conclusions: The co-testing modality is a feasible, effective and safe option for cervical cancer screening in urban population. Great importance should also be attached to 'genotypes excluding HPV16/18' and separate detection of each genotype when considering screening and vaccination strategy. Cervical cancer is a common gynecologic oncology and a global public health problem HPV testing has been incorporated in cervical cancer screening to triage cases with atypical cells of undetermined significance (ASCUS) cytology Recently, we conducted a prospective, population-based cervical cancer screening program in Buji Street, Longgang District, Shenzhen, China (Southern China). Buji Street is located in the middle of Shenzhen city, where has a rapid economic development, resulting in Shenzhen's high-mobility and dense population, but relatively backward medical care. No screening program had ever been organized in Buji Street, and the vast majority of the people had never been screened for cervical cancer, particularly via HPV testing. Given the limited data on cervical HPV infection and correlative cytology or histology findings, it's hard to establish effective cervical cancer prevention strategies.From this large cohort, we provide the study design, describe the prevalence of hrHPV genotypes, and analyze the association between cervical hrHPV genotypes and cytological abnormalities at baseline as well as histological results through 3-year follow-up. We hope this large-scale population-based study is instructive for the prevention of cervical cancer in urban areas similar to Buji street, Shenzhen, China.This prospective observational study was conducted in two phases: a baseline phase, and a three-year longitudinal phase Figure . Women a® Preservative Solution  and centralized to PUSH for cytology/HPV tests. 1.5mL SurePath specimen was prepared for HPV assay on a Cobas4800 system  per the instruction of manufacturer. The residual specimen was prepared for AutoCyte® thin-layer liquid-based cytology test . Cytology slides were reviewed by the senior cytologists from PUSH according to the Bethesda classification system (TBS) 2014 After written informed consent was given, a brief medical history was acquired. Routine gynecological examination was performed by PUSH gynecologists, and the specimens of cervical exfoliated cells were brushed at the same time. Cervical exfoliated cell samples were obtained via a standardized sampling procedure by removing cervical secretions and rotating brush 3 times. Afterwards, the specimen on the clinician-sampling brush was placed in a small bottle containing 12mL of SurePathWomen met any of the results (1) HPV16 and/or HPV18 positivity or (2) Other hrHPV positivity and cytology ≥ASCUS or (3) HPV negative and cytology ≥LSIL were called back for colposcopy-directed biopsy according to the Preventive Oncology International (POI) microbiopsy protocol Women had positive screening results (hrHPV-positive or LBC ≥LSIL) and without a histology of CIN2+ in the baseline phase were eligible for the three-year histological follow-up phase Figure . Follow-P-value <0.05 was considered statistically significant.Since women with a result of Other hrHPV positivity/ NILM cytology did not meet the criteria for either immediate colposcopy or return to routine screening and would be deferred to 6-12 months follow-up . Among them, 9,469 (93.0%) were considered NILM, 717 (7.0%) were diagnosed as abnormal cytology, 421 (4.1%) were classified as ASCUS, 29 (0.3%) were affected by AGC, 158 (1.6%) were diagnosed as LSIL, 47 (0.5%) were categorized as ASC-H, and 62 (0.6%) were positive for HSIL , followed by HPV16 (1.6%) and HPV18 (0.6%) Table . The enrtrend<0.0001), similar results were showed for HPV16 positivity and HPV16 and/or HPV18 positivity  than Other hrHPV infection. However, among HPV-positive women, 75.8% (322/425) of abnormal cytology and 50.9% (29/57) HSIL cytology were attributed to Other hrHPV infection.The overall hrHPV positivity rate among women with abnormal cytology was 59.3% (425 of 717), which was approximately 7.4 times greater than that among women with normal cytology . The prevalence of hrHPV increased with cytological severity, from 7.5% among women with NILM to 91.9% among those with HSIL (Ptrend <0.0001). Similar results were showed for HPV16 positivity and HPV16 and/or HPV18 positivity (Ptrend <0.0001). For women with histological results of ≤CIN1 and CIN2, the most prevalent infection type was Other hrHPV ; for women diagnosed as CIN3+, the most frequent genotype was HPV16 (47.4%), followed by Other hrHPV (43.9%). Of the 12 cases diagnosed as CIN2+ during 3-year follow-up phase, all of them were infected with Other hrHPV at baseline. Hierarchical typing results grouped as HPV16, HPV18, Other hrHPV, and negative showed a significant linear association with