Many genotypes of human papillomaviruses (HPVs) may lead to morphological changes in cells, resulting in various atypical cells, such as multinucleated cells (MNCs) and koilocytes, in the cervix. However, the relationships between the profiles of HPV genotypes and MNCs are not exactly known. Thus, this study comprehensively profiles the HPV genotypes in MNCs using a microdissection method. HPV genotypes and MNCs were detected in 651 cases with an abnormal Pap smear by liquid-based cytology. Specific HPV genotypes were also detected, including HPV16, 34, and 56, which might be associated with MNCs. This result suggests that the high-risk HPV genotypes, such as HPV16 and 56, are associated with the atypical changes in MNC morphology from normal cervical cells. The results also show that MNCs may be a predictor of squamous intraepithelial lesion. Over 100 genotypes of human papillomavirus (HPV) have been confirmed to be associated with tumorigenic diseases, including benign (warts) and malignant lesions (carcinomas) . Some ofThe screening tools for cervical cancer are the Papanicolaou (Pap) and HPV tests. These tests permit a detailed observation of the cytopathology of cervical smears ,14. In gA total of 1053 samples with cervical intraepithelial neoplasia (CIN) were obtained from patients during follow-ups at the Genki Plaza Medical Center for Health Care, Tokyo, Japan between 2014 and 2018. The mean patient age was 38 years . The data on HPV vaccination history were not collected. Of the 1053 samples, 651 SurePath™  liquid-based cytology (LBC) samples had atypical squamous cells of undetermined significance (ASC-US) + based on the Bethesda system . The 651After obtaining written informed consent from the subjects, the samples were collected. The Ethics Committee on Human Research of the Kyorin University (2019-10) and Gunma Paz University (PAZ18-37) approved the study protocol, and it was implemented in accordance with the approved guidelines.HPV genotyping was performed on the whole LBC samples to assess the association between the cytological signs on Pap smears and HPV genotypes. DNA from the residual liquid of the whole LBC samples were collected using the hot sodium hydroxide method . Cell peMD was used to detect HPVs directly from MNCs on Pap smear slides ,20. The Each cell pellet was mounted in a single thin layer on a microscope slide and fixed with 95% ethanol. Two cytotechnologists reviewed the Pap smear slides, and certain MNCs were selected for sampling. The selected cells were individually photographed; then, the slides were soaked in xylene to remove the cover slip. Then, the xylene was washed away with 100% ethanol. Subsequently, under a stereomicroscope, the chosen cells were collected by gently picking up the edge of the cell with the point of a 27-G injection needle. The cells attached to the point of the needle were precisely transferred to each tube containing the alkaline lysis solution. DNA isolation and Uniplex E6/E7 PCR were performed for the HPV genotyping of the MD samples, as previously described.p-values were estimated in each step. In all cases, a p-value of less than 0.05 was considered statistically significant.SPSS version 25.0  was used for the statistical analyses. The differences between the groups were examined using residual analysis. In addition, a stepwise logistic regression with backward elimination and a likelihood ratio test for model building were used to evaluate the HPV genotypes associated with MNCs. The goodness-of-fit was assessed using the Hosmer–Lemeshow test. All of the HPV genotypes were entered into the model; then, the nonsignificant genotypes were removed from the model one at a time. Moreover, the odds ratio (OR), confidence intervals (CIs), and p < 0.01). We estimated the correlation of the positive rate for MNCs with high-risk (p < 0.01). As shown in p < 0.01 and p < 0.05, respectively). The positive rate for MNCs were 60.0% (3/5) and 50.0% (3/6) in HPV35- and HPV45-positive cases, respectively. However, no significant relationships among HPV35, HPV45, and MNCs were found.The relationships among the MNCs, cytologic classifications, and HPV genotypes are summarized in igh-risk  and othep > 0.05). MNCs were significantly associated with the presence of HPV16 , HPV39 , HPV56 , HPV34 , and HPV67 .Subsequently, a binomial logistic regression analysis was used to determine the relationships between the detected HPV genotypes and atypical cells. The model was fit (Hosmer–Lemeshow test; p < 0.01. MD was performed in cases with MNC (Based on the correlation between the MNCs and HPV genotypes, MD-PCR was performed on the genotypes that showed with MNC . Table 4We studied the relationships between MNC, cytopathological classification by Bethesda system, and HPV infection profiles in cervical smears stained using the Pap and microdissection methods. The main highlights of this study are as follows: First, MNCs were most frequently found in LSIL . Second,In general, the malignancy grade of cervical cells may be evaluated by nuclear atypia and nuclear/cytoplasm rates . Based oHPV profiling was also performed in the koilocytes in the cervical smears using MD . VariousIn general, MNCs are found in