Cytology (smears and liquid-based) identified that 34.6% of the participants had low-grade squamous intraepithelial lesions (LSILs), and 3.5% had high-grade squamous intraepithelial lesions (HSILs). Neither HPV nor lesions were associated with low CD4+ counts . Of the women, 60% were infected in the cervix and 45% in the anal canal, with an agreement of at least one genotype in 90%. The HR-HPV types associated with HSILs were HPV66, 33, 52, 51, 45, 18, and 68.The incidence of anal intraepithelial neoplasias associated with HPV is rising worldwide. In the general population, this pathology is rare, but individuals living with HIV/AIDS are at a significantly higher risk. We aimed to study HPV infection and performed cytological screening to study the epidemiological and behavioral determinants in a group of men and women living with HIV from a region in Mexico with high HIV incidence. This was a cross-sectional study including adults living with HIV/AIDS performed in Merida (Mexico). We invited patients of public HIV/STD clinics and those affiliated with social organizations of people living with HIV to participate in the study. Participants responded to an instrument to assess their risky behaviors and clinical history. Swabs from the anal canal and cervix and anal cytology specimens were obtained by medical staff from women and by self-sampling from men. For the 200 participants, 169 men and 31 women, anal HPV PCR tests resulted in 59.8% positivity (62.6% of men and 45.2% of women), and 17 genotypes were identified. The most frequent high-risk (HR) types for the anal canal were: HPV33 (35.3%), HPV58 (20.6%), HPV66 (18.6%), HPV45 (17.6%), and HPV16 (14.7%). Multiple genotypes were found in over 80% of the participants. Receptive anal intercourse in the previous 12 months, inconsistent condom use, and detectable HIV titers (≥50 cc/mL) were associated with HPV infection ( Human papillomavirus (HPV) is the most common sexually transmitted viral infection worldwide. The WHO has estimated that all sexually active subjects will be infected at some point in their lives. As these are mostly subclinical infections, the relevance of HPV infection is not only due to its high incidence, but also because many genotypes are associated with cancer development. Worldwide, 4.5% of all cancers are associated with HPV infection. The relative contribution of genotypes HPV16/18 and HPV6/11/16/18/31/33/45/52/58 was also estimated to represent 4.5% of all cancers worldwide [Persistent infection with oncogenic HPV genotypes is the main risk factor for developing anal intraepithelial neoplasia (AIN). AIN is an infrequent pathology that has risen in incidence in the last few decades, with people living with HIV/AIDS being at higher risk . Unlike HPV infection in HIV patients is reportedly common and presents with multiple genotypes. The genotype distribution varies geographically. In Latin America, studies on HPV infection in the anal canal and associated pathologies are still limited.We aimed to study HPV infection and perform cytological screening to generate knowledge regarding the anal HPV epidemiology and behavioral determinants in a group of men and women living with HIV from a region in Mexico with high HIV incidence.This was a cross-sectional study including adults living with HIV/AIDS performed in Merida . The project was approved by the Ethics Committee Board of the Research Center “Dr. Hideyo Noguchi” (protocol number record CEI-0014-2016). The internal academic register is CIR-B-2017-0003.Patients of public HIV/STD clinics were invited to participate through printed fliers, and patients affiliated to four social organizations of people living with HIV/AIDS were invited through in-person group sessions or talks. The inclusion criteria for participation were: adults (>18 years old) with a HIV+ diagnosis who provided informed consent.Two anal canal samples were obtained from the male participants for a conventional Pap smear and liquid-based cytology. Samples from male participants were obtained with a cytobrush by self-sampling, following instructions provided by the staff. In the case of women, two samples from the anal canal and one from the cervix were obtained with Dacron swabs and a cytobrush, respectively, by trained personnel during pelvic examinations; self-sampling was not adopted by women. Conventional Pap-smear slides from the anal canal samples were obtained and fixed with cytospray on site and, together with liquid-based cytology specimens, were transported to an external laboratory for processing.After the anal Pap-smear slides were prepared, the remaining anal cells from the sampling device were recovered in saline solution, which was supplemented with an equal volume of absolute ethanol to produce a final 50% ethanol concentration; cervical cells were directly introduced in 50% ethanol at room temperature. All cell samples in ethanol were transported to the virology laboratory at room temperature for processing for the molecular detection of HPV, as described below.Anal cells from all participants and cervical cells from women were washed with PBS solution to remove the ethanol, and DNA extraction was performed with a DNAeasy Blood and Tissue Kit  following the manufacturer’s instructions.®, Waltham, MA, USA); the following cycling conditions were applied: 40 cycles of 95 °C for 30 s, annealing at 55 °C for 30 s and 72 °C for 30 s, and final elongation at 72 °C for 10 min. Amplification products were visualized in agarose gels.Human β-globin was amplified as an internal quality control with end-point PCR, using 1 μM oligonucleotides PC04 and GH20 , as reported in [, as repo®), reaching a volume of 50 µL, and subjected the mix to the following cycling conditions: 40 cycles of 95 °C for 1 min; annealing at 55 °C for 1 min and 72 °C for 1 min; and final elongation at 72 °C for 5 min. Amplification products were visualized in acrylamide 8% gels.HPV detection was performed by the amplification of the conserved L1 gene region with end-point PCR, using oligonucleotide mix PGMY11/09 . We addeAffordable HPV genotyping was performed with multiplex nested end-point PCR following Sotlar et al.’s  protocolPap smears and liquid-based cytology specimens from the anal canal were evaluated in an external laboratory by a certified cytopathologist.p < 0.05). The final database is available from the authors upon reasonable request.Participants responded to a survey instrument to assess their epidemiological risk factors, including sociodemographic information; sexual behavior; self-reported clinical history; tobacco, drug, and alcohol use; CD4+ status; and HIV viral load. Data recovered from the survey and laboratory results were combined and analyzed in SPSS database software using the chi-square test and odds ratio  to adjust the items. The same group was allowed to undergo testing in the most comfortable position for anal sampling before the project started .In this project, 204 HIV+ persons volunteered to participate; after 4 were excluded for not adhering to the inclusion criteria, 200 participants were finally included in the study, and all participants provided informed consent.The 200 included participants comprised 169 men and 31 women. The age distribution among the participants was between 18 and 72 years, with a mean of 37 years of age.The occupation data showed that 69.5% (139/200) were employed, 9% (18/200) were unemployed, 9% (18/200) were students, 9.5% (19/200) were homemakers, and 3% (6/200) were retired. Regarding marital status, 80.5% (161/200) were single; 9% (18/200) were married; and 10.5% (21/200) were divorced, widowed, or other.Regarding tobacco exposure, 33% (66/200) were smokers, and 35% (70/200) indicated passive exposure (cohabitating with a smoker). People who injected drugs represented only 4% of the cohort (8/200).All participants were sexually active; the average age at first intercourse was 16 years old for men (range 4–30 years), and 15 years old for women (range 6–25 years); young ages may indicate child abuse (data not shown). The age ranges and averages at first intercourse with a male partner were the same for both men and women.Overall, according to the analysis of sexual practices, 83% (166/200) reported that they had engaged in anoreceptive intercourse, with an average of 41 sexual partners in a lifetime and 5 sexual partners in the last 12 months. Active oral sex was reported by 79.5% (159/200) of all participants, with an average of 11 partners in the last 12 months.In men, 94% (159/169) reported having sex with men during their lifetime . Anoreceptive sexual practices were reported by 87% (148/169) of them, with an average of 7 sexual partners in the last 12 months. In the case of women, 42% (18/31) reported anal intercourse, 61%  with 1–2 sexual partners in the last 12 months.Lifetime consistent condom use during oral sex was reported by only 4.5% , and 55.3% (99/179) never used a condom for oral practices; the rest varied in the frequency of use. For receptive anal intercourse, lifetime consistent condom use was reported by 14.2% ; 48.3% (85/176) used a condom most of the time, 27.8% (49/176) rarely, and 9.6% (17/176) never.The condom use data for various sexual practices over the previous 12 months according to sex of the participant are presented in Trichomonas was not reported in the studied population.A history of at least one sexually transmitted infection (STI) was reported by 75.5% (151/200) of the participants, and 24.5% (49/200) reported never having had an STI. The most frequently reported STIs were syphilis 28% (55/200), perianal condyloma 25.5% (51/200), gonorrhea 16% (32/200), genital warts 16% (32/200), genital herpes 14.5% (29/200), pubic pediculosis 15% (29/200), urethritis 12% (24/200), hepatitis B 9.5% (19/200), and HPV 7.5% (15/200). Other reported STIs were hepatitis C and molluscum contagiosum  . TrichomInformation concerning HIV viral load was obtained from 80.5% of the participants . Of these, 61.5% (99/161) had an undetectable viral load or lower than 50 copies/mL; 26% (42/161) had a viral load ranging from 50 to 1000 copies/mL; and 12.4% (20/161) had viral loads ranging from 1000 to 20,000 copies/mL.3; 41.6% (67/161) had >200 to 500 cells/mm3; and 37.26% (60/161) had CD4+ counts > 500 cells/mm3.CD4+ counts were available for 161 participants (data for 39 participants were not available). Of these, 21.1% (34/161) had CD4+ counts lower than 200 cells/mmAll samples from men were obtained by self-sampling following the method established in the pilot, and all samples from women were obtained by staff. Anal samples were collected from all 200 participants  and cervix samples from all 31 female participants.