histological severity  results for CIN2+/CIN3+ detected at baseline and at cumulative baseline plus 3-year follow-up were estimated in Table The distribution and infection patterns of HPV types in various areas provides a scientific basis for the establishment of HPV-based screening strategies and vaccination programs The issue whether extending genotyping and individually detecting 'Other hrHPV' or not is still under debate and of increasing importance To fit the change, policies should be adjusted to focus more attention on extended genotypes excluding HPV16/18, which is useful for the development of tailored HPV vaccines targeting Other hrHPV genotypes and for the adaption of screening algorithms for increasingly vaccinated cohorts. Moreover, the current HPV assays may require a modification, such as adding more previously low prevalence hrHPV subtypes and separating each genotype Detailed information on prevalence of each combination of HPV/cytology results was also described. The prevalence of HPV-positive/normal cytology result was reported ranged from 1.9% to 9.8% Here was 7.5%) .5% 7. ThIn addition to assessing results of HPV and/or cytology, it is valuable to know the risk for high-grade lesions conferred by specific genotypes when evaluating screening strategies. The 2019 ASCCP guidelines have moved from result-based management to risk-based management, and recommend that women having ≥4% risk of CIN3+ immediately of their positive screening test are referred for colposcopy and potential biopsy, whereas those having risk below that threshold require retesting within 1 year In this program, women with a result of HPV negative/ LSIL cytology were referred to immediate colposcopy. However, there was no high-grade lesion found in this subgroup through 3-year follow-up. A prior study documented that risk for follow-up high-grade lesions in this subgroup was low and also that no cervical cancer was diagnosed, thus repeating co-testing after one year was an appropriate option for these women Although cervical cancer screening guidelines have recommended management measures for women with each positive HPV/cytology co-testing results based on the principle of “equal management of equal risks''There are several limitations of this study. Firstly, there was no records of using vaccination, occupations, birth control, smoking history, and number of sexual partners in the baseline data collected; despite that, most of the women enrolled in the population may be unvaccinated against HPV given that HPV vaccines were approved in mainland China since 2016, and have not been incorporated to National Immunization Program yet, thus the coverage is low to nonexistent. In addition, there might be some verification bias since not all the women underwent biopsy, for instance, some women with a result of HPV negative/ (NILM or ASCUS) were referred to routine screening, and some women infected with Other hrHPV not producing a detectable cytological abnormality (≥ASCUS) would be deferred to 6-12 months follow-up. However, cumulative histological results through 1-year or 3-year follow-up were included in the analysis.An important strength of this study lies in the large-scale, and well-organization of the screening program, as well as the standard screening procedures, such as the standardized sampling process, the uniform requirements for colposcopy referral, and the standard POI protocol for colposcopy/biopsy. Another strength is the stratification of the association between HPV genotypes and histological grades by age groups and cytology status. Moreover, comprehensive and complete data, ranging from HPV prevalence and cytology outcome to histological follow-up results, were provided in this large prospective study, which provides necessary information for the clinical management of different subgroups of women based on HPV and cytology results. Furthermore, we also tried our best to obtain the 3-year follow-up data, due to the high mobility and dense population in highly urbanized Buji Street, it is arduous and essential to obtain such longitudinal data.Based on a large cohort of women with cytology/HPV results and follow-up histology, this study described a screening modality of hrHPV/cytology co-testing in urban China, and explored the potential role of hrHPV testing and genotyping in risk assessment by different cytological categories, providing necessary information for cervical screening strategies and a new insight in assessing the potential efficacy of regional vaccinations. Our data support the guidelines using genotyping for HPV16/18 as a triage test since its high risk for cervical diseases. Moreover, our results highlight the importance of 'hrHPV genotypes excluding HPV16/18' and separate detection of these genotypes similar to HPV16/18 when considering triage and vaccination strategy given its considerably high prevalence and significant contribution to a high proportion of cervical abnormalities. The screening model of HPV/cytology co-testing is suitable for urban areas similar to Buji street, Shenzhen, China.


