Reproductive health changes can occur following infection with Human papillomavirus. HPV is the most prevalent sexually transmitted infection causing a variety of clinical manifestations ranging from warts to cancer. This study aimed to explore the reproductive concerns of women infected with HPV.In this qualitative study, we used the conventional content analysis approach, with the aid of MAXQDA.10 software, to analyze data extracted from the face-to-face semi-structured interviews with 20 Iranian HPV-positive women (sampled by maximum variation purposive sampling). The accuracy of this research was ensured according to the four criteria proposed by Guba and Lincoln.Exploring participants' reproductive concerns, three main categories were identified from the interviews including concerns about fertility potential, pregnancy and non-pregnancy reproductive issues. HPV-positive women concerned about reduced female/ male fertility due to HPV, the impact of the HPV on the fetal health, adverse pregnancy outcomes such as miscarriage and preterm delivery, and mother-to-child transmission of HPV during breastfeeding. HPV-positive women with abnormal cytology results were anxious that becoming pregnant or taking hormonal contraception might worsen their abnormalities. Most married women were reluctant to use a condom. Participants requested further information about the potential reproductive risks of the HPV vaccine. They also wanted to know about the safety of HPV vaccine during pregnancy and breastfeeding.HPV-positive women had some reproductive concerns that should be considered in the designing of educational-consulting interventions. Women need to be better understood and informed about the impact of HPV on their reproductive health. Health care providers may lack knowledge about these specific areas, and they could benefit from additional up-to-date information to address women's reproductive concerns. HPV is the most prevalent sexually transmitted infection among men and women of reproductive age worldwide. The role of HPV in cervical cancer is well known. HPV infections are correlated substantially with multiple reproductive system abnormalities. HPV can be a threat to the reproductive health of patients. Reproductive health refers to “a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity in all matters pertaining to the reproductive system and to its functions and processes, as defined by the World Health Organization (WHO) and the United Nations Fund for Population Activities (UNFPA).In this qualitative study, we conducted face-to-face semi-structured interviews with 20 Iranian HPV-positive women to explore their reproductive concerns. The conventional content analysis approach, with the aid of MAXQDA.10 software, was used to analyze data extracted from the interviews.HPV-positive women identified some reproductive concerns such as worrying about reduced male and female fertility potential, the impact of HPV on fetal health, negative pregnancy outcomes (miscarriage and preterm delivery), and the safety of breastfeeding. HPV-positive women who had abnormal cells in their cervical cytology results were anxious that becoming pregnant or taking hormonal contraception might worsen their health condition. Most participants were reluctant to use a condom in spite of being recommended to use it. Women also asked about the potential reproductive risks of the HPV vaccine. HPV-positive women need to be better understood and informed about the impact of HPV on human reproductive in educational-consulting interventions.HPV is the most prevalent sexually transmitted infection among men and women of reproductive age worldwide , and itsHPV infections are significantly associated with reproductive function abnormalities . In compTseng et al. found the overall frequency of HPV transmission from mothers to neonates was 39.7%, and a meaningfully higher rate of infection was observed when infants were delivered vaginally compared with cesarean . There iTo date, limited qualitative studies have explored adverse psychological responses to HPV diagnosis. Uncertainty about the psychological effects of a positive HPV test highlights the need for further research in this area . Most ofGiven the effect HPV may have on women of reproductive age, the provision of support and interventions for infected women requires a deep understanding of their concerns. The current cervical cancer screening recommendation in Iran is co-testing (HPV testing and Pap smear) strategy for all women aged 30–59, with any marital status, every five years , which lThe design of the present study is qualitative. It was conducted based on the conventional content analysis approach to understand the reproductive concerns of HPV-positive women by exploring their feelings, experiences, and perceptions .This study was carried out from September 2018 to December 2019 at the referral gynecology-oncology outpatient clinic of Valiasr  serving a large population of women from across the country (nearly 40 women daily). The clinic is equipped with colposcopy (two gynecology beds) and directed by oncologist-gynecologist SHSH (the last author) and her five colleagues.A coordinator of Valiasr clinic sent all women who tested positive for HPV (either only high-risk HPV or both high-risk/low-risk strains) to the interviewer  in the calm, convenient room to provide them with information about the purpose and methods of the study. Women were eligible for interview if they were over 18 with a heterosexual partnership ; had no severe disease  and were willing to share their experiences. A maximum variation purposive sampling was used to recruit information-rich candidates with diverse age, marital status, education, and socioeconomic status. In total, 20 Persian-speaking women with different ethnic, cultural, and religious backgrounds were included. Two invited women refused to participate because they prefer not to discuss HPV. Since the clinic is crowded, all participants interviewed during their waiting hours. Semi-structured one-to-one interviews were conducted using an interview guide (Appendix 1) started with the demographic background and reproductive and screening history. Three pilot-interviews were done (included in the study) to improve questions. Memos aided to design the next questions of the subsequent interviews. Besides, field notes were written during the interviews. Face-to-face in-depth interviews with participants' consent were recorded (lasted between 35 and 90 min), transcribed verbatim, and collected until data saturation was reached over fifteen months.The data analysis was performed concurrently with data collection, using a qualitative content analysis approach described by Burnard et al.  using MAThe accuracy of this qualitative research was ensured according to the four criteria proposed by Guba and Lincoln, namely credibility, dependability, confirmability, and transferability , 32. TheThis study was undertaken as a part of a Ph.D. thesis in Reproductive Health, which was reviewed and approved by the Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.FNM.REC.1397.139). Moreover, Valiasr hospital managers willingly facilitated the study. Written informed consent was obtained from all the participants. Direct quotes that are representative of the participants have been presented. Only the quotes used in the current manuscript have been translated into English.The characteristics of 20 women interviewed demonstrate the heterogeneity of the sample Table . Half ofMany women reported seeking information about fertility and pregnancy from a range of sources, including the Internet , their healthcare provider, and other women with HPV. They indicated that finding up-to-date trustworthy information was challenging. Most women preferred information provided by formal websites. Participants identified many fertility and childbearing concerns. The three main categories extracted from the interviews were "concerns about fertility potential," "pregnancy concerns," and "non-pregnancy reproductive concerns" Table .Table 2EOne of the most repeated concerns of HPV-positive women was fear of fertility impairment in both male and female patients mostly expressed by younger women who had pregnancy plans."We've been told to use a condom. Does this virus get into my husband's sperm? Isn't it weakening his fertility? I'm worried, it's because of this infection that I haven't got pregnant these years" (P.10).Some participants had questions about the presence of the virus in semen, sperm, and penis skin. They showed concerns about the effect of HPV on male fertility.Women with mixed HPV types whose husbands had genital warts (GWs) were mainly concerned about the importance of removing warts before conception."You know why I'm worried? If I had a baby now, I wouldn't care about HPV. I'm 37. I have to get pregnant soon. I may lose my chance of getting pregnant. Does the virus affect my ovaries?" (P.10).Participants were anxious and needed more information about the effect of papillomavirus on hormones and the reproductive system.I'm convinced that I am going to get cancer and I might never be able to have a baby in the future. I'm depressed because I'm not sure I can preserve my ability to get pregnant." (P.7).Some expressed fear and anxiety about losing their fertility due to getting cancer following persistent high-risk HPV."We've been trying to conceive for six months with no luck. We did IUI (Intra Uterine Injection) twice. I asked my doctor; she said the virus could be in the semen." (P.8).Few women attributed the failure of assisted reproductive techniques (ART) to HPV."I'm not sorry that I'm not getting married at all. I'm heartbroken that I don't know why, from the moment I find out [about HPV], I can feel … how much I wanted to have a baby. They (doctors) say there may not be a problem with this operation [LEEP], but there's. It can narrow the cervix and make it hard to get pregnant. All of those things run through your head." (P.12).Women were concerned that therapeutic procedures , and conization) might impact their ability to get pregnant. A single woman who was about to have LEEP surgery for her cervical intraepithelial neoplasia (CIN)-2, expressed her concern as follows:"When can I start trying to conceive after a LEEP?" (P.19).A wife planning to get pregnant after a LEEP asked:"I was spotting and lethargic every time I was vaccinated. Does HPV vaccine affect the period? I've read on Instagram that HPV vaccine can cause infertility" (P.14)."Can Gardasil shots cause missed periods?" (P.16).Few women were concerned about the potential effect of HPV vaccine on menstruation and fertility.Pregnancy for women infected with HPV had some challenges, mostly over personal health. Participants also expressed worries about fetal harm and adverse pregnancy outcomes including miscarriage, preterm delivery and cesarean section."I wanted to get pregnant, so I went to a specialist for a checkup. Now that my test results  came abnormal, I think it's not an appropriate time for me to get pregnant. I'm afraid pregnancy will make my results worse. I'm going to wait for the virus to go away, and then get pregnant." (P.1)."I wanted to get pregnant. Then it happened [high-risk HPV and Atypical Squamous Cells of Undetermined Significance (ASC-US)], and I can't think of pregnancy anymore. My doctor said I could get pregnant, but I'm afraid I'll get pregnant, and my immune system will go down, and then my abnormal cells will grow." (P.6).Most HPV-positive women with abnormal cytology results were anxious that weakening the immune system during pregnancy could lead to the virus persistence in their body, and worsen cervical abnormalities. This fear was so great that some who had decided to become pregnant soon changed their pregnancy plans, postponing them until their cytology and HPV results return to normal."Is colposcopy allowed in pregnancy? Could I be treated during pregnancy, if I get a serious precancerous condition?" (P.6).Some participants expressed concern about the safety of diagnostic and therapeutic procedures during pregnancy. They were worried that becoming pregnant would deprive them of timely treatment."Pregnancy causes warts to multiply or get more noticeable. They're disgusting." (P.2).Women with mixed HPV genotypes worried about increasing their warts during pregnancy. These thoughts discouraged and frustrated them."If I get warts, what treatments are available for pregnant women? Can I freeze it?" (P.10).They also had questions about preferred GWs treatments during pregnancy."I had a miscarriage last year. Was it from this virus? My Pap smear was always normal. What if