β-globin  was amplified in 194/200 of the anal samples, confirming that 97% of the samples were adequate for molecular testing. Those six samples  without β-globin amplification were considered inadequate and were not further considered for HPV detection or typing. The HPV L1 gene was detected in 116/194 of the adequate anal samples; therefore, 59.8% tested positive for HPV. According to the participants’ sex, 45.2% (14/31) of women and 62.6% (102/163) of men were HPV-positive.The HPV typing method included HR-HPV types 16, 18, 31, 33, 45, 52, 58, 59, 35, 68, 39, and 66 and LR-HPV types 6/11, 42, 43, and 44. HPV genotypes were identified in 112 (96.6%) of the 116 positive samples. The remaining four HPV positives (3.4%) had non-identifiable genotypes (not included in the 18 genotype primers used). In general, the most frequent genotypes detected were LR-HPV 6/11 (36.3%) and HR-HPV33 (35.3%). HR-HPV 16, 18, 45, 51, and 66 were found at frequencies of 14.7, 13.7, 17.6, 13.7, and 18.6%, respectively. LR-HPV 43, 24, and 44 were also found  .Notably, multiple genotypes were detected in 80.2% of the anal samples, and 75% (85/112 identifiable genotypes) had at least one HR genotype.HPV was detected in cervix samples from 60% of the women . All corresponded to at least one HR-HPV type, with HPV52 being the most frequent, found in 27.8% (5/18), followed by types 16, 18, 58, and 33, each found in 16.7% (3/18). Of the cervix samples from HPV-positive women, 89% (16/18) were infected with more than one genotype. In comparison, only 38.7% (12/31) were positive in the anal canal, and 10 had an infection in both anatomical regions, with a concordance of at least one genotype in most of these cases (9/10) .Available anal Pap smears and anal liquid-based cytology specimens were interpreted by an external cytopathology laboratory. In total, 197 anal cytopathology results were obtained for conventional anal Pap smears (three slides missing or broken) from 167 men (99%) and 30 women (97%), as follows: LSILs were found in 3 women and 33 men, and HSILs were found in 4 men.For liquid-based cytology, a total of 108 results were available (92 missing) from 14 women and 94 men; among these, anal lesions were reported as follows: LSILs were found in 3 women and 33 men, and HSIL was found in 3 men.To identify the agreement of the cytology methods, we included only a subset of the population. The group of 116 HPV PCR positives resulted in an overall good agreement , though The genotypes present in HSIL cases were as follows: the first patient had a unique infection with HPV66; the second patient was infected with HPV52 and 33; the third patient had HPV 45, 33, 44, 51, and 66; and the fourth patient had HPV18, 6/11, 42, 68, 66, and 51.p < 0.0001). Also significantly associated with HPV infection was having a detectable HIV viral load (>50 cc/mL) (p = 0.006). Smoking was marginally significant (p = 0.043). Age at first intercourse, CD4+ counts (<200 cells/mm3), and injectable drug use were not associated with HPV infection  were not associated with anal intraepithelial lesions detected with either cytology technique .Additional analyses showed that a detectable HIV viral load (>50 cc/mL) and low CD4+ counts , independently of their HIV status, as shown in a meta-analysis by Lin et al. , which iIn Mexico, few studies on anal HPV are available. We reported close to 60% HPV+ positivity for anal canal samples in the total population, corresponding to 62.6% of men. In Mexico City (in the center of Mexico) a study in a group of 31 HIV+ MSM reported 74.19% (23/31) HPV anal infections , and a sThe presence of HR-HPV types in the cited studies from Mexico in HIV+ MSM varied from 28 to 86% ,14. The A wide variety of genotypes were identified in the anal canal, but we had a particular interest in HR-HPV 16, 18, and 58, though their frequencies varied geographically (11–50%). A study performed in HIV+ MSM from Mexico reported 22.2%, 17.2%, and 13.9% prevalence for HPV 16, 58, and 18, respectively, and 48.8% for LR-HPV6/11 . AnotherThe results of this study are comparable to those of other international studies. In Hungary, HPV 16 was the most frequent at 42.5%, followed by types 18 (22.5%) and 58 (11.3%); the frequency of low-risk HPV6/11 was 49.1% in a population including HIV+ MSM . In compIn fact, HR-HPV genotypes have been found in 85% of AIN cases, as shown in a meta-analysis by Machalek et al. , with HPInterestingly, in our study, the most frequent HR-HPV genotype in all men was HPV33 (33.3%), contrary to the abovementioned studies reporting low frequencies for this genotype 5–7%) ,17,18. I% ,17,18.Regarding LR-HPV types, anal infections in HIV+ MSM are frequent and responsible for low-grade lesions, condyloma, and warts, which can be recurrent or also associated with other HR types ,24. In oOur study also included women; although this group was small, their participation was highly valuable as, to our knowledge, this is the first study to work with HIV+ women in Mexico. Previous studies have reported the prevalence of anal infections in HIV+ women to range from 31% in Zimbabwe to 90% in the United States ,26. In tWomen living with HIV and those with a history of cervical cancer are considered at a higher risk of developing anal pathologies; the anatomical proximity of the affected regions may play a role . In a prIt is pertinent to mention that the genotypes included in the currently available HPV vaccine  are 6, 11, 16, and 18, and that this vaccine has been shown to be safe and immunogenic in up to 98% of HIV+ MSM . In MexiThe other genotypes identified in our population  are covered by the Gardasil 9 nonavalent version, in addition to types 6, 11, 16, and 18. As of 2022, this vaccine is not available in Mexico; however, internationally, the vaccination of HIV+ men and women has shown benefits, and routine vaccination will hopefully become a reality in the near future for this vulnerable population.Despite the possible cross-protection of the multivalent vaccines, it is important to note that infection with multiple genotypes in HIV+ cases is very frequent. In our study, the infection of the anal canal with multiple genotypes was frequent, accounting for 80% of all HPV-positive participants, similar to other studies in Latin America, where the rate of multiple infections ranged from 55 to 80.5% ,30.Risk factors for anal HPV infection include tobacco use, a higher number of sexual partners, sexual debut at an earlier age, and low condom use during anoreceptive intercourse . In our 3 were not significantly associated with HPV infection or with cytological abnormalities (p > 0.05). On the contrary, detectable HIV RNA loads were indeed significantly associated with HPV infection , p > 0.05, data not shown). This could have been an effect of the population size.Immunological and HIV therapy are related to HPV and lesion development, as evidenced in published meta-analysis ; howeverAnal cytology as a primary screening method for the detection of AIN is suggested as part of the triage of HIV+ MSM. A 2019 meta-analysis by Gonçalves et al.  includinIn the development of our study, we faced the challenge that it is not common to find trained personnel for interpreting anal cytology in our region. Most HIV patients nationwide receive healthcare in public centers (CAPASITS—Centro Ambulatorio para la Atencion del VIH/SIDA e ITS), and the national guidelines for HIV care published by the Mexican health minister  include High-resolution anoscopy is considered the gold standard for the detection of anal cancer, and although official uniform international documents are lacking, several algorithms have been proposed . Expert Finally, follow-ups for cases that are at risk of developing AIN is urged, including counseling and educational strategies to address risk factors.In this study, we identified several risk factors associated with HPV infection in the anal canal and showed that HR-HPV types and co-infection with multiple types were frequent in the studied population; high-grade lesions, although infrequent, could be associated with genotypes considered uncommon in the general population and should not be underestimated. Women were of particularly high interest to us and should be prioritized in future research.