other infections due to Chlamydia or human herpes virus type 2 ,23,24. TThere are some limitations in this study. First, using the MD method it is possible that there was a contamination of HPV genotypes between the MNCs and other cells in the smears because the PCR method used in this study was specific and highly sensitive. Meanwhile, the positive rate of MD-PCR for MNCs was not very high because the target cell was a single cell, and the viral load might have been below the detection limit. Another possible explanation is the influence of PCR inhibitors on microscope slides. Thus, it may be necessary to consider microscope slides that are suitable for MD-PCR. It was also not demonstrated whether specific HPV genotypes were detected from microdissected MNCs on the histological specimens. Thus, it is unclear whether MNCs were detected in CIN tissues, even if they were significantly associated with LSIL. However, the contamination of adjacent cells is inevitable when single cells are isolated using LCM . DespiteIn conclusion, the present study showed that MNCs may be a predictor of cervical cancer cells. However, other atypical cells, such as binucleated cells, parakeratotic cells, giant cells, and smudge cells, were also found in the cervical smears. To predict the progression of candidates into cervical cancer cells, further studies may be needed to assess the profiles of HPV infection in these cells.


We did not find a significant difference in terms of prevalence of HPV-16 infection between vaccinated and non-vaccinated . Our results support the adoption of nonavalent HPV-vaccine to prevent the most prevalent infections caused by HPV-16 and -31 genotypes and underscore the need of surveillance to implement tailored vaccination programs and preventive strategies.The assessment of human papillomavirus (HPV) genotype dynamics could support the adoption of more tailored preventive actions against cervical cancer. The aim of the study was to describe the prevalence of HPV infection, HPV genotype distribution, and the epidemiological characteristics of women with ASC-US cytology since the introduction of HPV-DNA testing in Sardinia , (March 2016–December 2020). Specimens were tested by RT-PCR for 14 high-risk HPV genotypes. A total of 1186 patients were enrolled, with a median (IQR) age of 41 (38–48) years. Of these women, 48.1% were positive for at least one HPV genotype; 311 (26.2%) women were vaccinated with a median (IQR) age of 38 (30/47) years. The percentage of prevalence of HPV-16, -31, -66, -56, and -51 was 36.3%, 18.7%, 11.9%, 11.4% and 10.7%, respectively. The highest prevalence of infection was found in women aged <41 years, and single women. Moreover, women aged >41 years (OR: 0.51, 95% CI: 0.31–0.86;  Cervical cancer is the fourth most prevalent cancer in women worldwide; its annual incidence and mortality were equal to ~641,127 cases and >341,831 deaths in 2020 : HPV-DNAThe estimation of the prevalence of HPV infections, as well as the distribution of HPV genotypes following the vaccination of young and adult cohorts, could tailor future preventive strategies.The aim of the present study was describing the prevalence of Hr-HPV genotypes in a cohort of women attending a Sardinian  regional screening program, from March 2016 to December 2020; moreover, epidemiological factors associated with clinical progression of cervical lesions were evaluated.The study participants were recruited in Sassari, northern Sardinia . The regional screening program includes the Pap (Papanicolaou) test followed by the HPV-DNA testing in case of ASC-US , ASC-H , and ACG  results . Based oCervical specimens were collected with cervix-brush and suspended in a 20 mL preservation solution, PreservCyt transport medium . A liquid-based cytology was performed for all participants: those diagnosed with ASCUS according to the TBS-2001 classification  underwenNucleic acid extraction was performed using a commercially available extraction kit QIAamp DNA Mini Kit,  or GeneAll RibospinTM vRD II  ,12.HPV genotyping was conducted using the commercial kit Anyplex II HPV HR detection kit  , a multiMicrosoft Excel software  was used to collect demographic, epidemiological, clinical, and virological variables. Qualitative variables are described with absolute and relative (percentage) frequencies, whereas quantitative variables are summarized with means  or medians , depending on their parametric distribution. Comparisons between vaccinated and non-vaccinated patients were performed with a chi-squared or Fisher’s exact test for qualitative variables. Logistic regression analysis was carried out to assess the relationship between a severe cervical lesion (CIN2+) and demographic, epidemiological, and clinical covariates.A two-tailed p-value less than 0.05 was deemed statistically significant. All statistical analyses were performed with the statistical software STATA version 17 .A total of 1217 patients underwent HPV-DNA testing during the period March 2016 and December 2020. The median (IQR) age of the cohort was 41 (31–48) years. The majority were single  and ~27% (324/1186) were married. The majority attended high and middle school . More than half  were administered