I get pregnant and have a miscarriage again?" (P.4).Most women planning to have children in the future were concerned about the implications of infection on their  child. women interviewed mentioned the association between genital HPV infection and various maternal and fetal variables and pregnancy complications such as miscarriage and premature delivery."My sister's doctor removed abnormal cells from her cervix. After that, she became pregnant and her baby was born in the 35th week." (P.3).Women stated treatments like LEEP or conization might compromise their ability to carry a child to term by weakening the lining of the cervix."My doctor said if I had warts, I'd have a cesarean section. Thank god I did not have warts and I gave birth naturally." (P.2).Considering cesarean delivery for women with HPV was another matter posed by the participants. Some of them mistakenly believed that having genital warts was an indication of cesarean delivery to avoid perinatal development of laryngeal papillomatosis in the newborn.Misconceptions among healthcare professionals revealed in this regard which has caused concern among women."I worry about passing HPV to the baby during pregnancy and childbirth. What would I do to stop that?" (P.4).Fetal health was the main concern about pregnancy raised by most women interviewed. Participants also reported that they often found it challenging to find adequate information about this issue. They needed to know what to do to protect their child from the infection. In this regard, one of the participants planning to get pregnant soon stated:"Doctor gave me Podophyllin, saying I shouldn't get pregnant while using it. I'm afraid I'm pregnant. I want to know which wart-removing medicine is safe in pregnancy." (P.4).Since anogenital warts can proliferate during pregnancy, removal of warts during pregnancy was another issue that was raised. Women with GWs revealed information needs surrounding the teratogenicity of some wart-removing treatments. They felt anxious about any threats that might pose to the fetus.They also wanted to know if intercourse during pregnancy would increase the chance of HPV transmission to the fetus."I asked about vaccination and pregnancy. Doctor recommended postponing vaccination until after pregnancy. But I'd rather to get vaccinated first." (P.2).Almost all women interviewed reported being advised to take the HPV vaccine. Participants knew little about the safety of the HPV vaccine (mostly Gardasil) in pregnancy. Women who wanted to get pregnant preferred not to become pregnant until they completed the vaccine series.Some reproductive concerns unrelated to pregnancy have reported in the third category. Five challenges discussed in this category were breastfeeding, contraception method, premature menopause, cervical cancer, and familial cancer."Does HPV affect breast milk, I mean, just like a diet? Does breastfeeding cause mouth warts? Should we avoid breastfeeding?" (P.3).Two women mentioned the likelihood of passing HPV to a child through breast milk or early nursing."We use the pull-out  to prevent pregnancy, but sometimes I use emergency pills. I don't know if they can weaken my immune system. I read online that birth pills may induce cervical cancer. Are the emergency pills as harmful as the COCs?" (P.17)."I've taken LD (low dose COC) pills after my daughter was born, which is about nine years. They said I have to stop taking them. I don't know what to do. My husband does not use a condom." (P.11).To avoid unplanned pregnancies, women with HPV needed more information to choose preferable contraception. As long-term use of birth control pills increases the cervical cancer risk for women with persistent HPV, users recommended changing their contraception method. Women were worried about the negative impacts of combined oral contraceptives (COCs) and levonorgestrel (LNG) pills on their cellular changes.Some women in a monogamous relationship reported they (or their husbands) are reluctant to use a condom. They wanted to know why using a condom is essential for HPV-positive patients when they already had HPV."I have noticed my periods becoming infrequent. I've tested positive for HPV 16. I feel that my periods get missed and irregular due to this disease. Could this virus cause me to menopause? I'm worried I'm in menopause. I don't like to get there." (P.18).Another stated: "I had a hysterectomy last year (because of fibroids and heavy periods). They hadn't removed my ovaries. I also had a high-risk HPV 53, but I got vaccinated, I'm worried about the effect of HPV on my ovaries." (P.15).A few women over 40 who had no pregnancy intention mentioned concerns about early menopause. Testing positive for high-risk HPV genotypes and abnormal cytology have made these participants fearful of premature ovarian insufficiency."You don't know what is going on because you can't take Pap smear or colposcopy. I was scared. My gynecologist performed so-called Girly Pap smear. She took a cotton swab sample from the end of the vagina for HPV typing and cytology." (P.16).One of the most common concerns of women tested positive for HPV was cervical cancer that has described in a different manuscript as the psychological response to HPV diagnosis. A context-specific finding that seems specific to societies that adhere to cultural principles was fear of cervical cancer in virgin single women. Since vaginal virginity is a matter of prestige in most parts of Iran, they reported engaging in sexual intercourse without vaginal penetration. Two virgin women have expressed concern about cervical cancer following an ascending HPV infection from the perineum to the cervix. After getting genital warts and learning about HPV-related cancers, they were worried about cervical cancer. One revealed that:"My sister had a mastectomy last year. I wonder her cancer was associated with HPV. We both have HPV. I'm scared what if it's a familial thing, and then I might get cancer too." (P.9).Few women were anxious about the possible association between HPV and history of cancer in their female family members."My mother died of cancer. Did it have anything to do with HPV?" (P.20).Another woman with CIN-2 and high-risk types mentioned:The present study aimed to shed some light on the reproductive concerns of women infected with HPV and found they have reproductive concerns and informational needs over the effects of HPV infections on male and female fertility potential, the success of ART techniques (including IVF and IUI), fetal and newborn health, mother health and pregnancy outcomes. Our participants had questions and worries about the presence of HPV in the semen and the impact of HPV infection upon male fertility. Although concerns over male subfertility in HPV-positive men has received far less attention in previous qualitative studies, concerns about female fertility were generally mentioned in previous studies , 34. OOne study, conducted in Lithuania, has identified HPV-52 as the most common form of HPV in couples undergoing IVF1. Interestingly, the two participants who expressed the most concern over infertility were diagnosed with HPV-52. We believe more research should be conducted on the relationship between HPV-52 and infertility. Worries about the failure of ART procedures, including IVF and IUI, due to HPV infections do not seem to be unfounded, according to literature –11. HealFertility fears about the HPV-vaccination among women diagnosed with HPV have been reported in a qualitative study . Our parIn agreement with other studies, our participants mentioned fertility concerns about conservative treatment for CIN like LEEP and conization , 24, 34.Pregnancy concerns were the second category extracted from the interview data. Women mentioned that worrying about the adverse effects of pregnancy on their health was the main reason they decided not to get pregnant. In a mixed-method study conducted in the United States, 30 of 94 women who planned on getting pregnant indicated that their HPV test results would change their future pregnancy plans . Women iIn line with our findings, two studies also indicated concerns about the association between HPV infections and adverse pregnancy outcomes such as spontaneous abortion and preterm delivery , 34. In Almost all interviewed women had been recommended to use a condom. Most married reported that they (or their husbands) are reluctant to use a condom. They wanted to know why constantly using condoms is essential while they already are HPV-infected. Caregivers should also explain why they recommend using a condom.Some women with a history of taking hormonal contraceptives pointed out they have been recommended to stop taking combined hormonal pills. HPV-positive women raise some concerns about choosing their contraceptive method. Although, to conclude causality between COCs and HPV lesions, more studies are needed , particuSuch richness from the interviews revealed unexpected concerns such as fear of having an ovarian tumor and getting cancer because of a family history of cancer. They considered HPV to be a familial carcinogen factor. Similar concerns indicated by another study .The only context-specific finding was the fear of cervical cancer in single virgin women. Communities define 'virginity' in the different ways. In our society intact hymen determines someone's virginity. In this Islamic-Iranian context HPV-positive virgin women avoid vaginal examination. To reassure these women, doctors take a so-called "Girly Pap smear" from the upper part of the vagina with a cotton swab. Our findings revealed misconceptions regarding the relationship between non-penetrative sex and STIs among single women. These women still followed up with the gynecologist but did not have a proper pap-smear and hence may miss out on the opportunity of being diagnosed properly.In previous studies, the reproductive concerns of HPV-positive people had been discussed as a part of emotional and psychological responses to HPV diagnosis. Our findings are noteworthy because as far as we are aware, this is the only qualitative study in which reproductive concerns of HPV-positive women have received particular attention. Furthermore, women with diversity in HPV genotypes (both high-risk and low-risk) were interviewed. As queries about sexual health were part of the interview content, they will be discussed in another manuscript entitled "Sexual life of HPV-positive women." One of the limitations is that the study was conducted among women attending one colposcopy clinic. However, this referral clinic is likely to reflect other clinics in Iran since it covers a varied population. Possibly women who were interested in the topic decided to participate, and it applies to all qualitative research. We should comment on the possible impact of having a highly educated sample (~ 70% with university education) as another limitation. Unlike previous studies that women with genital warts are often excluded, a subset of our participants was HPV-positive women who had GWs. It is worth noting that these women may have additional concerns that must not be overlooked. Moreover, the relative weight or importance of themes and categories is not always apparent. The credibility of the process is demonstrated in the sentiments stated in the data set, suggesting that the extracted concerns may be transferrable to other settings. An additional strength is that this paper complies with the COREQ checklist designed for the reporting of qualitative studies .The findings of this qualitative study suggest HPV-positive women's concerns about the possible effect of HPV on male and female fertility, pregnancy outcomes, mother and child health, breastfeeding and contraceptive methods. Most of these concerns were based on actual proven correlations and should be addressed. These concerns need to be taken into account when Health care providers are counseling HPV-positive women. Medical professionals need to take women's informational needs more seriously to become one step closer to helping these women improve their reproductive health. Extracted concerns which are based on misinformation, such as recommending C-section for women with GWs or fear of HPV vaccine, require increasing education /awareness for patients and/or healthcare providers. Moreover, health care providers could benefit from additional training to be prepared to mitigate HPV-positive women's reproductive concerns. Women need to have better informational resources about these sensitive topics so that they can make informed decisions about having children. Some women may also benefit from referral to a specialist in the context of assisted reproduction.