A 16-year-old girl with no history of renal disease had a fever of 38 °C after her second HPV vaccination and was identified as positive for proteinuria. As she maintained urinary protein of 3.10 g/gCr and 5–9 urinary red blood cells/HPF, a renal biopsy was performed and small spikes on PAM staining with the granular deposition of IgG1++ and IgG3+ on the glomerular capillary wall were discovered by immunofluorescence, although PLA2R immunostaining was negative. Analysis by electron microscope showed electron density deposition in the form of fine particles under the epithelium. The diagnosis was secondary membranous nephropathy stage II. Immunostaining with the anti-p16 INK4a antibody was positive for glomerular cells, and Western blot analysis of urinary protein showed a positive band for p16 INK4a. However, laser-microdissection mass spectrometry analysis of a paraffin section of glomeruli failed to detect HPV proteins. It is possible that the patient was already infected with HPV and administration of the HPV vaccine may have caused secondary membranous nephropathy. SARS-CoV-2 vaccination was promoted during the COVID-19 pandemic; however, the effectiveness and side effects of the mRNA vaccines for SARS-CoV-2 were not fully elucidated. Gross hematuria in patients with IgA nephropathy and recurrence of nephrotic syndrome in patients with minimal change nephrotic syndrome (MCNS) were reported as side effects following SARS-CoV-2 vaccination ,4. Some Human papillomavirus HPV is a double-stranded DNA virus that can be divided into more than 100 types from the gene sequence of the surface capsid protein L1. High-risk HPV16, 18, 31, 33, 45, 51, 52, 56, 58, and 66 cause cervical cancer, and low-risk HPV6, 11, etc., cause condyloma acuminata ,8. In Ja2 98% (room air) without tonsillitis, lymphadenopathy, or neurological dysfunction. A urinalysis showed urinary protein, 1.46 g/gCr, 5–9 RBC/HPF, 1–4 WBC/HPF, cast (-), NAG 2.7 U/L, and selectivity index 0.121. A blood test showed WBC 8100 × 106/L, , Hb11.9 g/dL, and platelet 39.8 × 1010/L. Biochemistry data revealed TP 5.5 g/dL, Alb 3.5 g/dL, UN 11.0 mg/dL, Cr 0.47 mg/dL, eGFR 147.8 mL/min/1.73 m2, and CRP 0.01 mg/dL. Immunological tests found ANA (-), anti-dsDNA antibody (-), anti-SM antibody (-), IgG 544 mg/dL, IgA 112.4 mg/dL, IgM 134.7 mg/dL, IgE 34.3 mg/dL, C3 141.9 mg/dL, C4 23.2 mg/dL, ASO 14 IU/mL, lupus anticoagulant negative, anti-CLβ2GP1 antibody 1.2 U/mL, anti-SS A/B antibody (−/−), MPO-ANCA (-), PR3-ANCA (-), anti-GBM antibody (-), HBs antigen (-), HCV antibody (-), and D dimer 0.3μg/mL. These data indicate that there were no possibilities of lupus nephritis, other autoimmune diseases, or antiphospholipid syndrome.A 16-year-old girl had a fever of 39.0 °C for 4–5 days after the second HPV vaccination, even though she had no fever following the first HPV vaccination one month prior. She was prescribed acetaminophen, and she developed proteinuria for the first time 5 days after receiving the second vaccination. Her fever was resolved spontaneously, and proteinuria was not checked until the next school year in a urine checkup, which showed proteinuria 3+ and occult blood. She was hospitalized for examination by renal biopsy. She had a history of allergy to pollen. Her family history included cerebral hemorrhage in her maternal great-grandfather, multiple system atrophy in her grandfather, and hypertension in her grandmother. Physical findings on admission were blood pressure 120/82 mmHg, heart rate 115 bpm, body temperature 36.2 °C, SpOTwo samples of cortex were collected, including a total of 25 glomeruli without global sclerosis, mild mesangial cell proliferation in 10 glomeruli (40%), and normal tubulointerstitium and vascular system. PAM staining showed mild spikes with subepithelial immune complexes by AZAN staining, which were granularly stained along the capillary wall by fluorescent immunostaining for IgG .Among the IgG subclasses, IgG1 was the strongest, IgG3 was mildly stained , and imm® Histology, Roche Diagnostics KK, Tokyo, Japan), which is typically used as a surrogate marker of HPV infection in cervical cancer and oropharyngeal cancer [Immunohistochemistry was performed with the Leica auto-immune stain system using the antibody against the anti-p16-INK4a antibody , as mentioned previously , to deteThe angiotensin receptor blocker losartan 25 mg was administered to reduce the urinary protein, decreasing it from 3.1 g /gCr to 0.19 to 0.65 g /gCr. When the dose of losartan was reduced to 12.5 mg after 6 months, urinary protein increased slightly to 0.41 g/gCr .This study focuses on a report of membranous nephropathy in an adolescent girl after HPV vaccination, and the pathogenic mechanism is investigated.Membranous nephropathy is a major cause of nephrotic syndrome in middle-aged and elderly people but is rare in adolescents  . HistopaHPV is a sexually transmitted disease that is persistently transmitted locally to the cervix, and high-risk HPV types 16 and 18 cause about 70% of cervical cancers , and 22.In this study, the urinary protein was observed with a fever of 39 °C after two doses of a recombinant precipitated divalent HPV-like particle vaccine.As shown in The HPV vaccine Cervarix contains added aluminum hydroxide and monophosphoryl lipid A (MPL) derived from the cell membrane of Salmonella as an adjuvant. Aluminum oxyhydroxide  binds to viral DNA fragments and lipid A derivatives to form alum-nanoparticles, which are taken up by macrophages and form a granulomatous lesion called macrophagic myofasciitis (MMF) ,41. MMF We reported a case of young-onset IgG1-dominant and PLA2R-negative secondary membranous nephropathy after HPV vaccination. Since p16-INK4a was positive in glomerular and urinary proteins, she may have been infected with HPV, and administration of HPV envelop protein vaccines could be implicated in the development of secondary membranous nephropathy.It is important to check proteinuria after HPV vaccination. Secondary membranous nephropathy could occur after HPV vaccination with viral proteins. If it is possible to check the plasma antibody titers for HPV before vaccination, this may help to prevent the occurrence of membranous nephropathy.


We administrated measures of HPV knowledge that consisted of 29 questions. As a result, women who read the original text were more likely to correctly answer two questions on the general HPV knowledge subscale than women who read the summarized text. For the HPV testing knowledge subscale, there was a statistically significant difference in favor of women who read the original text for only one question. The final subscale, HPV vaccination knowledge questions, did not significantly differ across groups. Using BERT for text summarization has shown promising effectiveness in increasing women’s knowledge and awareness about HPV while saving their time.Despite the availability of online educational resources about human papillomavirus (HPV), many women around the world may be prevented from obtaining the necessary knowledge about HPV. One way to mitigate the lack of HPV knowledge is the use of auto-generated text summarization tools. This study compares the level of HPV knowledge between women who read an auto-generated summary of HPV made using the BERT deep learning model and women who read a long-form text of HPV. We randomly assigned 386 women to two conditions: half read an auto-generated summary text about HPV ( Globally, cervical cancer, cancer that starts in the cervical area, is the fourth most common type of cancer in women . Its preFrequent screening tests and HPV vaccines can help prevent cervical cancer. Previous studies have shown the association between health literacy and cancer screening; increasing health literacy can help individuals to understand the severity of the disease and, subsequently, make them more likely to adopt health preventive behaviors ,4. EarlyAttention ed: which research society? The research society must address the lack of awareness and knowledge about HPV, including in the areas of information systems and information technology. Most previous studies focusing on cervical cancer control in these two areas relied solely on promoting HPV vaccination using SMS, gamified web applications, or social media . DespiteOne fundamental challenge to women’s ability to acquire the necessary knowledge and awareness of HPV is the large amount of information. Machine learning techniques, such as deep learning to reduce information overload and focusing on the most valuable information through text summarization, have been validated in previous studies . To the Recently, several researchers have used deep learning and natural language processing (NLP) in the medical field ,20,21,22At the end of 2018, Google has built one such model, named BERT, that outperforms nearly all existing deep learning models in several NLP tasks ,24,25. BThe use of text summarization in the medical field has been reported in the literature . MedicalThe evaluation of text summarization techniques can be intrinsic or extrinsic. Using intrinsic evaluation, the quality of the summarized text is assessed based on metrics or attributes, such as ROUGE, accuracy, comprehensiveness, and relevancy ,36. The The goal of this research study is to determine the effect of using a deep learning text summarization technique to increase women’s knowledge of HPV. Thus, the main objective of this research article is to investigate whether the use of a deep learning summarization technique increases women’s knowledge about HPV. To do so, we compared the level of HPV knowledge between women who read an auto-generated summary of HPV made using the BERT deep learning model and women who read a long-form text about HPV.The original text was compiled from three different published articles to ensure that it contained the information required to answer all items on the HPV knowledge measure questionnaire ,39,40. NThe BERT model was used to auto-generate a summary of 20 sentences from the original text comprising 115 sentences. The resultant summarized text was comprised of 20 sentences, which is approximately 17% of the original text. No changes were made to the content of the auto-generated summary nor were its sentences and their structures altered or updated see .This study was approved by the Institutional Review Board (IRB) at Salem State University (IRB00006274). The study is an online randomized trial where participants were allocated into two groups. Group 1 read the summarized text while group 2 read the original text. The participants were recruited using the Amazon Mechanical Turk (MTurk) platform. MTurk participants are more attentive to instructions; therefore, MTurkers are suitable for recruitment in social science research . ParticiAccording to the Centers for Disease Control and Prevention (CDC), the age group for HPV-associated cancers in the US starts among women aged 20 and above . Thus, pWe measured the participants’ pre- and post-knowledge using the US version of Waller et al.’s (2013) HPV knowledge measure. The validity and reliability of the US version of the HPV knowledge measure are described and reported elsewhere . In the The study advertisement was placed on the MTurk platform. The study description and eligibility screening were presented on a separate survey that is linked to the MTurk portal, which was created in Qualtrics. A brief description of the tasks, the estimated time needed for completion, and the monetary reward upon completing the task were clarified to the participants before beginning the tasks. Women who were willing to participate in this study clicked on a link that directed them to complete a screening questionnaire to determine their eligibility . Those who were eligible were sent to an online consent form that described the purpose of the study. After signing the consent form, a pre-test survey was administered to the participants to determine their pre-existing knowledge about HPV. Participants were then assigned randomly to one of the two groups (group 1 or group 2). After filling out the pre-test survey, women in group 1 read the summarized text while women in group 2 read the original text. Next, women in both groups completed the post-test survey. To ensure that participants focused and paid attention to the task, two attention checks were added in each pre and post surveys. The attention check questions were: (1) choose “False” as an answer in the following question, and (2) choose “True” as an answer in the following question. Responses were also reviewed manually to verify the completeness of the questionnaires.Statistical analyses were performed using IBM Statistical Package of Social Sciences software . Descriptive statistics were computed using frequencies, percentages were used for categorical variables, and