oral contraceptives and 8.9% (105/1186) had undergone a gynecological intervention in the recent past .Three hundred and eleven (26.2%) women were vaccinated with a first HPV vaccine dose when their median (IQR) age was 38 (30–47) years and >66% and 27.7% were vaccinated with the tetravalent and nonavalent vaccine, respectively.HPV-DNA test was positive for at least one Hr-HPV genotype in almost half of the cases , with 15% (178/1186) showing infections caused by multiple genotypes.p-value: 0.01) .The most prevalent genotypes were HPV-16 , HPV-31 , HPV-66 , HPV-56 , HPV-51 , and HPV-39  . No state: 0.01)  (Table Se: 0.01) . HoweverThe most frequent genotypes in multiple infections were HPV-31 and -66.A higher prevalence of single infections was found in individuals with a negative cytology (~68%), and in those classified as CIN1 and CIN3 . Infections caused by more than one genotype were mainly found in CIN2 cases (>53%).A total of 617 women were followed-up and underwent a second pap-test after a median (IQR) period of 7 (8–9) months. Overall, most women registered a regression of lesion with a negative result , whereas 24.2% (149/614) and 3.6% (20/614) were classified as LSIL and HSIL, respectively.p-value < 0.001), whereas a higher prevalence was observed in women with a higher educational level . A statistically significant higher prevalence was described in women exposed to oral contraceptives .At baseline, a lower prevalence of infection was found in married women . A stratified analysis on HPV-16, the most prevalent and preventable genotype included in the vaccine, did not show a difference between the two groups   .p-value: 0.01). Moreover, a higher educational level seems to play a protective role , as well as parity   .The present study reported the results of HPV genotype distribution in women recruited into the cervical cancer screening program in Northern Sardinia, Italy, since the introduction of HPV-DNA testing in March 2016.The overall HPV prevalence in women with ASC-US cytology was ~48%, in accordance with other national and international surveys ,15, and Similar to other sexually transmitted diseases, the major HPV risk factors are related to sexual behaviors: age of the first sexual intercourse, number of sexual partners and habits . We founHPV-16 was confirmed as the most prevalent genotype, as previously reported for other HPV-related diseases . It was In a Swedish study, HPV types 16, 18, 31, 33, 45, and 52 were found in 689 of 808 screened invasive cervical cancers (85.3%). The addition of HPV types 35, 39, 51, 56, 58, 59, 66, and 68  increased prevalence by only 12 of 808 cases  . HPV scrWe found that CIN2 lesions are mainly related to infections caused by more than one genotype. However, our aim focused on the potential role of co-infection in the early phase or progression of lesions and did not find significant differences according to the cytological groups, due to the low rate of severe disease cases. Several studies did not describe a higher risk of severe dysplasia in women with multiple infections compared with women with a single genotype ,29. HoweThe target population could have affected the results on the effectiveness of HPV-vaccination, based on the higher prevalence of infections in the vaccinated group. However, the stratified analysis by HPV-16, the most prevalent and preventable genotype, did not show a significant difference between the vaccinated and non-vaccinated. The type of population enrolled in our study could explain some of the findings’ results. Firstly, the median age of vaccination was high. Real life studies showed that HPV vaccine effectiveness is highest when it is administrated before sexual debut . The AmeThe retrospective epidemiological design can raise several concerns. Although the present study reports the main epidemiological findings of the HPV screening program in ASC-US women since the introduction of HPV-DNA in 2016, our data are not representative of the general population, especially for the prevalence and distribution of HPV-genotypes, based on the adherence to the screening program. The small sample size during the follow-up, as well as the heterogeneous follow-up period, did not allow us to clearly identify the prognostic role of several demographic and clinical variables. Moreover, the low numbers of fully vaccinated patients did not allow us to assess the preventive role of the vaccine. Further studies would be needed to assess the epidemiological scenario in the vaccination era.Monitoring and genotype identification are crucial to promptly identify cross-protection and type-replacement, particularly after the implementation of a vaccine program. In line with WHO call for action to eliminate cervical cancer as a public health problem  future m