Cervical cancer is the second commonest female cancer in Sri Lanka. Two major drawbacks of the present cervical cancer screening programme are the suboptimal sensitivity of the pap smear and the low coverage. The objective of the study is to determine the feasibility of a new HPV/DNA test among 35 -years -old ever-married women in a district of Sri Lanka.stof July 2018 to 30th November 2018 in the public health divisions called Medical Officer of Health (MOH) areas of Kalutara district. The study population is comprised of ever-married women 35 years of age. Three women from each cluster (n = 413) were selected by consecutive sampling. A total of 918 women were recruited. HPV/DNA cervical specimen collection (n = 822) was carried out. Cervical specimens were tested by two cytoscreeners with the cobas 4800 PCR based screening machine. Clients’ perceptions and prevalence were assessed. The follow-up of women with positive HPV/DNA screening results was carried out. The operational and technical feasibility of the screening test were assessed. Data entry was done by using the statistical package IBM SPSS version 20.A community based descriptive cross-sectional study was conducted from 1n = 836). Clients’ perception was highly positive for HPV/DNA screening test procedure  and 99.6% (n = 819) of clients had mentioned that the HPV/DNA screening test is worthwhile to be incorporated into the National Cervical Cancer Screening program. The prevalence of HPV was 6.2% (95%CI: 6.18–6.22%). The coverage of the HPV/DNA screening was 89.5%(n = 822). Invalid results reported were 0.12% (n = 1). The percentage of HPV/DNA test positive women who underwent pap test within 3 months of the initial screening was 100% (n = 51), while the percentage of women who attempted to get a colposcopy within the 1 month of referral was 86.7% (n = 13).Overall response rate was 91.1% (HPV/DNA test implementation as a primary cervical cancer screening method is feasible among the 35- year age cohort of ever- married women in Kalutara district. It is necessary to further attempt alternative methods of cobas 4800 HPV/DNA test, which would be much suitable for resource-limited settings.The online version contains supplementary material available at 10.1186/s12889-021-10190-4. Cervical cancer is the 2nd leading cause of female cancer in Sri Lanka and women at risk for cervical cancer are more than 8.4 million . In Sri Cervical cancer is virtually associated with human papillomavirus infection (HPV) but HPV infection does not always cause cervical cancer. Most women even infected with high-risk HPV types never develop cervical cancer, as most of this infection regardless of HPV type is short-lived and the body eliminates them spontaneously in ≤2 years. A small percentage of women with high-risk HPV infection can have persistent infection and progress into pre-cancer and even fewer women will progress to have invasive cancer [More than a hundred and fifty HPV genotypes have been identified to date and nearly 60 different types of them are known to infect the human genital tract including cervix uteri. There are carcinogenic and non-carcinogenic genotypes and 10–15 carcinogenic genotypes are mainly associated with cervical cancer . Some caCervical cancer screening is a secondary preventive strategy for the early detection of abnormal tissues from the cervix. In 1996, the concept of the Well Woman Clinic (WWC) programme was introduced to Sri Lanka and cervical cancer screening through WWCs was based on only visual inspection of the cervix. Subsequently, in 1998 cervical cancer screening based on visual inspection of the cervix with pap smear was included in WWC services.The mean age of marriage for Sri Lankan female is 23.9 years . PersistNearly after 20 years of existence of the cervical cancer screening program, in contrast to the vigorous preventive measures, there is no marked reduction of incidence, of cervical cancer in Sri Lanka , 8 due tHPV/DNA test is used to detect cervicovaginal HPV infection in women and the sensitivity of the test to detect CIN II viral load is 92.9% . The objA community based descriptive cross-sectional study was conducted from 1st of July 2018 to 30th November 2018. The study population comprised of ever-married women 35 years of age in public health divisions called Medical Officer of Health (MOH) areas of Kalutara district. Women with; diagnosed invasive cervical cancer, pregnancy, ≤ 3 months in the postpartum period, hysterectomy, per vaginal bleeding, active infection at the time of examination evidenced by medical records or by visual inspection, currently on treatment