However, the existing methods for brucellosis diagnosis are time-consuming and expensive as they require a tedious experimental procedure and a sophisticated experimental device and performance. To overcome these defects, it is truly necessary to establish a real-time, on-site, and rapid detection method for human brucellosis. Here, a lateral flow immunoassay (LFIA) with a rapid, sensitive, and alternative diagnostic procedure for human brucellosis with a high degree of accuracy was developed based on blue silica nanoparticles (SiNPs), Staphylococcal protein A (SPA), and surface Lipopolysaccharide of Brucella spp. (LPS), which can be applied for rapid and feasible detection of human brucellosis. To our knowledge, this is the first report that uses blue SiNPs as a signal probe of LFIA for the rapid diagnosis of human brucellosis. The precursor of blue SiNPs@SPA such as colorless SiNPs and blue SiNPs was synthesized at first and then coupled with SPA onto the surface of blue SiNPs by covalent bond to prepare blue SiNPs@SPA as a capture signal to catch the antibody in the brucellosis-positive serum. When SPA was combined with the antibodies in the brucellosis-positive serum, it was captured by LPS on the test line, forming an antigen–antibody sandwich structure, resulting in the T line turning blue. Finally, the results showed that it is acceptable to use blue SiNPs as visible labels of LFIA, and standard brucellosis serum  could be detected at a dilution of 10−5 and the detection limit of this method was 0.01 IU/ml. Moreover, it also demonstrated good specificity and accuracy for the detection of real human serum samples. Above all, the blue SiNPs-based LFIA that we developed provides a rapid, highly accurate, and inexpensive on-site diagnosis of human brucellosis, and shows great promise in clinical diagnostics for other diseases.Brucellosis is a highly contagious zoonosis chronic infectious disease with a strong latent capability to endanger human health and economic development  A lateral flow immunoassay (LFIA) is a fast, simple, and economic method that is widely used in the field of rapid detection of various analytes. The labels used for LFIA include a series of particles, such as quantum dots , 2, lateBrucella spp. is a type of intracellular parasitic and facultative anaerobic Gram-negative short bacilli. According to the different susceptible hosts, Brucella spp. can be divided into six classical types, namely, Brucella abortus, Brucella melitensis, Brucella ovis, Brucella canis, Brucella neotomae, and Brucella suis  and Brucella ceti   , as well in 2010 , and ano in 2012 . The disd to 80% . Brucellery year . The maiStaphylococcal protein A (SPA), and surface lipopolysaccharide of Brucella spp. (LPS). At first, the precursor of blue SiNPs such as C.I. Reactive Blue 21 and colorless SiNPs was synthesized, and then C.I. Reactive Blue 21 was introduced onto the surface of colorless SiNPs in a given of amount of coupling substance to prepare blue SiNPs, followed by a layer of silica shell, which was formed by TEOS hydrolysis that was wrapped outside of SiNPs to avoid dye leakage. Afterwards, the amino group, which aims to combine with SPA by covalent bonding, was introduced to the surface of core-shell blue SiNPs via 3-aminopropyl triethoxysilane (APTES). After that, glutaraldehyde was used to activate the amino groups on the surface of blue SiNPs, and then SPA was added into above solution to obtain the blue SiNPs@SPA. The blue SiNPs@SPA as capture signal to catch the antibody in the brucellosis-positive serum. When SPA was combined with the antibodies in the brucellosis-positive serum, it was captured by LPS on the test line, forming an antigen–antibody sandwich structure, resulting in the T line turning blue. On this basis, the feasibility of this detection in human serum was also verified. We also discussed the feasibility of this work in human serum, and the results showed that it is acceptable to use blue SiNPs as visible labels of LFIA to diagnose human brucellosis in standard brucellosis serum and real human serum samples. Above all, a fast, one-step strategy and high-specificity method based on blue SiNPs for the diagnosis of human brucellosis has been developed, which can be applied in developing countries and rural areas.As little work using blue SiNPs as a reporter of LFIA to detect human brucellosis has been reported, here, an LFIA with a rapid, sensitive, and alternative diagnostic procedure for human brucellosis with a high degree of accuracy was developed based on blue SiNPs, SPA and goat anti-mouse IgG were provided by Beijing Bioss Biotechnology Co., Ltd. and (3-Aminopropyl) triethoxysilane (APTES) was provided by Shanghai Aladdin Bio-Chem Technology Co., LTD. Glutaraldehyde was obtained from Sigma-Aldrich ; bovine serum albumin (BSA) and tetraethyl orthosilicate (TEOS) were obtained from Shanghai Macklin Biochemical Co., Ltd. Anhydrous ethanol was obtained from Tianjin Xinbote Chemistry Co., Ltd. . Twenty five percent ammonia was purchased from Xilong Science Co., Ltd. Reagents used in indirect ELISA were purchased from Shanghai Jianglai Biotechnology Co., Ltd; phosphate buffered saline (PBS) buffers (pH 7.4) were obtained from Sangon Biotechnology Co., Ltd. . The glass fiber, releasing pad, nitrocellulose membrane, absorbing pad, and polyvinyl chloride sheet were obtained from Shanghai Jinbiao Biotechnology Co., Ltd. .Colorless SiNPs were synthesized by the Stöber method with a slight modification. A Stöber system containing 30 ml of anhydrous ethanol, 4 ml of water, and 5 ml of ammonia water was hydrolyzed for 30 min at 60°C. Then, 5 ml of TEOS was added slowly within 30 min, and the reaction continued for another 2 h. The final colorless SiNPs were isolated by centrifugation  and finally freeze-dried by a Lyophilizer.Dye-coated SiNPs were prepared by following a previous report with a slight modification . Briefly2O, 0.2 ml of TEOS, and 0.1 ml of NH3·H2O was hydrolyzed for 20 min, and then 5 ml of the above blue SiNPs was poured into the above solution and reacted for 12 h. After the reaction, the core-shell blue SiNPs were centrifuged and dispersed in pure water.A Stöber system that contained 10 ml of ethanol, 815 μl of HOne hundred milligrams of core-shell blue SiNPs was dissolved in 20 ml of ethanol under ultrasonic conditions at room temperature for 30 min. After that, the above prepared core-shell blue SiNPs were transferred into a three-necked flask containing 80 ml of ethanol, and 100 μl of APTES was added and refluxed at 60°C for 7 h. The resulting amino-modified blue SiNPs were washed four times with water.The SPA was combined with core-shell blue SiNPs through covalent crosslinking with a slight modification ; 2.5 ml −1 of FT-IR. After the dry samples were mixed with KBr, the samples were scanned at a speed of 2 cm−1.SiNPs and core-shell blue SiNPs were characterized by a transmission electron microscope (TEM) (200 kV) and a Fourier infrared transform spectrometer (FT-IR). KBr was set as the scanning background, and wavenumbers were set to 500–3,500 cmThe structure of the core-shell blue SiNPs-LFIA is shown in B. melitensis), brucellosis-positive control serum (B. melitensis), and negative control serum were purchased from Shanghai Jianglai Biotechnology Co., Ltd. The Brucellosis Prevention and Control Base, Chinese Centers for Disease Control and Prevention  donated a total of 102 human serum samples from their three clinical groups, including 69 human brucellosis serum samples, 24 negative control serum samples from healthy individuals, and 9 serum samples infected with other bacteria. All the serum samples were stored in a −80°C refrigerator. The serum agglutination test (SAT), the gold standard diagnostic method for brucellosis, was run following the Health Industry Standards of the People's Republic of China: Diagnosis for brucellosis GB/T (WS 269-2019), and each sample was run with three parallel tests. Relevant information on the serum samples is presented in Standard brucellosis serum , and each sample was run with three parallel tests. iELISA was run according to the previous protocol.2, as shown in 2 showed strong infrared absorption bands in the region of 800–1,180 cm−1, corresponding to Si-O-Si on the silica surface. As shown in the FT-IR absorption spectra of blue SiNPs-NH2, 3,450 and 1,610 cm−1 were attributed to the N-H bonds of the amino group. The above results were comparable with the blue SiNPs being successfully wrapped with an amino-modified silica shell; therefore, it is reasonable to believe that APTES was successfully conjugated with blue SiNPs, and amino groups were successfully coupled on blue SiNPs.FT-IR was used to characterize whether the amino group was modified on the surface of SiNPs. FT-IR provided direct proof for the functionalization of amino groups on the SiNPs, because FT-IR can detect the stretching vibration of chemical bonds on the surface of blue SiNPs and blue SiNPs-NHA TEM method was used to characterize the synthesized SiNPs. The TEM images of the colorless SiNPs and core-shell blue SiNPs are shown in Also, we tested the steadiness of blue SiNPs compared with colloidal gold nanoparticles in different pH values and different concentrations of salt solution . The resLFIA was employed to identify that SPA-conjugated core-shell blue SiNPs were successfully synthesized. The results could be observed by the naked eye, and it was obvious that the SPA-conjugated core-shell blue SiNPs were captured by a secondary antibody, and it showed a strong blue color at the C line . In contTo achieve the optimal response condition, four factors that would affect the final results were investigated. First of all, the amount of dye used in the synthesis of blue SiNPs was optimized. When too much dye was used, it was dissolved in the solvent and made it difficult for the blue SiNPs to be coated on the shell, which had been hydrolyzed by TEOS; however, if only a small amount of dye was used, then the color was not bright enough. So, it is necessary to find a suitable amount of dye that can not only make the color of blue SiNPs bright enough, but also prevent interferences. From Secondly, the sprayed concentration of LPS on the T line was optimized. It was obvious that the concentration of LPS affected the binding ability of LPS with antibodies in standard brucellosis serum. It can be seen from Next, we optimized the dosage of goat anti-mouse IgG. The sprayed concentration of goat anti-mouse IgG on the C line of the test strip could affect the binding ability of goat anti-mouse IgG with SPA. The goat anti-mouse IgG was printed in different concentrations  on the control line. It can be seen from Finally, we optimized the dosage of SPA. The concentration of SPA that coated on the surface of blue SiNPs could also affect the binding ability between SPA and antibodies in standard brucellosis serum. Experimental studies on the binding ability between SPA and LPS under different concentrations of SPA were carried out, and it can be seen from −1 to 10−6 IU/ml) were mixed with blue SiNPs, the sensitivity of the core-shell blue SiNPs-based LFIA for Brucella antibody was obtained. From −5, the T line still showed a blue color. According to the above results, we can conclude that our LFIA could detect Brucella spp. antibody at a level of 10−5 (0.01 IU/ml) in serum with no false positives.After the abovementioned experiment condition optimization procedure, the core-shell blue SiNPs-based LFIA was evaluated by standard brucellosis serum under optimal conditions. After the series of standard brucellosis serum  to determine whether the serum could be diagnosed as positive or negative; SAT followed the Health Industry Standards of the People's Republic of China: Diagnosis for brucellosis GB/T (WS 269-2019) to guarantee that there were no false positives, and each sample was run with three parallel tests to ensure accuracy. Indeed, the LFIA that we developed could detect antibodies in serum, in the whole blood, and even in plasma. Usually in a typical LFIA, the complex matrix can make the NC membrane change colors, which leads to a high background, especially in whole blood. However, due to the bright color of the blue SiNPs, even if the NC membrane turns red, the results were also easy to observe. In particular, the SiNPs can combine with a variety of dyes and appear in a variety of colors; therefore, the SiNPs can be more widely applied in the detection technology.We applied our developed LFIA to detect 69 positive brucellosis serum samples, 24 negative brucellosis serum samples, and nine non-brucellosis serum samples infected with