means and standard deviations were calculated for continuous variables. Normality of data was checked using Kolmogorov—Smirnov test. The scores of the HPV knowledge related outcomes were normally distributed for both groups, and thus, data were presented as means (SD). The assumption of homogeneity of variances was tested using Levene’s test for equality of variances. The test showed that the variances of the general HPV knowledge, HPV testing, and HPV vaccination in the two groups were not statistically significant  = 0.06, P = 0.81; F  = 0.08, P = 0.78; F  = 2.6; P = 0.12, respectively). Thus, this assumption is met.t-test was used to compare the mean scores of each HPV knowledge subscale and the total HPV knowledge score between groups 1 and 2 after the intervention. For those participants who reported having heard about HPV, HPV testing, and HPV vaccination at baseline, a general linear model was employed to compare mean scores of the total HPV knowledge after the intervention between the two groups with adjustment for the baseline scores. A p-value of <0.05 was considered statistically significant.The reliability scores of the three HPV knowledge subscales  were evaluated using Kuder—Richardson Formula 20 (KR-20). Cross tabulation and Chi-square statistical tests were performed to compare the proportions of correct responses for each item between the two groups. An independent The main outcome variables were general HPV knowledge, HPV testing knowledge, HPV vaccination knowledge, and total HPV knowledge. The scores for the first three variables were calculated. A score of 1 was given for a correct answer/response and a score of zero for an incorrect or “I do not know” response. The sum of the responses’ scores was calculated in each corresponding subscale, with a final score ranging from 0 to 16 for general HPV knowledge, from 0 to 6 for HPV testing knowledge, and from 0 to 7 for HPV vaccination knowledge. A total HPV knowledge score was then calculated by summing the scores of responses in each subscale; scores ranged from 0 to 29, with lower scores indicating poor knowledge.p > 0.05; see A total of 418 women were recruited to participate in this study. A total of 176 were excluded due to their incomplete responses . Thus, 242 responses were available for further analysis. The baseline characteristics of the participants are displayed in Comparisons between groups 1 and 2 according to participants’ answers to each item of the three subscales  after receiving the intervention are illustrated in p < 0.05). Group 1 gave significantly fewer correct answers than group 2 (81% vs. 92%) for the question about the HPV types. In contrast, group 2 gave significantly fewer correct answers than group 1 (38.8% vs. 68.6%) for the question concerning the necessity of treating HPV. There was no statistically significant difference between group 1 and group 2 for the remaining questions (p > 0.05). However, the rate of giving correct answers in the following questions: “HPV can cause cervical cancer”, “HPV can be passed on during sexual intercourse”, “Having sex at an early age increases the risk of getting HPV”, and “Most sexually active people will get HPV at some point in their lives” in group 1 were slightly higher than group 2 (96.7% vs. 92.6%), (94.2% vs. 91.7%), (90.9% vs. 86%), and (64.5% vs. 62.8%), respectively.Regarding the general HPV knowledge shown in , there wp < 0.05), with group 1 being slightly higher than group 2 (34.7% vs. 21.5%). Regarding the HPV vaccination knowledge items, shown in p > 0.05).Looking at the HPV testing knowledge questions presented in p < 0.05). There were no statistically significant differences in the mean scores between the two groups post the intervention on the general HPV knowledge subscale, HPV vaccination subscale, or total HPV knowledge (p > 0.05).A comparison of results of the three subscales and total HPV knowledge is summarized in N = 172), the mean score of the HPV knowledge were 22.07 and 22.43 for Groups 1 and 2, respectively. As shown in p = 0.443). It is important to note that all ANCOVA assumptions  were checked before we run the analysis as described in the Statistical Analysis section.When we examined the total HPV knowledge after the intervention, adjusted for total HPV knowledge at baseline, for those who have heard of HPV, HPV testing, and HPV vaccination at baseline  There are many types of HPV, and (2) HPV usually does not need any treatment. However, this discrepancy may be due to the relevant information required to answer these two questions not being addressed clearly in the auto-generated shortened summary. The level of summarization in our study was adjusted to 17% of the original text, which might have affected whether adequate details were present in the summary to answer these two questions. This suggests that human judgment might be needed to ensure the completeness and the comprehensiveness of the summarized text. The summarized text may present a way to increase women’s knowledge about their own personal health, as long as human judgment is used to ensure completeness of information.When the HPV testing knowledge subscale responses were analyzed, those who read the original text were more accurate for only one question: when you have an HPV test, you get the results the same day: . They were more likely to correctly answer “false” than those who read the auto-generated summary. The fact that only one question in the subscale differed between groups indicates that the use of auto generated summaries is likely effective. To further support this interpretation, the final subscale (HPV vaccination knowledge questions) did not differ as a function of what text was read.This work has implications for practical applications, including the use of auto-summarization tools for public health education material development, and the uses and limitations of the BERT model in the context of auto-summarizing health education articles. Summarizing health education articles using a deep learning approach is of added value for the development of health educational materials that support public health. Studies have shown that information overload can increase mental effort and be fatal when it hinders understanding medical and health-related information . The usaThe features of the BERT model are promising for those who are interested in using machine learning to increase public knowledge of various health conditions, above and beyond HPV. The BERT model, and the auto-generated summaries it produces, has promising implications to create educational materials for health interventions. In building different educational materials, it is useful to have a tool that can produce an auto-summary instead of having to manually summarize the text.The study has several limitations. It was an online study hosted on the MTurk platform, therefore only accessible to registered workers with an MTurk account. Moreover, the sample size slightly underpowered and may not have been able to detect statistically significant differences for small effects. Despite the limitations of this study, auto-generated summarization is a promising tool to increasing public health knowledge on various diseases and developing future health interventions.Future studies should address two primary issues. First, different levels of text summarization should be investigated to determine the most effective and informative percentage of summarization. Second, the current study should be expanded to investigate the effectiveness of text summarization techniques in different contexts to provide further insights regarding the possible effects of using auto-generated summaries to gain knowledge and awareness. In addition, future studies could be directed towards integrating automatic text summarization with educational interventions and health behavioral changing models, which have been shown to be effective in cervical cancer prevention . The PreIncreasing women’s awareness and knowledge of HPV is a healthcare priority since many women globally suffer from this infection and its associated life-threatening diseases, such as cervical cancer. One way to achieve the goal of increasing women’s awareness and knowledge of HPV is the use of deep learning text summarization techniques, such as BERT. These techniques are used to summarize related medical articles to reduce the amount of text, and consequently, the time needed to read it. Our findings showed the efficiency of using auto-generated summaries in increasing women’s knowledge and awareness. More research must be done to investigate the effect of applying different machine learning algorithms and techniques in health education.


Despite the high burden of cervical cancer, access to preventive measures remains low in India. A single-dose immunisation schedule could facilitate the scale-up of human papillomavirus (HPV) vaccination, contributing to global elimination of cervical cancer. We projected the effect of single-dose quadrivalent HPV vaccination in India in comparison with no vaccination or to a two-dose schedule.In this modelling study, we adapted an HPV transmission model (EpiMetHeos) to Indian data on sexual behaviour , HPV prevalence data , and cervical cancer incidence data . Using the model, we projected the nationwide and state-specific effect of HPV vaccination on HPV prevalence and cervical cancer incidence, and lifetime risk of cervical cancer, for 100 years after the introduction of vaccination or in the first 50 vaccinated birth cohorts. Projections were derived under a two-dose vaccination scenario assuming life-long protection and under a single-dose vaccination scenario with protection duration assumptions derived from International Agency for Research on Cancer (IARC) India vaccine trial data, in combination with different vaccination coverages and catch-up vaccination age ranges. We used two thresholds to define cervical cancer elimination: an age-standardised incidence rate of less than 4 cases per 100 000 woman-years, and standardised lifetime risk of less than 250 cases per 100 000 women born.Assuming vaccination in girls aged 10 years, with 90% coverage, and life-long protection by two-dose or single-dose schedule, HPV vaccination could reduce the prevalence of HPV16 and HPV18 infection by 97% (80% UI 96–99) in 50 years, and the lifetime risk of cervical cancer by 71–78% from 1067 cases per 100 000 women born under a no vaccination scenario to 311 (80% UI 284–339) cases per 100 000 women born in the short term and 233 (219–252) cases per 100 000 women born in the long term in vaccinated cohorts. Under this scenario, we projected that the age-standardised incidence rate threshold for elimination could be met across India (range across Indian states: 1·6 cases [80% UI 1·5–1·7] to 4·0 cases [3·8–4·4] per 100 000 woman-years), while the complementary threshold based on standardised lifetime risk was attainable in 17 (68%) of 25 states, but not nationwide (range across Indian states: 207 cases [80% UI 194–223] to 477 cases [447–514] per 100 000 women born). Under the considered assumptions of waning vaccine protection, single-dose vaccination was projected to have a 21–100% higher per-dose efficiency than two-dose vaccination. Single-dose vaccination with catch-up for girls and women aged 11–20 years was more impactful than two-dose vaccination without catch-up, with reduction of 39–65% versus 38% in lifetime risk of cervical cancer across the ten catch-up birth cohorts and the first ten routine vaccination birth cohorts.Our evidence-based projections suggest that scaling up cervical cancer prevention through single-dose HPV vaccination could substantially reduce cervical cancer burden in India.The Bill & Melinda Gates Foundation. With approximately 604 000 new cases and 342 000 deaths worldwide in 2020, cervical cancer is the fourth most common cancer in women.Evidence before this studyNo formal literature search was done before the start of the study. The very high burden of cervical cancer in India underpins the urgency to upscale key interventions, including vaccination against HPV, cervical cancer screening, appropriate treatment, and palliative care, as recommended in the WHO strategy towards the elimination of cervical cancer as a public health problem. Modelling studies focusing on low-income and middle-income countries suggest that HPV vaccination could help these countries reach the WHO elimination threshold. There is also growing evidence that a similar effect could be achieved with single-dose HPV vaccination. Notably, the IARC India trial with a quadrivalent vaccine, and the Costa Rica Trial with a bivalent vaccine, have shown comparable efficacy between single-dose and multi-dose schedules, with sustained antibody levels for at least 11 years after vaccination. These findings are