for HPV infection, diagnosed physical or mental retardation or disease status and women who did not reside within the district continuously for ≥3 months before the date of the survey were excluded from the study at the field and clinic settings.For the calculation of the sample size to assess the present prevalence, we assumed that the expected prevalence of HPV was 3.3%  and the A Public Health Midwife (PHM) area was taken as a cluster. In the district, there are 413 PHM areas. Eligible Families Register/s was the sampling frame. Three women were selected from each cluster from the list of 35- year age cohort ever-married women population prepared from relevant area Eligible Families Register/s according to the ascending order of register numbers. We selected the first subject from the list by using a lot method and then two more subjects were selected forward and consecutively. A total number of 918 women were recruited to the study after applying possible exclusion criteria in the field setting. The number of women who approached clinics for HPV/DNA specimen collection was 836 (91.1%), therefore the number of non-respondents was 82 (8.9%). After applying the rest of the exclusion criteria at the clinic setting 822 (89.5%) women were subjected to a specimen collection.Information regarding socio-demographic characteristics and client perceptions for new HPV/DNA test implementation were gathered by using an interviewer-administered questionnaire, developed for this study and attached as an additional file Public Health Midwives (PHMM) were uniformly trained to locate households. A two-day training session was conducted for research assistants (RA)  to familiarize them with proceedings. They were uniformly trained to administer an interviewer- administered questionnaire with socio-demographic characteristics of participants and it was administered at a separate and neutral place within the house. Age was calculated using the date of birth by recall or by using the National Identity Card and approximated to the last completed year. Once a questionnaire was completed, the participant was given a clinic appointment card with a reference number and invited to attend the clinic.Staff training was done to collect cervical HPV/DNA specimens. Videos and guidelines were used for staff training. Instruction regarding accurate numbering of specimens, completion of specimen request forms, and preparation for transport were also included in the training sessions. Two cyto-screeners were uniformly trained for specimen barcoding, to handle the machine and report writing to ensure the quality of performance. Colposcopists too were uniformly trained to ensure the quality of performance.n = 822) collection was carried out by Medical Officers (MOO) or Public Health Nursing Sisters (PHNSS) at WWCs. Cusco’s speculum was inserted to visualize the cervix before obtaining the HPV/DNA cervical specimen. HPV/DNA specimens obtained from the cervix using special broom-like devices were separately placed into HPV/DNA specimen collection containers with cell collection media/thinprep (20 ml). Cervical specimens were packed and transported to the laboratory District General Hospital (DGH), Kalutara. Prepared guidelines were strictly adhered to during data collection, barcoding, and preparation for transport and attached as an additional file HPV/DNA cervical specimenn = 82 collectVariables used to assess the feasibility were; client perceptions for the new HPV/DNA test and it’s positive follow-up, the overall prevalence of cervicovaginal HPV infection and it’s subgroup analysis, HPV/DNA screen positive follow-up results according to the algorithm of Asia Oceania guidelines for the implementation of programs for cervical cancer prevention and control  and opern = 822) at community WWCs (n = 89) in Kalutara district by RAs. The descriptive longitudinal study design was used to describe the follow-up of women with positive HPV/DNA screening results. All HPV/DNA positives (n = 51) were subjected to pap smear at community WWCs and pap screening was carried out at District General Hospital, Kalutara, and National Institute of Health Science (NIHS), Kalutara. HPV/DNA positive and cytology result ≥ Atypical Squamous Cells of Undetermined Significance (ASCUS) were referred to colposcopy either at Kethumathie Maternity Hospital, Panadura, or Family Health Bureau, Colombo.Client perceptions for the new HPV/DNA test and it’s positive follow-up were gathered by using an interviewer-administered questionnaire  were women, who attended for an HPV test, and non-respondents  were women, who had an interview but did not attend for an HPV test. A total number of 822 women were subjected to HPV/DNA specimen collection after excluding 14 women at the clinic setting  between respondents and non-respondents according to nationality and