other bacteria samples, and all the serum samples were verified by SAT. The sensitivity, specificity, and accuracy of core-shell blue SiNPs-based LFIA were assessed completely. From the results, we can see that with our method, 61 positive brucellosis serum samples were identified as “positive” correctly, eight positive brucellosis serum samples were wrongly identified as “negative,” 2 negative serum samples were wrongly identified as positive, and 31 of the negative samples were identified as “negative” correctly, as described in Furthermore, we also compared our methods with iELISA to evaluate the sensitivity and specificity. Compared with our developed method, iELISA was also used to detect the same 102 serum samples, and the resulting ROC curve and the results of iELISA are shown in As a new nanomaterial, SiNPs have significant advantages, namely, stability and feasibility, compared with other nanoparticles. Based on their stability, bioavailability, colorfulness, sensitivity, and specificity, SiNPs have shown great promise in immunology , virus dThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Research Ethics Committee of Medicine, the School of Public Health, Jilin University, permit number: JLU2014-0303.2.4. The patients/participants provided their written informed consent to participate in this study.LG, JL, and KX conceived and designed the study. LG, DW, FL, and YZ performed the assay of experimental detection. YW, JZ, and LZ analyzed the data. LG drafted the manuscript. JL, JW, and XS provided constructive opinions and suggestions. KX, JL, and XS reviewed and made improvements in the manuscript. All authors have read and approved the final version of this manuscript.This work was financially supported by the Department of Education of Jilin Province, China , the National Natural Science Foundation of China (Grant Numbers: 82173572 and 81401721), the Science and Technology Development Bureau of Jilin Province, China (Grant Number: 2018010195JC), the Jilin Provincial Development and Reform Commission (Grant Number: 2020C038-7), the Jilin Provincial Health and Family Planning Commission (Grant Number: 2017J074), and the Graduate Innovation Fund of Jilin University (Grant Number: 101832020CX275).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pretreatment of COS-7 cells with C14 before addition of HPV-16-PsV was associated with more potent anti-HPV activity than simultaneous deposition on COS-7 of HPV-16-PsV and C14, suggesting that C14 is more effective in preventing HPV attachment to target cells than post-HPV adsorption viral events. Overall, these in vitro studies suggest that the monoterpenoid zinc tetra-ascorbo-camphorate molecule may be suitable for further clinical evaluations as potential microbicide or therapeutic drug.Zinc tetra-ascorbo-camphorate (or drug “C14”) is a synthetic monoterpenoid derivative that has potent anti-HIV-1 activity in vitro. In this study, we evaluated the in vitro antiviral properties of C14 against human papillomavirus (HPV). Inhibition assay of HPV-16-pseudovirus (PsVs) adsorption on COS-7 cells by C14 was used. C14 inhibited HPV-16-PsVs adsorption with IC Terpenoid; Camphor derivates; L-ascorbic acid conjugate; Zn metal; HPV; HPV-16; Pseudovirus; Inhibition assay. Indeed,a priori devoid of cellular toxicity  encoding the luciferase protein. After the infection of COS-7 with this HPV-16-PsVs, cells are washed to remove the unbound PsVs and incubated with the luciferin, the specific substrate of the luciferase. The luminescence generated by the enzymatic activity of the luciferase carried by HPV-16-PsVs on the luciferin substrate is measured by a luminometer. Therefore, adsorption of HPV-16-PSVs by COS-7 cells is associated with luminescence, while lacks of luminescence indicates the absence of cellular uptake. Dilution of PsVs that yielded at least 80% of the positive control luminescence signal on the luminometer , after adding 50 μL of luciferin substrate and after adjusted with background luminescence, were selected as suitable and therefore selected as the working dilution for neutralization experiments.The viral lifecycle of HPV is closely linked to the differentiation process of the stratified squamous epithelium of the mucosa . These pnfection . These vnfection . These rnfection . In the lsewhere . Brieflylsewhere , 24, 25.lsewhere . The pla2.34 cells/well) in Dulbecco's Modified Eagle Medium , supplemented with 10% Fetal Bovine Serum , 100 IU/ml penicillin and 100 μg/ml streptomycin  and 50 μg/mL of Fungizone™ Amphotericin B (Thermo Fisher Scientific) in 96-well plates and incubated 24 h in 5% CO2 at 37 °C. In the first experimentation, COS-7 cells were preincubated during 3 h in set of C14 dilutions (C14 diluted in non-supplemented DMEM). After the preincubation time, the dilutions of C14 were removed and replaced by HPV-16-PsVs diluted in non-supplemented DMEM. In the second experimentation, the C14 stock solution and HPV-16-PsVs stock were mixed, in the same tube, with non-supplemented DMEM, until reaching the desire concentration for C14 and HPV-16-PsVs into the inoculum (100 μL). Infected cells were grown overnight at 37 °C and then fed with 100 μL of supplemented DMEM. After an additional 24 h of growth at 37 °C, the medium was removed, cells were washed with Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) and 100 μL of Pierce™ Firefly Luciferase One-Step Glow Assay solution (Thermo Fisher Scientific) was added to each and plate was incubated in the dark, for 15 min. Cells lysates were harvested and transferred to a LumiNunc™ 96-well white microplates and the luciferase enzyme activity was measured as described above. A well containing only the pGL4.10 [luc2] (1 μg) served as negative control  and a background luminescence control well was constituted by only COS-7 cells (no PsV or reporter plasmid). The inhibition of the luminescence signal over 80% was considered as effective inhibition of the PsV transduction of the COS-7 cells. A pool of sera from individuals vaccinated by the Gardasil-9® vaccine (Merck & Co. Inc.) constituted the positive control for the HPV-16-PsVs-based inhibition assay . All experiments were performed in triplicate.Serial dilutions of the stock solution of C14 diluted to tenth covering an appropriate concentration range  were subjected to HPV-16-PsVs-based papillomavirus inhibition assay. Briefly, COS-7 cells were pre-plated (102.450) corresponding to the C14 concentration (μM) that causes 50% cytotoxicity on COS-7 cells and inhibitory concentration 50 (IC50), corresponding to the C14 concentration (μM) that induces 50% inhibition of HPV-16-PsVs activity in luciferase assay in COS-7 cells, were calculated using a dose–response–inhibition analysis on GraphPad Prism v5.04 software . Then the therapeutic indexes (TI = CC50/IC50) were calculated.The cytotoxicity of the C14 derivative against COS-7 cells was assessed using MTT assay (Sigma-Aldrich), as previously described . The per33.150 was >0.00515 M of C14.Therefore, the intrinsic toxicity of C14 derivate concentrations up to 5,000 μg/mL was evaluated by using a colorimetric cell viability assay. COS-7 cells were exposed to serial five-fold dilutions of C14 stock solution for 24 h. The viability index corresponding to the ratio of viable cells after the treatment on the fraction of viable mock-exposed cells was calculated. Cells treated by a solution of PBS-azide 0.1% were used as a positive control for toxicity (data not shown). The viability index corresponding to the ratio of viable cells after the treatment on the fraction of viable mock-exposed cells, was calculated. As shown in 3.250 was 2.8 μg/mL (2.9 μM) of C14 , and TIs ranged from >960 to >3,330.50 reaching 8.1 μg/mL (8.3 μM) of C14 (95% CI: [6.1–12.1]), and TIs ranged from >410 to >820.When PsVs was added simultaneously with serial dilutions of C14, the inhibition curve was displaced on the right, with estimated IC450 ranging between 2.9 and 8.3 μM and TIs between >410 to >3,300. Pre-treatment of COS-7 by C14 before adding HPV-16-PsVs was associated with more potent anti-HPV activity than simultaneous deposition on COS-7 of HPV-16-PsVs and C14, suggesting that C14 would act in early stages of the infection by preventing the attachment of HPV on binding sites in target cells, rather than acting directly on the viral post adsorption events. Our observations complement and extent the previous demonstration of in vitro efficacy of C14 against against both R5-and X4- HIV-1   , 37, 38 lovirus] .in vitro HPV inhibition tests. Indeed, at this time, HPVs cannot be cultured in vitro, nor replicate in a conventional monolayer cell cultures [Our observations report, to our knowledge for the first time, on the antiviral activity of a terpenoid against HPV. This may simply be due to the technical difficulties of cultures , 41. HowPoxviridae family [Papoviridae family to which HPV belongs. Otherwise, L-ascorbic acid is well known to have anti-inflammatory properties and has demonstrated numerous effects on the immune system [2+-camphorate or hydrated species of zinc-camphorate, which are highly reactive with proteins, as previously described [The mechanism of action of C14 against HPV-16 is yet unknown. It was previously demonstrated that C14 harbors potent HIV-1 entry inhibition activity and/or targets pre-integrative step of viral cycle, indicating that C14 inhibits the initial stages of the HIV replication cycle . In our e family , which ie system , and maye system , 50. Fure system  likely aescribed , 52. In 50 ranging between 7.3 and 15.9 μM and selectivity indexes between 170 to 2,500. The simultaneous treatment was more efficient that the post-infection treatment, suggesting that a direct inactivation of viral particles or inhibition of virus replication at the initial phases of the viral replication cycle could be involved. Finally, the cytotoxicity for host primary cells of a non-specific antiviral compound is a major issue. For example, the nonoxynol-9, a non-specific surfactant, which destroys HIV-1 particles in vitro, caused lesions in the vaginal epithelium in vivo and increased the probability of being infected with HIV-1 [in vitro and in vivo against HPV (IC50: 1–20 ng/mL) [50: 3 μg/mL) and HSV-2 (IC50: 0.9–3.6 μg/mL) with high TIs [in vitro against HPV (IC50: 2.8 μg/mL), HIV (IC50: 1μM) [50: 7.3–15.9 μM) [The risk of HPV infection can be reduced by complementary or even synergistic strategies combining the use of microbicidal molecules and prophylactic vaccines for primary prevention, and early detection and treatment of high risk-HPV-associated lesions . We prevth HIV-1 . In addith HIV-1 , and comth HIV-1 . Such stth HIV-1 , 55, 56.th HIV-1 . Carrage0 ng/mL) , HIV (IChigh TIs . Similar15.9 μM)  with higin vitro is also observed in vivo, as for example using the HPV-PsVs vaginal challenge in mice [Our study obviously has many limitations. Concerning HPVs, we only evaluated the adsorption inhibition of HPV-16-PsVs on COS-7 cells, and these first observations should be verified with other oncogenic HPV genotypes. The molecular action mechanism of C14 against HPV-16 remains unknown. Further analytical works such as liquid chromatography coupled with mass spectrometry (LC-MS) could be helpful to fully understand the molecular interactions of C14 with HPV-16 viral particle and what happens to C14 after having interacted with the viral particle. In addition, the antiviral activity of monoterpene unit and ascorbic acid, which are the component units of C14, should be examined. Furthermore, our study clearly lacks the demonstration in an animal model that the antiviral activity of C14 molecule  in mice , 24, 56.5Together, our results demonstrate that the C14 is a promising microbicide molecule that should be advanced for further in vivo evaluation and clinical testing.Ralph Sydney Mboumba Bouassa: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Bernard Gombert; Gabin Mwande-Maguene; Aurèle Mannarini: Conceived and designed the experiments; Performed the experiments.Laurent Bélec: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare the following conflict of interests: Ralph Sydney Mboumba Bouassa, Gabin Mwande-Maguene and Laurent Bélec report no conflicts of interest. Bernard Gombert, the chief executive officer of MGB Pharma, Nîmes, France, gave the C14 molecule for the study. Aurèle Mannarini is chemist adviser for MGB Pharma, and discussed the interpretation of the results. Bernard Gombert and Aurèle Mannarini did not play a role in the study design, data collection and analysis, as well as decision to publish.No additional information is available for this paper.