Objective: To examine self-rated health and activities of daily living (ADLs) limitations among American Indian and Alaska Native (AI/AN) veterans relative to white veterans. Methods: We use the 2010 National Survey of Veterans and limit the sample to veterans who identify as AI/AN or non-Hispanic white. We calculated descriptive statistics, confidence intervals, and used logistic regression. Results: AI/AN veterans are younger, have lower levels of income, and have higher levels of exposure to combat and environmental hazards compared to white veterans. We found that AI/AN veterans are significantly more likely to report fair/poor health controlling for socioeconomic status and experience an ADL controlling for age, health behaviors, socioeconomic status, and military factors. Discussion: The results indicate that AI/AN veterans are a disadvantaged population in terms of their health and disability compared to white veterans. AI/AN veterans may require additional support from family members and/or Veteran Affairs to address ADLs. American Indian and Alaska Native peoples (henceforth AI/ANs) have participated in the US military since the American Revolution, and their substantial contributions have been noted . Indeed,The military is a large federal provider of employment, occupational training, and educational opportunities; post-service benefits, military experience, socialization, and training are expected to generally positively impact civilian socioeconomic attainment . With siAmerican Indian and Alaska Native veterans serve disproportionately in the US military. About 12,000 AI/ANs served in the US military during World War I, and in World War II, more than 44,000 AI/ANs served in the military . Much liThe literature suggests that the major motivations for AI/ANs to enlist in the military seem to be pursuit of economic or educational benefits, allegiance to the United States, and family, tribal, or cultural values and traditions. Indeed, extant research ranging from studies on World War II to Desert Storm suggests that AI/ANs join the military because of the prospect for economic or educational advancement after service . A qualiTribal and cultural values have also been cited as a potential reason for AI/AN participation in the armed forces and are articulated as a desire to participate in the warrior tradition of one’s tribe or nation, which, as noted earlier, is a similar motivation as other participating groups . The modIn general, AI/AN peoples have strikingly worse health outcomes than whites. AI/ANs have high rates of chronic illness and have a mortality disadvantage at every age compared to whites . AmericaUnfortunately, in addition to experiencing a health disadvantage compared to whites, AI/ANs often have lower socioeconomic status than whites, which negatively influences health outcomes . AmericaSimilar to the literature from the general population, AI/AN male veterans seem to carry a higher burden of disease and have lower socioeconomic status relative to white male veterans. Among male veterans, AI/AN veterans tend to have higher rates of mental health disparities than whites . ApproxiDespite efforts by the federal government to address historical disadvantages , AI/ANs In light of the disproportionate rates of high military participation and of high rates of chronic illness, we are interested in exploring veteran health through examining the self-rated health and ADL limitations of AI/AN veterans compared to white veterans in a nationally representative sample of veterans. As AI/AN veterans differ systematically from white veterans in their socioeconomic status , health The data for this analysis came from the 2010 National Survey of Veterans (henceforth NSV). The NSV was commissioned by the Department of VA to plan for future programs that meet the needs of veterans . The NSVexcellent, very good, good, fair, or poor.” Consistent with past research regarding the health of veterans , we used two separate measures of health: self-rated health and ADLs. Self-rated health has been widely documented to be a reliable overall measure of health due to its high concordance with physicians’ reports and because it is a highly reliable predictor of mortality . Respondns e.g., , we dichnes test . We usedWe also used ADLs to measure health. Activities of daily living measure the ability to live and function independently. They describe collective fundamental skills that are required to independently care for oneself . UltimatIn our analysis, we included basic demographic information and socioeconomic variables as controls. Specifically, we have years of age at survey coded continuously. Additionally, we included the gender of the veteran, with male coded “1” and female coded “0.” To maximize the sample size among AI/AN veterans, we included female veterans. As socioeconomic status is one way that AI/ANs are disadvantaged compared to whites, we included two measures. First, we included total household income as the following categorical variable: (1) $0–$34,999 as the reference category, (2) $35,000–$74,999, and (3) $75,000+. Similarly, we coded education as a categorical variable with (1) less than high school as the reference category, (2) high school, (3) some college, (4) college, and (5) professional degree. Similarly, we included marital status as a categorical variable with (1) married as the reference, (2) divorced, (3) widowed, and (4) never married.Perhaps the greatest strength of the NSV compared to other large surveys in analyzing veterans is the detailed information collected regarding military service and individual health behaviors. We coded