religion. There was no significant difference (p > 0.05) between respondents and non-respondents according to the occupational status of women. The majority of women, who participated in the study were Sinhala  and Buddhist , while the non-participation among Muslims  and Islamic  women were high. Working women population were higher among non-respondents  than respondents   and 99.6% (n = 819) of clients were mentioned that the HPV/DNA screening test should be incorporated into the National Cervical Cancer Screening programme in Sri Lanka (Table n = 818) were aware of screen positive follow-up of the HPV/DNA screening test and according to them the possibility to repeat a conventional pap test within 6 weeks in the case of HPV/DNA test positive result was 100% (n = 822)  Table .Table 2n = 14), 0.24%(n = 2) and 4.14%(n = 34) respectively , while the prevalence of the high risk HPV serotype infection 16, 18 and 12 pooled risk  were 1.7%(n = 51) were Sinhala  and Buddhist . The educational level of screen-positive women was ≤ grade 5 in 17.6% (n = 9), while the income was Rs ≤ 15,000 among 25.5%(n = 13) of screen-positive women , while the percentage of cytology ≥ASCUS among HPV/DNA screened positive for serotypes 16 and 18, only serotypes 12 pooled high risk and serotype 16 with 12 pooled high risk was 31.25% (n = 5), 26.5%(n = 9) and 100%(n = 1) respectively.The overall percentage of cytological abnormality ≥ ASCUS among women who were tested positive for HPV/DNA was 29.4% (n = 12), who were positive for HPV/DNA with cytological abnormality ≥ ASCUS. Among women with biopsy tissue abnormality 83.3% (n = 10) were CIN I and CIN II, while 16.7% (n = 2) were CIN III.The biopsy tissue abnormality was found among 80% of women (n = 5), while the percentage of biopsy tissue abnormality among HPV/DNA screened positive for only serotypes 12 pooled high risk and serotype 16 with 12 pooled high risk positive was 17.6% (n = 6) and 100% (n = 1) respectively . The percentage of invalid results reported was 0.12% (n = 1). The management adherence percentage of HPV/DNA positives for pap screening within 3 months of the initial screening was 100%(n = 51), while the percentage of women who attempted to get a colposcopy within 1 month of referral was 86.7% (n = 13).The coverage of the HPV/DNA screening test was 89.5%(The length of the clinic HPV/DNA specimen obtaining procedure per client (minutes) at a community WWC by a Medical Officer (MO) and Public Health Nursing Sister (PHNS) was 10 min and 13 min respectively, while the procedure meantime of the cytoscreener per specimen (including report writing) was 3.5 min. The mean time duration for specimen collection and transportation to the laboratory was 7 days, while the report delivery to the relevant MOH office following specimen collection was 13 days.Well Woman Clinics are provided services related to prevention and early detection of some selected non-communicable diseases including cancers in women. In the beginning, cervical cancer screening through WWCs was based on only visual inspection of the cervix, and in 1998 cervical cancer screening based on pap smear was included in WWC services. One major drawback of the present program is, suboptimal sensitivity (53%) . TherefoThe clinical usefulness of HPV triage for women with ASCUS cytology even with the carefully validated test was limited by the fact on average 43.7% were high-risk HPV positive, while the prevalence of CIN II or worse was only 5.1%, in a study conducted in the United States . Even ifHigh positive client perceptions for the HPV/DNA screening and it’s positive follow-up indicated adequate community awareness, field, and clinic staff performance in the district. The overall prevalence of high-risk cervicovaginal infection was 6.2%. When the proportion of women tested positive for high-risk HPV/DNA in a country is at least 1%, it indicates a good quality standard for the HPV/DNA screening test as a cervical cancer screening method .Follow-up of women with positive HPV/DNA screening was carried out according to the algorithm of Asia Oceania guidelines for the implementation of programs for cervical cancer prevention and control . The impAll HPV/DNA screened positives were referred for a pap test at a community WWC. A positive follow-up register was strictly maintained at each MOH level to minimize the loss to follow-up. All HPV/DNA screened positives with cytology results ≥ASCUS were referred to colposcopy. HPV/DNA, pap test, and colposcopy screening facility were offered to participants free of charge. Free test reports were incentives to participants. Inadequate colposcopic facilities within the district was one of the biggest challenges ever faced in the present study. Some referred clients for colposcopy had to repeatedly visit the procedure due to rush at the clinic as that was a combined