supported by recent evidence from vaccine trials with randomised single-dose groups, such as the KEN SHE and DoRIS trials. From April, 2022, the WHO Strategic Advisory Group of Experts on Immunization recommended single-dose or two-dose schedules for the primary target group of girls aged 9–14 years and girls and women aged 15–20 years.Added value of this studyWe projected the effectiveness and efficiency of quadrivalent single-dose HPV vaccination in India using evidence-based scenarios of single-dose long-term protection derived from the most up-to-date efficacy and immunogenicity data from the IARC India HPV vaccine trial. To our knowledge, we provide the first projections of the effect of HPV vaccination both at national and state-specific levels in India to account for differing cervical cancer risk across the country. We found that single-dose vaccination with long-lasting protection and 90% coverage would prevent up to 78% of cases of cervical cancer among vaccinated birth cohorts across the country, with the greatest relative reduction in cases estimated to occur in states with high cervical cancer incidence. The WHO elimination threshold for cervical cancer (age-standardised incidence rate: 4 cases per 100 000 woman-years) could be achieved by single-dose vaccination across India, whereas the threshold proposed on the basis of standardised lifetime risk (250 cases per 100 000 women born) was projected to only be achieved in 17 (68%) of 25 Indian states . We found single-dose vaccination to be more efficient than two-dose vaccination, preventing at least 21% more cancer cases per dose.Implications of all the available evidenceThese projections will help Indian national and subnational health authorities secure resources to introduce a nationwide HPV immunisation programme, monitor the local effect of HPV vaccination, and plan additional preventive interventions, such as cervical cancer screening, in particular in states with high cancer incidence. This study complements existing evidence that single-dose HPV vaccination could be an effective and efficient strategy for cervical cancer prevention in India and other low-income and middle-income settings. Finally, our projections suggest that the higher vaccination efficiency under a single-dose schedule could free up resources for a broader target vaccination population. Catch-up vaccination in girls age 11–15 years and older, possibly up to age 20 years, could be considered.Among 2005–14 birth cohorts worldwide, the largest burden of cervical cancer is projected in low-income and middle-income countries, with close to a sixth of cases projected to be in India, making it the country with the highest expected burden.9Introduction of HPV vaccination into national immunisation programmes in India, and other low-income and middle-income countries, would be facilitated if a single dose of HPV vaccine were shown to be effective in preventing cervical cancer. Vaccine trials have shown that a single-dose schedule provides non-inferior efficacy against persistent HPV infection compared with multi-dose schedules, with lower, but sustained, antibody levels up to at least 11 years after vaccination.Here, we model the effect of single-dose quadrivalent HPV vaccination on HPV infection and cervical cancer in India, at the national and state-specific levels. We also compare the effect of single-dose HPV vaccination relative to no vaccination and the alternative of a two-dose vaccination, and also consider the effect of catch-up vaccinations in different age ranges.In this modelling study, to project the effect of HPV vaccination in India, we adapted a previously published HPV transmission modelvs female), and sexual activity risk group , which determines their probability of establishing stable and one-off heterosexual partnerships. Transmission of different high-risk HPV types was assumed to be independent and governed by type-specific natural history parameters.We divided the model population (n=20 000) into strata by age, sex  and the Indian National Centre for Disease Informatics and Research .Simulations using the 100 best-fitting parameter sets were used to derive the mean and the 10th and 90th percentiles  of the model outcomes. We weighted state-specific outcomes by corresponding population sizes to obtain outcomes nationwide and by high or low cancer incidence cluster. Model outcomes over time were derived for up to 100 years after the start of the vaccination programme, whereas model outcomes by cohorts were derived for the period up to the birth cohort vaccinated 50 years after the start of the vaccination programme.We assessed reduction in HPV prevalence over time for HPV16 and HPV18; HPV16, HPV18, HPV31, HPV33, and HPV45; and any high-risk HPV type for girls and women aged 15–40 years. We assessed reduction in the cumulative risk of HPV infection by 5-year birth cohort, and for HPV16, HPV18, HPV31, HPV33, and HPV45. Reduction in the first 5-year vaccinated cohort is referred to as a short-term effects. Reduction in the 5-year cohort vaccinated 46–50 years after the start of vaccination is referred to as a long-term effects. A Lexis diagram illustrating the 5-year birth cohorts is shown in the We assessed lifetime risk of cervical cancer (in cases per 100 000 women born) by 5-year birth cohort using a recently published method .7 BaseliTo assess progress towards cervical cancer elimination and to compare countries, we derived the age-standardised incidence rate (ASIR) of cervical cancer on the basis of the world standard population.Finally, we assessed the relative per-dose efficiency to prevent cervical cancer, which we defined as the additional proportion of cervical cancer cases prevented per dose under single-dose versus two-dose vaccination. For example, in scenarios with the same efficacy under single-dose and two-dose schedules, relative efficiency would be 100%. In the equation, this reads as ([efficiency of single-dose strategy] / [efficiency of two-dose strategy]) – 100%, where the efficiency of a given strategy is defined as (percentage relative reduction in lifetime risk) / (doses required). Details for computation of model outcomes are in the We considered scenarios of no vaccination and single-dose and two-dose quadrivalent HPV vaccination targeting high-risk types HPV16 and HPV18 (and low-risk types HPV 6 and HPV 11). Based on available evidence, we assumed life-long protection by two-dose vaccination throughout all scenarios.In addition to lifelong vaccine protection for both single-dose and two-dose vaccination, referred to hereafter as assumption A, we considered four alternative assumptions B–E;  with lowIn combination with the different vaccine protection assumptions, we considered alternative vaccination coverage of 60–100% and catch-up vaccination in women and girls from age 11 up to ages 15, 20, 25, or 30 years, and modelled these catch-up scenarios by their age at the at start of vaccination. The same vaccine efficacy was assumed for all catch-up cohorts, although vaccine effectiveness in catch-up cohorts might be lower because the vaccine can only prevent newly acquired HPV infections. Catch-up vaccination was modelled with 60% coverage or with the same coverage as routine vaccination in the base-case scenario . As an analysis to study a possible optimal strategy under a scenario with a restricted number of vaccine doses, we compared the scenario of single-dose vaccination catch-up to age 20 years with the scenario with two-dose vaccination without catch-up vaccination. An overview of all vaccination scenarios is shown in the Because access to cervical cancer screening in India is very low, we assumed no screening throughout all scenarios.The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.In the base-case scenario, assuming life-long protection from HPV16 and HPV18 infection under both single-dose and two-dose schedules, routine vaccination in girls aged 10 years with 90% coverage (without catch-up vaccination) was projected to reduce the prevalence of HPV16 and HPV18 infection by 63% (80% UI 57–69) among women aged 15–40 years in India in the 20 years since the start of the vaccination programme (from 3·7% [3·1–4·3] at baseline to 1·4% [1·1–1·6] at 20 years), and by 97% (96–99) by 50 years after the start of the programme  in the short term, which further increased to 78% (78–79) in the long term , table 1As a result of the projected reduction in cervical cancer risk, the WHO elimination threshold could already be attained in the low cancer incidence states in the short term and across all states in the long term, with ASIRs ranging between 1·6 cases (80% UI 1·5–1·7) and 4·0 cases (3·8–4·4) per 100 000 woman-years . Due to We then explored the sensitivity of the model estimates to variations in single-dose protection and vaccination coverage. In our projections, two-dose vaccination with life-long protection led to higher impact than did single-dose vaccination with waning protection . Under tSome scenarios with reduced coverage and waning single-dose protection also allowed attainment of the ASIR elimination threshold . For exaIn the base-case scenario, without catch-up vaccination, women beyond the eligible age of routine vaccination benefited slightly from routine vaccination in younger cohorts. Among women aged 11–30 years at the start of the vaccination programme, we projected that the nationwide lifetime risk of cervical cancer would be reduced by 3% , whereas the same catch-up in women aged 26–30 years is estimated to reduce lifetime risk by only 14% 12–16; .Finally, we compared two alternative strategies allocating the same number of doses over a 10-year period: targeting only ten routine cohorts with two-dose vaccination without catch-up, or reallocating the second dose from the routine cohorts to include catch-up in the ten birth cohorts aged 11–20 years at the start of the vaccination programme. We estimated that the two-dose strategy without catch-up (and 90% coverage in the routine cohort) would only reduce the lifetime risk of cervical cancer in the 20 birth cohorts by 38% , we found that single-dose HPV vaccination with catch-up vaccination in girls aged 11–20 years could be cost-effective under the WHO recommended cost-effectiveness threshold of one-times gross domestic product per capita.Our projections also support previous modelling studiesTo complement our assessment of progress towards elimination, we also provide effect projections in terms of lifetime risk indicators. As recently suggested,A major strength of this modelling study is the amount of local data and evidence that make our results context specific. First, we used data from multiple HPV prevalence surveys, and state-specific data on cervical cancer incidence and sexual behaviour in our model to capture the varying epidemiology of cervical cancer across India. To our knowledge, we are the first group to project the effect of HPV vaccination in India with state-specific granularity. Second, we explored uncertainty on single-dose protection duration by using efficacy and immunogenicity data from the IARC India HPV vaccine trial (unpublished),33Our study has some limitations. First, our estimates regarding cervical cancer elimination depend on the assumed baseline risk of cervical cancer. The nationwide baseline risk reported here (11 cases per 100 000 woman-years) is lower than that in GLOBOCAN (18·0 cases per 100 000 woman-years),In summary, in this modelling study, we projected that, under a range of plausible conditions of coverage and vaccine protection, single-dose HPV vaccination could be more efficient in decreasing the burden of cervical cancer in India than a two-dose strategy, and could eliminate cervical cancer as a public health problem. Emerging evidence from ongoing studies and new vaccine trials will be crucial to periodically reassess the potential benefits of single-dose vaccination and whether booster vaccination is needed at an older age.External researchers can make written requests to the IARC for sharing of data regarding the IARC India HPV vaccine trial. Requests will be assessed on a case-by-case basis in consultation with lead investigators and co-investigators. A brief analysis plan and data request will be required and reviewed by the investigators for approval of data sharing. When requests are approved, anonymised data will be sent electronically in password protected files. All data sharing will abide by rules and policies defined by the involved parties. Data sharing mechanisms will ensure that the rights and privacy of individuals participating in research will be protected at all times. Data are from public sources that are listed in the JB has received support to his institution from IARC/WHO outside of the submitted work. All other authors declare no competing interests.