these questions in the following manner and consistent with previous research . First, We used Stata statistical software to conduct descriptive and multivariable analyses . We calcDescriptive statistics are presented in In terms of period of service, our sample of AI/AN veterans served post-Vietnam (54%), Vietnam era (27%), and WWII-Vietnam era (19%), and for our sample of white veterans, 37% served in the post-Vietnam era, followed by WWII-Vietnam era (34%), and then Vietnam era (29%). In our sample, Army was the most common branch to have served followed by the Navy, Air Force, and Marines. The majority of our sample had a military age of entry 20 years and below. Sixty-seven percent of the AI/AN sample served for 0–4 years, compared to 74% of whites. Approximately 40–45% of AI/AN veterans had combat experiences, whether that be served in combat/war zone or were exposed to death/trauma, versus approximately 33% of whites. Veterans who reported definitely/probably exposed to environmental hazards accounted for 39% of the AI/AN sample and 22% of the white sample.As an additional sensitivity analysis (results available upon request), we analyzed if there were significant differences in risk of reporting “fair” or “poor” health or 1+ ADL across age and, specifically, in older adulthood. To investigate this issue, we fit three interaction models for each outcome only controlling for gender. For the first, we interacted AI/AN with age (specified continuously) and found that the interaction was not significant for self-rated health or ADLs. For the second model, we created a dummy variable with “1” coded as those who were 50 and older and “0” if otherwise. Again, the interaction term was not statistically significant suggesting that the health disparities were not pronounced among older adults. Finally, we coded an additional dummy variable with those aged 65+ coded “1” and those 64 and younger coded “0.” Once again, the interaction model was not statistically significant, suggesting there were little differences in the extent of the health disparities across age or older adulthood.American Indian and Alaska Native peoples have a long history of participating in the US military and continue to be present today. Indeed, a disproportionately high percentage of veterans, relative to their nonveteran population, identify as AI/AN , 2017. EThe inclusion of ADLs allows us to examine an aspect of quality of life for AI/AN veterans—the ability to independently care for oneself. As with the majority of literature, ADLs are often focused on elderly populations ; howeverIn terms of the general description of the study sample, AI/AN veterans have a higher prevalence of reported fair/poor health, reporting one or more ADLs, and are younger than white veterans. AI/AN veterans have lower levels of income with 48% of the sample having a household income of $0 to $34,999 compared to 33% of white veterans. For our health behavior measures, AI/AN and white veterans have similar trends of current and past smoking behavior and similar use of the VA healthcare services (for further description of AI/AN veteran healthcare service use, see Our analysis uncovered significant differences between whites and AI/ANs in self-rated health in our early models, which is supported by previous literature that has also found that AI/AN men are more likely to report fair/poor health relative to white men . In our Our results for ADL has revealed an important health indicator, where across all our logistic regression models, AI/AN veterans have approximately 2 times the odds of experiencing an ADL relative to white veterans. It further bolsters the idea that racial/ethnic minority veterans are in poorer health than their white veteran counterparts . This fiThere are some important limitations to our study. Most importantly, the dataset we used was cross-sectional, giving us only a snapshot of an incredibly complex life course process of health status for veterans . HoweverAmerican Indian and Alaska Native peoples have a long history of honorably serving in the US military yet experience some of the highest health disparities. This analysis demonstrates significant differences between whites and AI/ANs self-rated health and ADLs. Our findings correspond with past research that increasingly documents that the military is not immune from racial/ethnic inequities in health. Even when controlling for such variables like age and socioeconomic status, AI/AN veterans are still more likely to have poorer self-rated health, and AI/ANs have approximately 2 times the odds of reporting an ADL limitation or impairment relative to white veterans, net of controls for age, health behaviors, socioeconomic status, and military factors. The literature demonstrates that AI/AN veterans are being institutionally underserved and do not have the same access to health care as their white counterparts. Although future research is needed on AI/AN veteran outcomes, including more that focuses on their legacy of resiliency which our data could not speak to, one thing is abundantly clear: AI/AN veterans’ health outcomes must be addressed as a piece of honoring their sacrifice to the United States and its military.