clinic. Some women were reluctant to visit the Family Health Bureau, Colombo colposcopy clinic due to the traveling inconvenience.n = 5), while the percentage of HPV/DNA screened positive for serotypes 12 pooled high risk was 26.5%(n = 9). Human papillomavirus serotypes 16 and 18 had a greater absolute risk of biopsy tissue abnormality  therefore the carcinogenicity of these 2 serotypes were high, while the percentage of HPV/DNA screened positive for serotypes 12 pooled high-risk serotypes was 17.6%(n = 6) with a low risk of carcinogenicity, which gives a similar pattern to the world [HPV/DNA screened positive for serotypes 16 and 18 had a greater percentage of cytological abnormalities ≥ ASCUS 31.25%, %, n = 5,he world .HPV/DNA screening as a primary cervical cancer screening will reduce the need for pap tests as only HPV/DNA positives are referred for pap cytological triage, therefore the work burden of cytoscreeners and Consultant Histopathologists would be reduced. In addition to that, it will reduce the colposcopy burden in a country by 40–50% as only HPV/DNA screened positives with cytology result ≥ ASCUS are referred for colposcopy procedure. Moreover prolonging the screening interval is recommended for HPV/DNA screening, because of its high sensitivity  and thern = 822). The percentage of invalid results reported was 0.12% (n = 1), which was a good indicator for adequate staff training for the HPV/DNA screening implementation in the district. All HPV/DNA screened positive women  who were referred had undergone a pap test within 3 months of initial screening , while out of fifteen women who were referred for colposcopy 13 were attempted for a colposcopy procedure  within 1 month of referral, which was showed good quality standards of cervical cancer screening [Operational and technical feasibility indicators of the new HPV/DNA screening implementation were developed based on the literature review. A panel of experts reviewed and selected indicators for the study to minimize the selection bias associated with indicators. As the first author recruited subjects after carefully applying exclusion criteria for HPV/DNA screening the coverage of the HPV/DNA screening was high . If the machine runs for 28 working days per month, annually one cobas 4800 machine can screen a maximum of 63,168 specimens, which would be adequate to screen the whole Western province 35- year age cohort women population . According to the mid-year population estimates for 2017 in Sri Lanka, the total number of 35- year age cohort women population assumed by 1% of the total population was 214,440. One procedure cost was Rs. 2881.95(USD 15.6). Therefore the country has to spend Rs. 618 million rupees (USD 3.35 million) annually to incorporate cobas 4800 HPV/DNA screening test with “thinprep cell collection media/Liquid Based Cytology (LBC)” to screen all 35-year age cohort women population in Sri Lanka.Simple inexpensive approaches for cervical cancer screening by cobas 4800 test in low-resource settings were successfully attempted in some other countries. In a research study, each woman was subjected to HPV/DNA specimen collection with a swab placed into a sarstedt tube and a cytobrush placed into a “thinprep cell collection media” The rates of the agreement were reported very high (> 90%) for any high-risk HPV (HR-HPV) types, HR-HPV 16, HR-HPV 18 and HR-HPV 12 high pooled risk serotypes .In some countries, dry transport in dry tubes was successfully attempted , and fouThis study was restricted to one district out of 25 districts in Sri Lanka due to logistic constraints. The population characteristics and the public health infrastructure of the district favored the generalizability of the research findings to the whole country.When implementing a new screening test into the National programme, in a low resource setting like Sri Lanka, needs to always concentrate on the economic feasibility and cost-effectiveness. Therefore, needs to determine the most appropriate method for Sri Lanka by assessing other alternative methods of HPV/DNA testing by cobas 4800 screening machine.The present study has explored, that the HPV/DNA screening test is feasible to be incorporated into the National Cervical Cancer Screening programme as a primary cervical cancer screening method. It is recommended to further assess alternative methods of cobas 4800 HPV/DNA testing to determine the most appropriate method for a resource-limited setting.Additional file 1 Interviewer Administered Questionnaire. Basic Information & Background characteristic of the participants, Socio-demographic characteristic of participants & Participant’s perception of the new HPV/DNA screening procedure.Additional file 2. Guideline for HPV/DNA cervical specimen collection for health staff.Additional file 3 Number of women recruited into the study (n = 918).