Cervical cancer is a major concern to women’s health, being the fourth most common cancer worldwide. A great percentage of these cancer is consequence of an HPV infection, namely from specific genotypes such as 16/18. Portuguese screening program subjects women to a reflex cytology triage every 5 years. Aptima® HPV is a screening test which presents better specificity than other tests which are used in Portugal (Hybrid Capture® 2 and Cobas® 4800) and still have a comparable sensitivity. The present study aims to estimate the number of diagnostic tests and costs that are avoided using Aptima® HPV compared to the use of two other tests, Hybrid Capture® 2 and Cobas® 4800, within the cervical cancer screening programme in Portugal.A model, consisting of a decision-tree, was developed to represent the full Portuguese screening program for cervical cancer. This model is used to compare the costs resulting from using Aptima® HPV test versus the other tests used in Portugal, during 2 years. Other outcomes such as the number of additional tests and exams were also computed. This comparison considers the performance of each test (sensitivity and specificity) and assumes an equal price for every test compared.Cost savings resulting from the use of Aptima® HPV are estimated at approximately €382 million versus Hybrid Capture® 2 and €2.8 million versus Cobas® 4800. Moreover, Aptima® HPV prevents 265,443 and 269,856 additional tests and exams when compared with Hybrid Capture® 2 and Cobas® 4800.The use of Aptima® HPV resulted in lower costs as well as less additional test and exams. These values result from the greater specificity of Aptima® HPV, which signals less false positive cases and consequently avoids carrying out additional tests.The online version contains supplementary material available at 10.1186/s12905-023-02219-0. Cervical cancer is the fourth most common cancer in women worldwide . ApproxiScreening programs are essential to reduce the incidence of cervical cancer, as well as the associated morbidity and mortality . CervicaData from prospective clinical trials have shown that HPV testing is associated with 60 to 70% higher protection against invasive cervical carcinomas compared to cytology . AccordiThe Aptima® HPV assay detects E6/E7 viral messenger RNA (mRNA) of 14 high-risk types of HPV (16/18/31/33/35/39/45/51/52/56/58/59/66/68) ) . In anotAccording to the available evidence, testing with Aptima® originates less false-positive results than DNA-based testing assays. Although sensitivity is a key measure to evaluate the effectiveness of HPV tests, improved specificity is also important to avoid unnecessary use of health resources . The preTo represent the full Portuguese screening program, a decision-tree model was developed. This model consisted of nodes for each step of the screening routine and directed edges which guide the women during this process. Probabilities are assigned to the edges to describe how a generic patient would be guided along the screening process. By considering a cohort composed of Portuguese women aged between 25 and 60, we can estimate the expected number of women who followed each path of the decision tree and quantify the difference in terms of costs and exams performed for each HPV test considered. More details about the model and its design are provided further.The Portuguese Methodological Guidelines for Economic Evaluation Studies of Health Technologies were followed in this study. These guidelines provide guidance to conduct economic evaluation studies of medicines and medical devices . The NatTable The HPV mRNA test Aptima® HPV was compared with two HPV DNA tests (Hybrid Capture® 2 and Cobas® 4800). Hybrid Capture® 2 and Cobas® 4800 were selected as comparators for this study since the first one (Hybrid Capture®) is the standard of care for HPV testing , and the second one (Cobas® 4800) is the most used screening strategy in Portugal . Women with a colposcopy showing conclusive results with high-grade histological lesions undergo biopsy. Women undergo transformation zone (TZ) excision if they have a) inconclusive results in the colposcopy or b) a colposcopy with conclusive results without high-grade histological lesions after cytology High Grade Squamous Intraepithelial Lesion (HSIL). After biopsy results, women with CIN < 2 are followed-up and re-tested for HPV after 12 months (HPV-negative women return to routine screening and HPV-positive women undergo new colposcopy). Women with CIN ≥ 2 initiate treatment. Women with inconclusive results or conclusive results without high-grade histological lesions in the new colposcopy undergo TZ excision. Women with conclusive results with high-grade histological lesions in the new colposcopy repeat biopsy  and two mRNA-based HPV tests   Euros, €The comparison between Aptima® HPV versus Hybrid Capture® 2 or Cobas® 4800 was performed considering the number of HPV tests and other diagnostic tests  that are avoided with an HPV test versus another. Cost savings resulting from the avoidance of carrying out further HPV tests and other diagnostic tests were estimated to assess the budget impact of using the Aptima® HPV test instead of the other two HPV tests. To compare these tests, it is considered that all women included in the cohort are tested. Since the process is cyclic and may last for a few years, we present the results for the scenarios where each woman is followed: (a) during a 2-year period and (b) until the first colposcopy. Additionally, a scenario where each woman is followed during a single year is presented in the sensitivity analysis.The main source of uncertainty in the analytical model is associated with the transition probabilities; therefore, a deterministic sensitivity analysis (DSA) was used to investigate the sensitivity of the results of the base case scenario after variations of these input parameters. According to the 2019 “Human Papillomavirus and Related Diseases Report” from the ICO/IARC Information Centre on HPV and Cancer, the prevalence of HPV infection among women with normal cervical cytology in Portugal ranges from 10.5 to 25.4% . As suchScreening for cervical cancer with Aptima® HPV avoids carrying out 265,443 and 269,856 additional diagnostic tests  after the first HPV test compared to Hybrid Capture® 2 and Cobas® 4800, respectively  Table , for theIn addition, screening with Aptima® HPV also avoids carrying out further HPV tests and other diagnostic tests, and therefore generating cost savings, in every Portuguese geographic region individually considered Table .Table 6AWhen the analysis was conducted considering only the diagnostic procedures carried out until the first colposcopy, the Aptima® HPV test still avoided diagnostic tests and saved costs compared to Hybrid Capture® 2 and Cobas® 4800 Table .Table 7AThe sensitivity analysis showed that the use of Aptima® HPV is also associated with fewer tests and lower costs compared to the use of Hybrid Capture® 2 and Cobas® 4800 when both the prevalence rates of HPV infection Table  were varThe number of tests and costs avoided by Aptima® HPV compared to Hybrid Capture® 2 and Cobas® 4800 have not significantly changed when the lower rate of prevalence (10.5%) of HPV infection was assumed. However, the results have significantly changed when the higher value of prevalence (25.4%) was used  compared to those associated with the use of Hybrid Capture® 2 (Qiagen) and Cobas® 4800 (Roche Diagnosis) within the cervical cancer screening programme in Portugal. Based on the available evidence, Aptima® HPV avoids additional HPV tests and other diagnostic tests , therefore generating significant cost savings when compared to the other two HPV tests considered in this analysis. The cervical cancer screening strategy is homogeneous throughout all national territory, leading to cost savings in all regions. By assuming that the purchase prices of the three HPV tests are equal, the cost savings associated with the use of Aptima® HPV results essentially from its improved specificity compared to the other two HPV tests. In addition to that, the need of HPV genotyping upon the use of the Hybrid Capture® 2 test contributes to further increase the amount of costs saved with Aptima® HPV versus the former.This analysis is in line with the methodology of a previous study that compared the cost-effectiveness of high-risk HPV testing using a DNA-based assay and a mRNA-based assay under the US cervical cancer screening guidelines . The samAccording to the results of this 2-year time horizon analysis, the number of diagnostic tests avoided by Aptima® HPV versus Hybrid Capture® 2  was lower than the number resulting from the comparison between Aptima® HPV and Cobas® 4800 . However, the costs difference between Aptima® HPV and Hybrid Capture® 2 was greater. The reason is that Hybrid Capture® 2 test does not allow to differentiate HPV genotypes and therefore the cost of genotyping needed to be added to Hybrid Capture® 2, making it the most expensive option. The number of cytology procedures avoided by Aptima® HPV versus Hybrid Capture® 2 was greater than versus Cobas® 4800. This difference is a consequence of the performance of each HPV test—sensitivity and specificity. Hybrid Capture® 2 is associated with an increased probability of a patient with an HPV+ test being infected by other serotypes compared to Aptima® HPV and Cobas® 4800. On the contrary, Cobas® 4800 is associated with an increased probability of a patient with an HPV+ test being infected by 16/18 serotypes, leading to the need to perform more colposcopies and biopsies to confirm the diagnosis.According to the decision tree model, the proportion of women who are submitted to HPV re-test after the first colposcopy is low. Only women for whom the first colposcopy had conclusive results without high-grade histological, or those with CIN < 2 are re-tested for HPV in a follow-up after 12 months. Moreover, both colposcopy and biopsy were assumed to be 100% sensitive and 100% specific. Therefore, both the number of tests avoided, and the costs saved by Aptima® HPV compared to the other two HPV tests have not significantly changed when the analysis was carried out only until the first colposcopy.The sensitivity analysis showed that the conclusions obtained in the base case scenario were robust, since the direction of the results remained unchanged in every alternative scenario. Additionally, the time horizon and the discount rate were shown to