Background: The mucosal high-risk (HR) human papillomavirus (HPV) is associated with oropharyngeal carcinogenesis. Aims of this study were to evaluate the prevalence of HR-HPV infection in laryngeal squamous cell carcinoma (LSCC) from different subsites, and the clinico-biological meaning of p16 overexpression. Methods: Ninety-seven LSCCs submitted to primary surgery (n = 75) or to post-irradiation salvage laryngectomy (n = 22) were evaluated for HR-HPV DNA and RNA using Luminex-based assays. p16 immunohistochemistry was performed. Results: HR-HPV DNA from HPV16 was detected in seven cases (8.75%), without significant differences between supraglottic and glottic lesions. HPV RNA was never detected. p16 overexpression correlated with HR-HPV DNA, but the kappa agreement score was poor. HPV DNA showed no impact on prognosis. p16 overexpression was associated with a better survival  in primarily operated cases, while an inverse association with OS was observed in the salvage surgery group. Conclusions: HR-HPV infection appears to have a marginal role in LSCC independent of the anatomical subsite. p16 expression is deregulated in LSCC independent of HPV but displays a prognostic role in patients submitted to primary surgery. The negative predictive role of p16 overexpression in patients undergoing salvage surgery deserves more investigations for validation and elucidation of its clinical relevance. Human papillomaviruses (HPVs) are known to infect cutaneous and mucosal epithelia. Some types of HPV from genus alpha, in particular HPV16, have been associated with many human cancers, i.e., cervical, oropharyngeal, anal, vulvar, and penile  and are The role of HPV in laryngeal carcinogenesis, postulated back in the 1980s , is lessHowever, the mere detection of HPV DNA is not indicative of a cancerogenic role of HPV in a certain malignancy. Only detection methods able to demonstrate transcription of E6 and E7 viral oncogenes, such as mRNA detection essays, can give some evidence of an active role of the virus . Studiesp16 is an onco-suppressor that negatively regulates cell cycle progression by inhibiting cyclin-dependent kinases, frequently inactivated in many human malignancies ,22,23,24The role of p16 as a standalone prognostic marker, disentangled by its relevance as a surrogate marker for HPV, has been investigated in breast cancer, where its expression seems related to a worse prognosis , and in The main aim of the present study was to investigate the presence of HPV oncogenes in laryngeal cancers through DNA and mRNA detection assays in the same samples, as well as to investigate the role of p16 as a surrogate marker of HPV infection and/or prognostic marker in LSCC. A secondary aim was to verify the hypothesis of a specific HPV carcinogenic role in the supraglottic/marginal larynx.3).Ninety-seven LSCC patients submitted to primary and salvage surgery at Policlinico Gemelli Hospital (Catholic University of the Sacred Heart), Rome, between 1998 and 2013 were included. Data about smoking and alcohol consumption had been collected. All patients had undergone a careful work up, as described elsewhere ,38,39, aThe present study was approved by the local ethical committee of Fondazione Policlinico Universitario A. Gemelli (Prot Mol.HN sf33976/16 rif. A. 20363/13 id. 204).Slides, prepared at the Institute of Microbiology of Policlinico Gemelli according to the HPV-AHEAD protocol, were sent to the laboratories of the IARC Infections and Cancer Biology group at Lyon, France, for HPV DNA analysis. DNA was extracted after an overnight incubation of the paraffin tissue sections in a digestion buffer .HPV DNA positivity was determined by a type-specific multiplex genotyping (TS-MPG) assay combining multiplex PCR and bead-based Luminex technology . The test uses type-specific primers targeting the E7 region of 19 different hr-HPV  and 2 low risk HPV (HPV 6 and 11) genotypes. *I mRNA. Two primers for the amplification of β-globin were included as a positive control for template DNA quality. After PCR amplification, 10 μL of each reaction mixture was analysed by multiplex HPV genotyping (MPG) using Luminex technology  as described previously . All HPV*I mRNA assay developed for 20 HR- or pHR-HPV types was applied for the detection of viral transcripts. The assay amplifies a 65–75 base pair amplicon of HPV and an 81 base pair amplicon of ubiquitin C (ubC) cDNA. Total RNA was purified from three pooled sections of the same tissue block using the Pure Link FFPE Total RNA Isolation Kit  as described previously . RT-PCR The HPV RNA analysis was performed at the German Cancer Research Center laboratories in Heidelberg, Germany.P16 expression was evaluated by IHC through a monoclonal antibody . FFPE sections were deparaffinized with xylene and rehydrated with alcohol. Antigens were retrieved with a 10 min cycle in epitope retrieval solution . The material was then cooled down at room temperature for 20 min. Endogenous peroxidase was blocked with 5 min immersion in 3% hydrogen peroxide solution. Then a 30 min incubation with the p16 antibody at room temperature was performed, followed by an incubation with a secondary goat anti-mouse for p16. Finally, the sample has been developed with a chromogen substrate solution for 10 min  and then stained with haematoxylin (Dako), dehydrated, mounted on slides with a permanent mounting medium and covered. Immunoreactivity was evaluated under microscopic vision. Scoring was performed by an expert pathologist, and the semiquantitative method used included only nuclear staining using the German immunoreactive score of Remmele and Stegner (IRS). An intensity score  and an extension score  were attributed to each case. A composite IRS score accounting for both was then calculated by multiplying the sheer numbers (0–12). IHC procedures were performed at the Institute of Histopathology, Policlinico Gemelli, Rome, Italy.Two IRS cut-offs  were tested with regard to prediction of HPV infection and/or prognosis. The first one was already described in the literature for the evaluation of p16 expression ; we also2 test. To assess the reliability of p16 IHC as a diagnostic test for HPV-driven carcinogenesis we computed the Kappa value as previously described [Statistical analysis was performed using JMP software, release 7.0.1, from the SAS Institute. The correlation between p16 IHC and HPV status was verified using a χescribed ,25. SurvThe study includes 97 LSCC patients. For every patient, we analysed just one tumour sample coming from the main surgery, which in 75 cases (77.3%) was the primary treatment (with or without adjuvant therapy), and in 22 cases (22.7%) was performed for salvage after recurrence. Seventeen samples resulted negative for ß-globin and were excluded for viral DNA/RNA analyses . p = 0.505). All the HPV DNA-positive cases were further tested for viral RNA but found none of them was positive for HPV RNA, although all seven samples resulted positive for the RNA quality control marker, ubiquitin C (ubC). In 2 out of 97 cases, p16 IHC could not be evaluated for insufficient FFPE tissue. Of the remaining 95 samples, 48.42% were positive for p16 overexpression with an IRS cut-off ≥4 (moderate intensity in at least 11–50% of cells), while only 31.57% were positive with a cut-off ≥6 (moderate intensity in 51–80% of cells or strong intensity in 11–50% of cells).Among the 80 cases tested for HPV DNA, 7 (8.75%) tested positive for HR-HPV16 DNA. No correlation was found between the subsite and HPV DNA positivity (p = 0.007 at Likelihood Ratio test) but not when considering the composite IRS score ≥ 6 (p = 0.0733 at Likelihood Ratio test). We then checked the reliability of p16 IHC as a surrogate marker for HPV DNA detection, obtaining a poor agreement (Kappa = 0.14 with IRS score ≥ 4 and Kappa = 0.133 with IRS score ≥ 6), deriving from the high number of false-positive cases  (p = 0.86 at Log-Rank) (p = 0.93 at Log-Rank).Next, we evaluated the impact of HPV DNA detection on prognosis, even if transcriptionally active HPV was demonstrated through mRNA testing in none of the cases. HPV DNA detection was not associated with differences in overall survival (OS) (og-Rank) A, relapsog-Rank) D, or disp = 0.21 at Log-Rank; IRS ≥ 6 cut-off p = 0.97 at Log-Rank) , but higher p16 expression with the IRS ≥ 4 cut-off was associated with a significantly better RFS   B,C or DSog-Rank) E,F.n = 75) and post-irradiation recurrences (n = 22) were analysed separately, p16 positivity at IHC, with IRS > 4 as cut-off, was associated with a better OS in primarily operated cases , and, surprisingly, with worse overall survival in salvage surgeries after irradiation  (When primary cases (og-Rank) .p = 0.049 at Log-Rank; IRS ≥ 6 cut-off p = 0.26 at Log-Rank)  . LSCC is the only head and neck site with decreasing survival rates in recent decades ,45. SuchRegardless, in the larynx more than in the oropharynx, where the role of HPV infection is proven, the transcriptional activity and in particular the expression of HPV oncogenes E6 and E7 appears fundamental to hypothesize a carcinogenic role. Data from the literature indicate without doubt that the prevalence of HPV-driven carcinogenesis in the larynx estimated with an mRNA-based approach is much lower , rangingDespite the absence of HR-HPV mRNA in the present series, suggesting that the HPV in the DNA positive cases may have not been transcriptionally active, we tested HPV-DNA in relation to oncological outcomes anyway, finding no statistically significant impact on OS, RFS, and DSS. In the literature, whichever is the method used for HPV testing in LSCC, evidence about a prognostic/predictive role outside the oropharynx is lacking as well ,14,49,50The absence of demonstrated predictive relevance of HPV infection in laryngeal oncology, makes the issue concerning the reliability and standardization of HPV detection methods in LSCC less critical than in OPSCC . HoweverInk4a, is an important cyclin-dependent kinase inhibitor. Loss of p16 expression, mostly an epigenetic suppression mediated by methylation, has been described, well before the demonstration of HPV-driven carcinogenesis, to be a frequent molecular abnormality in HNSCC [Apart from its potential role as a surrogate marker of HPV infection, p16, encoded by the antioncogene in HNSCC ,52,53 wiin HNSCC . Other sin HNSCC ,36. In tThe present series is not homogeneous as for primary treatment, and not either for time of cancer sample collection, including primary surgery specimens, and previously irradiated cancers, with specimens from salvage surgeries, collected after recurrence. This can be considered a bias, but also an advantage, as we could analyse the two groups separately. In primarily operated cases, p16 expression was strongly associated with longer RFS and OS, and appeared as a prognostic marker, substantially independent of HPV status. On the contrary, in the salvage surgery group, recurrent cancers with p16 overexpression seemed to show a significantly worse survival. The last finding is surprising and, taken together with the first one, potentially extremely interesting from the perspective of a molecular prediction of the response to different treatment modalities and ultimately in guiding treatment selection. Nevertheless, it needs further evaluation, as the salvage surgery group was small (22 patients). Furthermore, in this group, the question of whether p16 expression was the same as at diagnosis or changed between the diagnosis and the recurrence, maybe because of irradiation, remains unanswered.Our data, along with those reported by other researchers, do not support a strong role of HPV in supraglottic SCC, evidence further consolidated by the extremely low prevalence in cases studied with an mRNA-based approach. However, when considered as a standalone biomarker, we found p16 overexpression to be significantly associated with a longer RFS. The different impact of p16 on primarily operated and salvaged patients is extremely interesting for its potential role in treatment selection, but it needs further evaluation.