impact the results in a lower scale than the costs and prevalence of HPV. Noteworthy, the variation of the prevalence rate of HPV infection significantly affects the results of this study. Given that the sensitivity of Aptima® HPV is comparable to the one of Cobas® 4800 and Hybrid Capture® 2 (97.5% for both), the consequences of varying the prevalence rate of HPV infection are mainly explained by the specificity of each test . Since mRNA-based tests, such as Aptima® HPV, have greater specificity than DNA-based tests, the advantage of the formers are more noticeable when the prevalence of HPV infection in the population is lower. When the prevalence of HPV infection increases, the total number of false positives decreases . Thus, if there is a lower number of false positives, the number of subsequent diagnostic tests is also going to be lower. That is why the number of subsequent diagnostic tests avoided by Aptima® HPV versus Cobas® 4800 was lower in the sensitivity analysis with a higher prevalence rate of HPV infection  compared to the base case scenario analysis. The same rationale is applicable for the comparison between Aptima ® HPV and Hybrid Capture® 2.Considering the analysis of costs, the savings of Aptima ® HPV versus Cobas® 4800 are higher when the prevalence is lower due to the higher number of diagnostic tests avoided (as explained before). Regarding the comparison of Aptima® HPV versus Hybrid Capture® 2, an increase of the prevalence rate of HPV infection in Portugal leads to an increase in the total number of HPV genotyping tests. Because of this, the use of Aptima® HPV in this scenario generates greater cost savings versus Hybrid Capture® 2. Conversely, when the prevalence of HPV infection decreases the number of HPV genotyping tests avoided and cost savings decrease.The results of the present study have not significantly changed when the costs of the additional diagnostic tests  were varied, with little impact in the magnitude of the costs avoided by using Aptima® HPV.There are some limitations that should be pointed out. First, this cost analysis was based on an analytical model which simulates the Portuguese cervical cancer screening program. Although few steps of the screening program were obtained from literature, a panel of clinical experts was needed to describe the procedures used to follow-up women after their first HPV test. In addition, many transition probabilities were informed by the experts, since these data were not available in the scientific literature. Therefore, there are some uncertainties intrinsically associated with this decision tree model.Second, the performance characteristics of the HPV tests were retrieved from a study conducted in the United Kingdom, in which six HPV diagnostic tests have been compared, including Aptima® HPV, Cobas® 4800 and Hybrid Capture® 2 . StudiesThird, the prevalence of HPV infection among women with normal cytology varies considerably. In Europe, the rates of HPV infection prevalence range from 2.0 to 48.3% . The samFourth, in line with a similar study, it was assumed that patients would perfectly adhere to the screening programme, diagnosis and follow-up recommendations . HoweverFifth, only two HPV DNA-based tests were compared with Aptima® HPV. According to the consensus of the Portuguese Society of Gynecology, Hybrid Capture® 2 test is the standard of care on cervical cancer screening. The panel of the experts in gynecology which provided inputs for this study elected Cobas® 4800 as the most used screening strategy in Portugal. There are additional HPV tests available on the market which were not considered in this analysis, because they are rarely used. Nonetheless, the results of this study may need to be updated if the HPV tests considered as the standard of care in cervical cancer screening changes in the future.Sixth, we assume equal prices for the three tests compared. Although this is a standard practice in cost analysis, one must bear in mind this assumption when interpretating these results.In conclusion, the results of this study have shown that the use of Aptima® HPV is cost saving compared to the use of both Hybrid Capture® 2 and Cobas® 4800, supporting the replacement of the DNA HPV tests currently in use within the cervical cancer screening programme in Portugal by Aptima® HPV.Additional file 1. Supplemental material.


The precise grading and characterization of cervical intraepithelial neoplasia (CIN) has been the focus of pathologists for a long time. This study aimed to explore known strategies for the grading of CINs.After routine H&E review, 85 lesions graded CIN 1, 2, or 3 were investigated primarily by HPV RNAscope to detect HR-HPV and LR-HPV, in combination with an HPV-DNA test and P16/Ki67 immunohistochemistry (IHC). Then, the 85 cases were divided into a control group (49 cases) and a test group (36 cases). The former consisted of cases with consistency between morphology, HPV DNA detection and P16/Ki67 IHC. We used them to evaluate HPV RNA distribution patterns in CINs of different grades. The latter were ambiguous cases in which pathologists could not confirm the diagnosis because of inconsistencies between morphology, HPV DNA detection and P16/Ki67 IHC. We reassessed them by comparison to the pattern in the control group.The expression patterns of HPV mRNA signals were different in different CIN lesions. LSIL/CIN1 lesions were mostly expressed in superficial epithelium with diffuse clustered nuclear or cytoplasmic staining; HSIL/CIN2 were characterised by nuclear/cytoplasmic punctate or diffuse cluster nuclear staining in the mid-surface layer, and scattered nuclear/cytoplasmic punctate staining in basal and parabasal cells; whereas HSIL/CIN3 showed full-thickness nucleus/cytoplasmic scattered staining with a punctate pattern. According to the staining pattern, we corrected the diagnosis of 22 cases .Because of its distinct location pattern, HPV RNAscope has obvious advantages over the HPV-DNA test, and combined with P16/Ki67 IHC, it can help pathologists correctly grade CIN. In addition, it can effectively discriminate true CIN from normal or CIN mimic lesions, such as immature squamous metaplasia, atrophy, and inflammatory/reactive changes. Therefore, HPV RNAscope is a valuable auxiliary diagnostic test to avoid the overtreatment and undertreatment of CIN lesions.The online version contains supplementary material available at 10.1186/s13000-023-01308-w. Uterine cervical carcinoma (UCC) is one of the three major malignant tumours in gynaecology and seriously threatens the health of women. Approximately 14,480 women in the United States are diagnosed with cervical carcinoma, and 4,290 die from the disease [For patients to go from HPV infection to UCC involves a lengthy process that provides time for early detection and intervention , 4. CervAt present, the pathogenesis of cervical carcinoma is relatively clear, and a large number of UCC and CIN cases are related to a persistent infection of high-risk human papilloma virus (HR-HPV). In particular, HPV16 and HPV18 are the most common types, detected in approximately 50 and 20% of cervical squamous cell carcinomas, respectively . The HPVIn formalin-fixed paraffin-embedded (FFPE) tissue samples, the biomarkers P16 and Ki-67 are common and helpful tools widely used to guide CIN grading by pathologists . In geneAs stated earlier, persistent HR HPV infection plays a key role in the development of CIN and UCC. In recent years, a new detection method named HPV E6/E7 mRNA In Situ Hybridization (RNAscope) has appeared. In this study, we used HR-HPV and LR-HPV RNAscope to analyse 85 cervical lesions, compared them with the results of HPV DNA cervical cytology tests and P16/Ki67 immunohistochemistry, and performed the pathological diagnosis.We divided 85 CIN lesions into two groups: the control group (H&E morphology is consistent with HPV-DNA and P16/Ki67 immunotype) and the test group . Using these combined assays, we not only found that HPV RNAscope has different expression patterns in different grades of CIN, but more importantly, HPV RNAscope is an effective auxiliary diagnostic tool for difficult CIN grading cases. HPV RNAscope can provide a more accurate diagnosis of CIN lesions, which can help detect HPV types that have not been included in our routine detection, and can greatly reduce the overdiagnosis or underdiagnosis of cervical lesions.n = 29), CIN2 (n = 31), and CIN3 (n = 25) from 2019 to 2022. The age of the patients ranged from 20 to 64 years old, and they had no other diseases in female reproductive tract. The clinical data and pathological characteristics of 85 CIN cases are shown in Table This was a retrospective observational cohort study. We screened 85 cases of CIN lesions at the Third Affiliated Hospital of Guangzhou Medical University: CIN1 , which can examine 14 high-risk HPVs at one time, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Pap smear samples were collected in preserved fixative solution, and preserved slides were prepared, stained, and processed with an automated stainer. The TCT slide results were confirmed by two pathologists who specialize in cytology.2O2 followed by incubation with the secondary antibody. The reaction was detected by diaminobenzidine (DAB), and the slides were redyed with haematoxylin–eosin (H&E) staining. Meanwhile, positive and negative control slides were designed to ensure the effectiveness of the staining procedure. Finally, the slices were put into xylene solution for transparency, sealed with neutral gum, and observed under a bright field microscope.IHC for P16  and Ki67  was performed according to the manufacturer’s instructions. After deparaffinization and dehydration, antigen retrieval was performed by boiling the slides with ethylene diamine tetraacetic acid (EDTA) antigen retrieval solution in a pressure cooker for 15 min. Then, the sections were incubated with the antibody after blocking endogenous peroxidase with HAccording to the Lower Anogenital Squamous Terminology (LAST) , P16 posRNAscope® 2.5 HD Detection Reagent was purchased from Advanced Cell Diagnostics . Experimental procedures were strictly implemented in accordance with the instructions provided by ACD. Briefly, the samples were fully fixed , sliced, deparaffinized, and dehydrated in sequence. Sections were then treated serially with Pre-Treatment 1–3 solution and rinsed with distilled water after each Pre-Treatment step. The sections without a cover slip