Tobacco and alcohol use are risk factors for Squamous Cell Carcinoma of the Head and Neck (SCCHN); however, there is growing recognition of HPV as a risk factor for SCCHN. HPV‐related SCCHN is thought to affect mostly middle‐aged individuals but as the US population ages, it is important to evaluate the change in incidence of HPV‐ and non‐HPV‐related SCCHN in individuals who are ≥65 years old.This was a retrospective study using data from a population‐based cancer registry (SEER) to identify individuals ≥65 years old diagnosed with SCCHN between 2000 and 2016 also stratified by sex, race, and birth cohort. The subgroups of HPV‐associated and non‐HPV associated sites were analyzed independently. The incidence per year was calculated and joinpoint detection was used to identity significant changes in incidence trends and annual percent change (APC).p = <0.05). For HPV‐ and non‐HPV‐related SCCHN males had a higher annual rate compared to females, 54.5 versus 18.0 in non‐HPV‐related and 19.1 versus 4.4 in HPV‐related sites. For non‐HPV‐related sites there was a decrease in APC across all stratified groups. For HPV‐related sites there was an increase in APC across all stratified groups, especially males (APC 8.82% 2006–2016 p < 0.05) and White individuals (APC 8.19% 2006–2016 p < 0.05). When stratified by birth cohort, HPV‐related SCCHN sites had a higher APC in ages 65–69 (8.38% p < 0.05) and 70–74 (8.54% p < 0.05).For HPV‐associated sites from 2000 to 2016, there was an average annual rate of 10.8 per 100,000 individuals with an APC of 2.92% (Among the population ≥65 years old from 2000 to 2016, the incidence rate for HPV‐related SCCHN sites has increased across all stratified groups, especially in White individuals, males, and age groups 65–74. The incidence rate for non‐HPV‐related sites has decreased across all stratified groups during this time. This study reports results that are consistent with other studies demonstrating that the prevalence HPV associated of head and neck cancer overall is increasing in the elderly population age ≥65 years. This increase is occurring when stratified by race, sex, and birth cohort however, the White male population is experiencing the most significant annual percent increase in the incidence of HPV‐associated head and neck cancers. HPV‐associated head and neck cancers are no longer a disease associated with younger patients and the increasing prevalence of this disease in the elderly population is important when considering the unique challenges of treatment for this age group. By 2034, older adults will outnumber children for the first time in US history.The epidemiology of head and neck cancers has changed drastically in the past decades in the United States.It is important to consider the impact of the increasing prevalence of HPV‐related head and neck cancers in patients who are ≥65 years old because they represent a historically unique population in head and neck cancers. These patients have a higher prevalence of comorbidities at the time of diagnosis and they are more likely to develop treatment‐related comorbidities in the short term, such as pneumonia, weight loss, dysphagia, malnutrition, and dental issues, and in the long term >5 years after diagnosis such as weight loss, dysphagia, new hypertension, and anemia.2http://seer.cancer.gov). This study looked specifically at older adults defined as age ≥65 years at the time of diagnosis. The study population was stratified by race (Black or White) and sex  and birth cohort  for subgroup analysis  database spanning 2000–2016. SEER‐18 is derived from 18 cancer registries in the United States and covers 27.8% of the cancer cases in the United States  was calculated using the Joinpoint regression program using log‐linear regression and comparison of the incidence trends.3p < 0.05) per year and from 2003 to 2016 of −0.95% per year . For HPV‐related sites the opposite was observed with an increase in APC from 2000 to 2016 for males and females. For males the average annual rate was 19.1 per 100,000 (CI 18.8–19.4) compared to 4.4 per 100,000 (CI 4.3–4.6) in females. For males there was a joinpoint in 2006 with an APC of 4.53% (p < 0.05) from 2000 to 2006 and 8.82% (p < 0.05) from 2006 to 2016. For females there was a joinpoint in 2007 with a statistically significant APC of 4.91% from 2007 to 2016.When the cohort was stratified by sex the rate of both HPV‐related and non‐related SCCHN was greater among males compared to females. For Non‐HPV‐related sites the rate among males was 54.5 per 100,000 (CI 54.0–55.1) compared to 18.0 per 100,000 in females (CI 17.7–18.3). The APC decreased for both males and females from 2000 to 2016. For males there was a joinpoint in 2003 with a statistically significant APC of −4.29% from 2000 to 2003 and a non‐significant APC of −1.27% from 2003 to 20016. For females the APC from 2000 to 2016 was −1.09% (p < 0.05) for Black and 2.55% (p > 0.05) from 2000 to 2006 and 8.19% (p < 0.05) for White individuals.When the cohort was stratified by race the average annual crude rate was similar between White and Black individuals but the trends in APC varied. For non‐HPV‐related sites Black individuals had an average annual rate of 34.9 per 100,000 persons compared to 33.5 in White individuals. The APC for both White and Black individuals decreased from 2000 to 2016 with APC −2.33% for Black individuals and −3.52% from 2000 to 2003 and −0.85% from 2003 to 20016 for White individuals. For HPV‐related sites the average annual rate was 10.2 for Black individuals and 11.6 for White individuals. The APC was increased from 2000 to 2016 for both Black and White individuals with 4.15% (p < 0.05) and −5.27% (p < 0.05) for ages 75–79. The APC decreased from 2003 to 2016 with −1.68% (p < 0.05) for ages 65–69 and −0.33% for ages 75–79 which was non‐significant. For HPV‐related sites the average annual rate decreased as age increased with the greatest rate in age 65–69 of 14.2 (CI 13.9–14.5) and smallest in age 75–79 of 10.7 (CI 9.7–10.5). For all birth cohorts the APC was positive. For age 65–69 the APC was 8.38% from 2000 to 2016 (p < 0.05) and for age 70–74 there was a joinpoint in 2006 with a significant APC of 8.54% from 2006 to 2016 (p < 0.05). The later birth cohorts still had a positive APC, however, it was not as great as the younger birth cohorts. From 2000 to 2016 the APC was 4.08% for age 75–79 (p < 0.05) and 4.62% for age ≥80 (p < 0.05).The cohort was further stratified into birth cohorts, age 65–69, age 70–74, age 75–79, and age ≥80 (Table 4This study demonstrates that the APC in incidence rate is decreasing for non‐HPV‐associated squamous cell carcinomas of the head and neck when stratified by gender and race and also by birth cohort. Every birth cohort experienced a negative APC of the incidence rate which was most pronounced in birth cohort aged 65–69. SCCHN in HPV‐associated locations was found to have an increase in the APC of the incidence rate over the same time period. Males had a larger annual incidence rate compared to females by just over 4x as much as females. The overall positive APC from 2006 to 2016 appears to be mostly driven by White males suggesting that this demographic is experiencing the greatest increase in disease incidence.When the population was stratified by birth cohort, those in the 65‐ to 69‐year‐old and 70‐ to 74‐year‐old groups had the highest average annual incidence and APC when compared to those aged 75–79 and 80+. The trends are consistent with the study by Tota et al. from 2019 which forecasted that the incidence of oropharynx cancer would increase dramatically from 2016 to 2029 in individuals from 65 to 74 years of age.The reasons for this increase remain unclear due to the limitations of the database to quantify tobacco or alcohol use, sexual behavior, or other risk factors. One possibility is that this could be due to increased awareness of the association between HPV and HNC, however, a Swedish study looked at banked oropharyngeal tumor pathology samples from the 1970s onward and found that only 28% of these tumors were associated with HPV in the 1970s, which increased to 77% in the 2000s and 93% in the 2010s.The variation between men and women is also an interesting finding in this study. It is unclear why men have such a higher rate of HPV‐related cancers when compared to women; however, as in the studies mentioned above, changes in sexual practices could be contributing to this difference. A 2017 study in the Journal of Infectious Diseases by Lewis et al. demonstrated that the prevalence of HPV was significantly higher in males compared to females in cohorts 40–49 years old and 50–59 years old.There is also a differing annual rate of HPV‐associated SCCHN when stratified by race. White individuals had the highest APC from 2006 to 2016 compared to Black individuals. This pattern has been observed in multiple other studiesThe results of the study are important to demonstrate that the epidemiology of head and neck cancers among elderly patients is changing. The risk factor profile is changing with the decrease in tobacco resulting in a decline in non‐HPV‐related head and neck cancers and a change in sexual practices resulting in an increase in the incidence of HPV infection and HPV‐associated head and neck cancers. Older patients are often under‐represented in clinical trials due to their age and higher prevalence of comorbidities. Older adults with head and neck cancers have a higher risk of functional and cognitive impairment, depressive symptoms, and social isolation, which are all important factors when considering treatment plans for patients.The study had a number of limitations. In the SEER database, HPV status is limited to years 2010–2016 and only proportions of HPV+cases are reported rather than incidence rates of cases and as result a large number of cases are missing data and HPV status. Therefore, site‐specific codes of anatomical sites for SCCHN highly associated with HPV were used as a proxy for HPV‐associated cancers. Due to this limitation, assumption of HPV status is made by the location of the tumor and thus, there are likely HPV‐associated tumor cases that are not represented in the data and tumors that are not HPV‐associated that are represented in the HPV‐associated case count based on anatomical location. Additionally, there is not a way to stratify based on risk factors such as tobacco or alcohol use or sexual practices; so the reasons for the change in incidence are based on assumption of known risk factors from prior study data. This study also did not address stage of disease at diagnosis or changes in survival outcomes, which are important considerations to examine when determining the overall effect the change in prevalence or incidence of SCCHN.5This study reports results that are consistent with other studiesNone.Ethical approval was not sought for the study.


Few studies provide detailed findings about the health disparities of women being told by a physician whether they have ever had a human papillomavirus (HPV) infection. This study sought to characterize the prevalence and characteristics associated with women age 18 to 59 years in the United States who report being told they were infected with HPV. This study used data from the National Health and Nutritional Examination Survey. Descriptive statistics were computed on study variables and multiple logistic regression analyses were conducted to explore the association of the study variables with the outcome variable. Sampling weights were applied to produce national estimates of prevalence. The sample consisted of 1,669 females, representative of 75,107,170 females in the United States population. Around 11.5% reported being told that they had an HPV infection, of which 60.9% were White, and 82.9% were born in the United States. White women are 2.0 times more likely to be told they have HPV than Asian women and 2.8 times more likely than Black women. United States-born women were 2.1 times more likely told they had an HPV infection than those foreign born. This study found that among U.S. women, less than 12% reported ever having been told they have had an HPV infection. Epidemiologic findings suggest gaps between ever being told of a previous infection and being diagnosed with a clinically relevant HPV infection. Despite epidemiologic data indicating higher HPV prevalence among those less educated and women of color, these groups were less likely to report ever being told they have an HPV infection than White women, and those with a college degree suggesting communication gaps among these subgroups about HPV infection that might exist. Strategies to address potential gaps in communication among these subgroups can potentially reduce the economic burden and health disparities related to HPV infection. Other identified risk factors for HPV include a greater number of sexual partners, lower education level, lower income, smoking, and being uninsured.12 Prevalence is also lower among non-Hispanic Asian and higher among non-Hispanic Black women than non-Hispanic white and Hispanic.10However, the overall prevalence in women of being infected with low and high-risk genital HPV for adults 18 to 59 years of age remains about 40% and 20%, respectively.13 Another meta-analysis investigating the prevalence of HPV found that HPV was most common in Africa, Eastern Europe, and Latin America in those under age 25.14 Both studies identified a second peak of HPV in Africa and the Americas in women age 45 years and older.International epidemiology parallels findings seen in the United States. A 2007 global meta-analysis found that HPV was more common in women under the age of 35 and in those with abnormal cervical cytology or cervical cancer.Despite an extensive body of epidemiologic HPV literature, few studies provide detailed data about the prevalence of women being told by a physician about whether they have ever had an HPV infection. While many women with HPV never develop symptoms or problems, those with clinically apparent infections or who test positive on a screening exam should know if they have been infected. Knowledge about ever being told about an HPV infection likely affects whether a woman follows recommendations regarding cervical cancer screening, adheres to treatment, and shape attitudes toward HPV immunization. An understanding of the characteristics of women who reported having been told they had an HPV infection can help providers and public health officials to identify if potential communication gaps between women experiencing a clinically relevant infection and being told they have an infection exist. Addressing these gaps can facilitate strategies to improve communication about HPV infection. This study adds to the literature by using a national database to characterize the prevalence and characteristics associated with women in the United States who report ever being told they were infected with HPV. Understanding these associations can help clinicians and public health personnel to educational awareness programs that potentially improve screening and follow-up, decrease health disparities, and increase immunization rates.https://www.cdc.gov/nchs/nhanes/index.htm.This study used data from 2015 to 2016 National Health and Nutritional Examination Survey (NHANES) to examine the prevalence and characteristics related to women in the United States who report ever being told they had an HPV infection. NHANES uses a cross-sectional survey to monitor the health and nutritional status of the civilian noninstitutionalized United States population. The survey consists of interviews conducted in participants' homes and includes demographic, socioeconomic, dietary, and other health-related questions followed by standardized physical examinations. NHANES oversamples some populations to obtain more precise estimates for subgroups and to help assure a nationally representative sample. Data are publicly available and released in 2-year cycles. Details about NHANES design and data collection can be found at i.e., race, marital status, income, education, country of birth, citizenship, whether or not they received the HPV vaccine, and whether or not they have been told by MD that they had genital warts), and presented graphically. Cases were weighted by the full-sample 2-year interview weight for demographic characteristics computation to produce nationally representative estimates. For statistical tests, unweighted values of the sample were used. Chi-square tests were conducted on the association of the study variables with the outcome variable . Multiple logistic regression analyses were also performed to assess the independent effect of the study variables with the outcome. Statistical significance was set at alpha of 0.05 and statistical analyses were performed using SPSS 26. The National Center for Health Statistics Institutional Review Board reviewed and approved NHANES, and participants provided written informed consent.Included in the analyses of the 2015–2016 NHANES data were all females age 18 to 59 years that responded on the variable “Ever told by MD you had HPV.” Descriptive statistics were computed on study variables  representative of the United States population. Their age ranged from 18 to 59 years, of which 60.9% was White, 13.0% Black, and 10.3% Mexican American. The average age was 39.4 years (standard deviation = 11.9) and majority were born in the United States (85.9%) (p < 0.05).The study consisted of 75,107,170 females (unweighted  (85.9%) . The pro (85.9%) . For thoAmong the sample group, p < 0.05) than those married (7.4%) even after adjusting for age, race, ethnicity, marital status, educational level, and citizenship. Approximately 70% of the sample were born in the United States and 30% were born in other countries  (p < 0.05), and 286% greater odds than Asian , and 259% greater odds than those with some high school education but without high school diploma . In general, college educated women were 2.9 times more likely to be told they had an HPV infection than women that never completed high school. Country of citizenship also affected the prevalence of reporting an HPV infection with 9.1% of United States citizens reporting yes to ever having an infection compared with 5.0% of non-United States citizens  . The “otp < 0.05 . Those wcitizens .15 Health literacy links to improved adherence for both acute and chronic disease,16 making it important that women know if they have ever had an HPV infection.This study found that in a nationally representative study sample of United States women 18 to 59 years of age, less than 12% of United States women reported ever being told they had HPV. Knowledge of a previous infection correlates with HPV health literacy and knowledge.6 In contrast only 11.5% reported ever having an infection and while most infections are asymptomatic, there is at least a 1% incidence of sexually active women having genital warts at any one time and among women undergoing primary screening, about 8% test positive for oncogenic HPV.17 Genital warts are easily transmittable and the high rate of transmission generates a 50% lifetime risk of acquiring genital warts in sexually active individuals and provides additional epidemiologic evidence that infected women may not be aware of having had an HPV infection.18 High-risk HPV infections (HR-HPV) are associated with Papanicolaou (PAP) smear change19 and recent recommendations incorporate HPV testing into cervical cancer screening. These new screening recommendations incorporating HPV testing means more women will test positive for HPV. This implies that without better communication the gap between having an infection and an awareness of being infected might widen.Ideally women reporting ever having an infection should be a proxy for prevalence of a symptomatic HPV infection or a positive screening test. However, epidemiologic findings suggest that there are gaps between women experiencing a clinically relevant or symptomatic HPV infection and women reporting ever being told they have an HPV infection. In 2013–2014, the prevalence of any and high-risk genital HPV for adults 18 to 59 years of age was 45.2% and 25.1% in men, and 39.9% and 20.4% in women, respectively.20 In contrast, our results found that only 1.38% of Mexican American women reported ever being told they had an HPV infection. In comparison, 3.95% White women reported having been told about an HPV infection suggesting a greater infection awareness gap among Mexican American women being told they have HPV and either testing positive or showing clinical manifestations of an HPV infection. Also troubling was the finding that while Black women are disproportionately affected by HPV,21 only 1.14% of Black women reported ever being told they had an infection. The disparities between demographic subgroups reporting an infection and epidemiological data highlight that minority women are at greater risk for being unaware of experiencing an infection.Another study reported a prevalence of HR-HPV infection among Mexican American females attending a cervical cancer screening clinic of 13%.22 The persistent failure for most women to connect genital warts to HPV despite awareness campaigns23 highlights the need for better communication about the causal connection of HPV infection to genital warts.Other studies also identify discrepancies between prevalence and an awareness of being infected with HPV. For example, a 2006 study found that less than half of the women reporting genital warts treatment had heard of HPV, similar to our finding that only 43% of women with genital warts reported ever having had an HPV infection. This helps validate that gaps exist between experiencing a clinically apparent HPV infection and ever being told about an HPV infection suggesting that women health care providers may be missing important “teachable moments” related to educating women about HPV. Research supports that these opportunities can be important motivators for patient behavior change and adherence to treatment.11 Estimates for annual direct medical costs associated with the prevention and treatment of anogenital warts and cervical HPV-related disease is at least $4 billion and more than 4,000 women succumb annually to cervical cancers. It is critical that women infected with HPV be made aware of their