were then hybridized in HPV hybridization solution at 40 °C for 30 min in a HybEZ Oven . The hybridized probe signal was amplified through the serial application of Amp 1–6; wash buffer steps were performed after each step. The signal intensity was demonstrated by the application of diaminobenzidine (DAB) for 10 min at room temperature. Finally, sections were counterstained with haematoxylin, dehydrated through graded ethanol and xylene and then mounted for microscope observation.The HPV RNA signal appeared brown in the nucleus or cytoplasm on the sections, and normal epithelium was used as an internal negative control. Staining data were recorded as positive patterns based on factors such as signal intensity, the range of staining, the thickness of epithelial staining, and cytoplasm/nucleus staining. All of the slides were reviewed by two trained pathologists.P value < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS software (17.0). The expressed ratios and percentages were used to describe the measures in the study. The comparison of the enumeration data between the control group and test group was performed by the row × list chi-square test, and a n = 29), CIN2 group (n = 31) and CIN3 group (n = 25) according to the original pathological diagnosis. Then, according to the differences in P16/Ki-67 IHC and HPV-DNA results, the CIN lesions in the 3 groups were further separated into 2 groups: the control group and the test group. The control group included cases in which HPV DNA and P16/Ki67 phenotype matched the H&E morphology, whereas those that were not matched were included in the test group. Based on the different inconsistent combination of HPV-DNA and P16/Ki67 phenotypes, the test group was further divided into three groups: A , B , and C . The number of cases in each group is shown in Table First, all CIN lesions were divided into the CIN1 group n = 9, CIN2 gThere were 49 CINs in the control group, including 16 cases of CIN1, 15 cases of CIN2, and 18 cases of CIN3. HR-HPV RNAscope detection was performed in this group, and all 49 cases were positive (100%). In 16 cases of CIN1 lesions, a diffuse clustered expression pattern in nuclei or cytoplasm was observed; staining areas were mainly located on the upper 1/3–1/2 of the epithelium  staining pattern in CIN2 (86.7%) and CIN3 (88.9%) was significantly higher than in CIN1 (18.8%). In addition, the frequency of clustered expression on the superficial layer epithelium (SLE) staining of CIN1 (100%) was significantly higher than that of CIN3 (5.6%). In conclusion, the results of the control group showed that there were different expression patterns of HPV mRNA in different CIN lesions. The main staining features of LSIL/CIN1 were diffuse clustered nuclear and cytoplasmic staining of the superficial epithelium. HSIL/CIN2 showed nuclear/cytoplasmic punctate or diffuse cluster nuclear staining in the mid-surface layer and scattered nuclear/cytoplasmic punctate staining in basal and parabasal cells. HSIL/CIN3 showed full-thickness nucleus/cytoplasmic scattered punctate staining.HR-HPV and LR-HPV RNAscope detection was performed on 36 cases in the test group, of which 16 cases were HR-HPV positive and 1 case was LR-HPV positive, with a positive rate of 47.2%. The number of CIN1-CIN3 cases and RNAscope-positive cases in the test group are summarized in Table There were 13 CIN samples in the test-A group, in all of which the auxiliary examination showed P16-, Ki67 + and HPV + staining. The H&E morphology, P16/Ki67 phenotype and RNAscope expression patterns are shown in Fig. The phenotype of 8 original CIN samples in group Test-B was P16 + , Ki67- and HPV + . The H&E morphology, P16/Ki67 phenotype and HPV-RNAscope expression patterns are shown in Fig. According to the results of HPV DNA detection, the inconsistent cases of HPV and P16/Ki-67 staining were included in the Test-C group, including the cases with P16 + , Ki-67 + , HPV- or P16-, Ki-67-, and HPV + staining. There were 15 cases. In this group, 3 cases were positive for HPV-DNA, including 1 case of CIN1 positive for 12 other HR-HPV types and 2 cases of CIN2 positive for 16 types and 12 other HR-HPV types. H&E morphology, P16 and Ki67 IHC and RNAscope characteristics are shown in Fig. CIN diagnosis has always been puzzling for pathologists because of diagnostic subjectivity and uncertainty. For this reason, a number of auxiliary diagnostic techniques have been utilized. The most widely used are the HPV DNA test and P16/Ki67 immunohistochemistry. However, both of these tests have certain shortcomings. HPV DNA testing is conducted mainly through the collection of exfoliated cervical cells and uses the β-globin gene as an extraction and amplification control, based on real-time polymerase chain reaction (PCR) technology to detect whether the content of high-risk HPV DNA in cells exceeds the threshold –26. The The pathogenic role of the HPV genotype in the development of CIN and cervical carcinoma has been well established , 33. TheIn this study, 85 lesions graded CIN 1, 2, or 3 after routine H&E review were investigated primarily by detecting HR and LR-HPV E6/E7 mRNA in combination with an HPV-DNA test and P16/Ki-67 IHC. There are three major findings of the present study.First, there were different expression patterns of HPV E6/E7 mRNA in different CIN lesions. The main staining features of LSIL/CIN1 are diffuse clustered nuclear and cytoplasmic punctate expression in the superficial layer of epithelium (SLE + pattern). HSIL/CIN2 showed nuclear/cytoplasmic punctate or diffuse cluster nuclear staining in the mid-surface layer and scattered nuclear/cytoplasmic punctate staining in basal and parabasal cells (FTE/SLE + pattern). HSIL/CIN3 showed full-thickness nucleus/cytoplasmic scattered staining in the puncta (FTE + pattern). Interestingly, these different expression patterns maybe represented the different HPV infection phases in corresponding intraepithelial lesions. A large number of studies have shown that HPV infection is mainly divided into three stages: incubation, proliferation and transformation , 38. IniThe results show that the observation index of HPV RNAscope should focus on the location of mRNA particles expressed in tumour cells (including nucleus/cytoplasm and epithelium location), rather than the amount of cellular expression. The data show concordance between CIN grade and HPV infection stage, suggesting that HPV RNAscope may be a valuable auxiliary diagnostic test for CIN grading.Second, the HPV RNAscope assay is a highly sensitive and specific method to directly confirm the stage of HPV infection in lesions by microscopy. Compared to the HPV DNA test, which uses PCR technology to confirm whether the HPV DNA content is higher than the threshold, the HPV RNAscope assay can screen more cases of HPV infection in a more intuitive and simple way.In the data analysis of the Test-C group, we noticed that there were 3 cases of HSILs/CIN3 and 3 cases of HSILs/CIN2 having typical histological morphology, and P16/Ki67 was also in line with expectations , but HPV DNA was not detected in cervical cytology. However, we found scattered positive expression in tumour cell nuclei through HPV RNAscope in 4/5 cases (3 CIN3 and 1 CIN2). We speculated that HPV-DNA was negative in these 4 cases because the content did not reach the threshold during the testing process. However, under the microscope, we can see that even though the expression level is not high, HPV virus DNA has been clearly integrated into the host nucleus. This phenomenon demonstrates that HPV RNAscope has obvious advantages over the HPV-DNA test. In addition, HPV RNAscope also detected more high-risk or low-risk HPV phenotypes than HPV DNA tests. Therefore, we advocate that such cases need further testing with HPV RNAscope to determine HPV status and to assist our final diagnosis.Another point worth noting is that among the 24 cases with HPV-DNA positivity in the Test group, only 10 cases were positive by HPV RNAscope analysis, and the positive rate was 41.7%. We speculate that the results are mainly related to transient HPV infection because the virus is then cleared by the body’s own immune system –41. The Third, the HPV RNAscope assay can effectively discriminate between true CIN and normal or CIN mimic lesions, such as immature squamous metaplasia, atrophy, and inflammatory/reactive changes. In this study, we corrected 20 cases originally diagnosed as CIN lesions (8 cases were CIN1 and 12 cases were CIN2) as inflammatory or repair/reactive changes after reviewing the HPV RNAscope results. Additionally, 1 case was changed from CIN1 to CIN3, and 1 case was changed from CIN2 to CIN3. This knowledge can reduce the cases of both non-CIN lesions being mistaken for CIN and CIN not being recognized, thus avoiding the overtreatment and undertreatment of CIN lesions, respectively. In addition, the HPV RNAscope staining pattern may be the first diagnostic tool supporting the histological basis of CIN2.Inevitably, this study has some limitations. The main one was the relatively small sample size. In addition, we were unable to evaluate the risk of lesion progression associated with HPV E6/E7 mRNA because this was a cross-sectional study. It is necessary to conduct further prospective analysis and in-depth studies with a larger sample size in the future and strive to provide a more objective and convincing method for auxiliary cervical lesion classification.In conclusion, this study supports the viewpoint that HPV RNAscope can be used to assist in the grading of CINs. The positive expression pattern of HPV E6/E7 mRNA may reflect the stage of HPV infection and the developmental direction of CINs, providing the possibility for patients to achieve more precise treatment. In addition, newly trained or relatively inexperienced pathologists can rely on HPV RNAscope for the differential diagnosis of difficult CIN-related lesions, such as repair or immature squamous metaplasia. HPV RNAscope has great potential value for the pathological diagnosis of CINs and deserves further study. However, it is worth noting by pathologists that even though we have discussed the many advantages of HPV RNAscope above, morphology is the most important component of CIN lesions, and RNAscope is also carried out on the basis of morphology. Treating RNAscope as an auxiliary tool rather than a necessity can effectively prevent the occurrence of overdiagnosis/underdiagnosis of CINs.Additional file 1.