infection and its causal role to PAP smear abnormalities and cervical cancer. Women's awareness of having an HPV infection is associated with increased knowledge, a greater understanding that HPV causes cancer24 and links to better adherence with treatment.25 Research indicates that women who acknowledge having a previous infection are also more likely to have their children vaccinated.26The significant burden of HPV in terms of both cost and health underlines the importance of our findings. In the United States, an estimated 10% of the population have an active HPV infection, 4% have an infection that causes cytological abnormalities, and an additional 1% have an infection causing genital warts.There are several study limitations. The report of ever having an HPV infection relies on self-report and is subjected to recall bias. However, while women may fail to remember being told, this also suggests poor communication about HPV infection. Also, some women may prefer not to disclose a previous genital infection leading to an under-reporting about previous HPV infection. Since NHANES does not include populations considered at higher risk for HPV such as those institutionalized, incarcerated, and the homeless, this study does not report on these populations. Also, to identify potential gaps between women reporting ever been told they had HPV, epidemiologic findings were used to identify the prevalence of clinically evident infections or infections found by screening rather than chart or case review. While these estimates may not be exact, they consistently demonstrate discordant results between women with positive HPV test results and/or clinically evident genital warts and women reporting they have ever been told they had an HPV infection. Research studying women with positive HPV test results in a clinical setting and/or clinically evident genital warts and reporting being told they have an HPV infection will be helpful to confirm and further characterize potential gaps in awareness of experiencing an HPV infection. A strength of this study is that it represents the first to use a nationally representative sample to provide baseline data about the percentages and characteristics of women reporting about whether they have ever been told they have had an HPV infection.27–29Using a national database, this study found that 11.5% of U.S. women reported ever having been told they have had an HPV infection. Epidemiologic data suggest gaps between ever being told of a previous infection and ever being diagnosed with a clinically relevant HPV infection especially among less educated and minority women. Increasing the awareness of having had an HPV infection will likely require combined approaches with strategies targeted to improving provider communication and increasing consumer awareness. Each diagnosed HPV infection represents a “teachable moment” and failing to adequately inform patients represents a missed opportunity to enhance a woman's knowledge of HPV. Despite the consequences of HPV infections, many women remain unaware of HPV and would benefit from being told of an infection.


About 45:000 cancers are linked to HPV each year in the United States alone. The HPV vaccine prevents cancer and is highly effective, yet vaccination coverage remains low. Pharmacies can play a meaningful role in increasing HPV vaccination access due to their availability and convenience. However, little is known about pharmacists’ perceived barriers to HPV vaccination. The objective of this systematic review was to summarize existing literature on perceived barriers to administering HPV vaccination reported by pharmacists. Barriers identified from selected studies were synthesized and further grouped into patient, parental, (pharmacist’s) personal, and system/organization barrier groups. Six studies were included in this review. The cost of the HPV vaccine, insurance coverage and reimbursement were commonly reported perceived barriers. Adolescent HPV vaccination barriers related to parental concerns, beliefs, and inadequate knowledge about the HPV vaccine. Perceived (pharmacist’s) personal barriers were related to lack of information and knowledge about HPV vaccine and recommendations. At the system/organization level, barriers reported included lack of time/staff/space; difficulty in series completion; tracking and recall of patient; perceived competition with providers; and other responsibilities/vaccines taking precedence. Future strategies involving pharmacy settings in HPV-related cancer prevention efforts should consider research on multilevel pharmacy-driven interventions addressing barriers. Human Papillomavirus (HPV) is estimated to be the cause of 70% oropharynx, vaginal, and vulvar cancers, 60% of penile cancers, and 90% of anal and cervical cancers . HPV-assCommunity pharmacies have the potential to play a meaningful role in increasing HPV vaccination rates. The President’s Cancer Panel  and NatiDuring the ongoing COVID-19 pandemic, the U.S. Department of Health and Human Services (HHS) declared that pharmacists are allowed to administer childhood vaccines for children that are three years and above . Most stThis systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA) .Electronic searches were created and completed by a research team member and health sciences librarian (A.W.) experienced with systematic searches in the following databases: PubMed [NLM], Web of Science [Thomson Reuters], Cochrane Central Register of Controlled Trials [Wiley], and Scopus [Elsevier]. The search strategy was created first in PubMed , and theThe titles and abstracts were first screened independently for eligibility by two researchers . Then, the same researchers reviewed the full text of selected abstracts for further eligibility. Conflicts were resolved by a third researcher (J.M.M.). Study inclusion criteria include: (1) primary research article including qualitative, quantitative, and mixed-method studies, (2) reported outcome included perceived barrier to administering HPV vaccine (3) study population included pharmacists, pharmacy students, pharmacy technicians, and pharmacist representatives. Studies were excluded if the study objectives were unrelated to HPV vaccination. Two co-authors  abstracted and synthesized all findings. Barrier type categorization was adapted from the findings reported in the included studies ,16,17. SThe electronic database searches yielded 444 articles; 292 titles and abstracts were selected, and 13 full-text studies were reviewed for inclusion. In the end, 6 out of 13 studies were selected for this review using the exclusion and inclusion criteria ,18,19,20Patient: Four out of the six included studies reported patient-related barriers ,16,17,19Parental: Three studies included parent-related barriers ,18,20. A(Pharmacist’s) Personal: Three studies reported pharmacist’s personal barriers to HPV vaccination. ,18,19. ISystem/Organization: Five out of the six studies reviewed reported system/organization level barriers ,17,18,20At the organization level, other responsibilities/vaccines taking precedence over HPV vaccination was also reported as a barrier ,20. OtheIn one study, concerns that it may appear that pharmacists are in competition with health care providers to administer the vaccine was seen as a barrier . LiabiliCommunity pharmacies could potentially play an important role in improving HPV vaccination rates due to their availability and accessibility. This review presents a summary of the literature on pharmacists’ perceived barriers to providing the HPV vaccine in a pharmacy setting. Based on the findings from the six included studies, pharmacists’ perceived barriers to HPV vaccination uptake exist at the patient, parental, pharmacist’s , and system/organization levels. The existence of reported barriers at different levels suggests that efforts at improving HPV vaccination rates among pharmacies should be targeted at these levels.Barriers related to the cost of the HPV vaccine and insurance coverage were frequently cited in the articles reviewed for this paper ,18,19,20Parents play an important role in HPV vaccination for their adolescent children. Results from this review showed that pharmacists perceived parental concerns, beliefs, and lack of knowledge as barriers to HPV vaccination ,18,20. SPartnerships and collaboration agreements between health care providers and pharmacists, including strong provider recommendation and referral to pharmacies for subsequent HPV vaccine doses may increase parental awareness and vaccine uptake . PhysiciThree studies included in this review reported personal barriers among pharmacists to HPV vaccine administration ,18,19. TAt the organization level, remembering to screen  as well While the purpose of this review was to summarize existing literature related to the perceived barriers to administering HPV vaccination reported by pharmacists, some of the studies reported facilitators of HPV vaccination suggested by study participants ,18,20. MEvidence-based strategies to improve HPV vaccination rates that have previously been implemented in other settings, such as in primary care, may help to improve HPV vaccination in pharmacies. For instance, previous studies suggest that reminder and recall systems  and deciTo our knowledge, this is the first study to review previously reported perceptions of barriers to HPV vaccine administration among pharmacists. Numerous commentaries and professional organizations suggest that pharmacies could play an important role in improving HPV vaccine uptake. The studies included in this review varied greatly in terms of study design, which made the synthesis of findings challenging. The search strategy employed to identify eligible studies for this review, encompassed publications on studies conducted worldwide. However, all the eligible studies included in this review were conducted in the United States. The perceived barriers that pharmacists experience may vary geographically within and outside of the United States. Therefore, generalizability of results may be limited. Nevertheless, many of these barriers are, to some extent, universal and therefore may provide direction for those investigating HPV vaccination barriers in different health care delivery systems. This literature search yielded only a few studies, which indicates that additional research is needed to explore pharmacist’s perceived barriers, especially at the system/organization levels. Because most of the studies included in this review relied primarily on quantitative survey-based data collection methods, it limits the ability to thoroughly understand pharmacists’ perceived barriers to HPV vaccine administration. A more comprehensive understanding of barriers to HPV vaccine administration is needed, especially to better understand how these barriers may impact various pharmacies in utilizing differing immunization delivery practices. Future work should include in-depth qualitative analyses of barriers among pharmacists practicing in a wide variety of settings.In conclusion, pharmacies present an opportunity to increase HPV vaccination rates. Targeted public health efforts to increase HPV vaccination among pharmacies should consider a multilevel approach. Future strategies involving pharmacy settings in HPV-related cancer prevention efforts should consider research on pharmacy-driven interventions that addresses the barriers to administering the HPV vaccine at various levels.