Public acceptance of the HPV vaccine has not matched that of other common adolescent vaccines, and HPV vaccination rates remain below the Healthy People 2020 target of 80% compliance. The purpose of this study was to evaluate the capacity of nine pediatric clinics in a Federally Qualified Health Center organization to implement a systems-based intervention targeting office staff and providers using EHRs and a statewide immunization information system to increase HPV vaccination rates in girls and boys, ages 11 to 16 over a 16-month period. System changes included automated HPV prompts to staff, postcard reminders to parents when youths turned 11 or 12 years old, and monthly assessment of provider vaccination rates.During the intervention, 8960 patients (11–16 yo) were followed, with 48.8% girls (n = 4370) and 51.2% boys (n = 4590). For this study period, 80.5% of total patients received the first dose of the HPV vaccine and 47% received the second dose. For the first dose, 55.5% of 11 year old girls and 54.3% of 11 year old boys were vaccinated. For ages 12 to 16, first dose vaccination rates ranged from the lowest rate of 84.5% for 14 yo girls up to the highest rate of 90.5% for 13 yo boys. Logistic regression showed age was highly significantly associated with first dose completion  while males did not have a significant association with first dose completion compared to females. The intervention increased overall counts of first and second HPV vaccination rates. Over 34,000 cases of cancer are caused by the human papillomavirus (HPV) each year in the United States . CancersCurrent CDC recommendations for HPV vaccination are determined by the patient’s age when the initial HPV vaccine dose is administered. For adolescents starting the vaccination series between the ages of 9 and 14 years, two doses separated by 6–12 months are sufficient. For adolescents beginning the HPV vaccination series at 15 years of age or older, three doses are recommended. The second dose should be administered one to two months after the first dose and the third dose should be administered six months after the first dose .Acceptance of the HPV vaccine has not matched that of other common adolescent vaccines, and HPV vaccination rates remain below the Healthy People 2020 target of 80% compliance . AccordiIn 2014, increasing the rates of HPV vaccination was recognized as a public health priority and three related goals were identified: (1) improve clinical methods to maximize the recommendation and administration of the HPV vaccine; (2) increase acceptance of HPV vaccination; and (3) improve HPV vaccine availability . EvidencPreliminary research has found promising results using systems-based interventions in a variety of settings including urban hospital primary care clinics; urban private pediatric practices; urban and suburban clinics associated with academic hospitals; urban and suburban primary care clinics; and Federally Qualified Health Centers (FQHCs), with the majority of interventions targeting girls . The aimThis is a systems-based intervention to increase HPV vaccination in boys and girls in a FQHC organization with 30 clinic offices providing medical and dental services to patients in Charlotte, Hendry, and Lee Counties in southwest (SW) Florida. Nine pediatric clinics participated in this intervention. Funding for this project was from the Florida Comprehensive Cancer Control Program (FCCCP) within the Florida Department of Health (FDOH). FDOH acknowledges and values the continued improvement of HPV vaccination rates and is committed to enhanced collaboration to implement sustainable health system interventions . This stUsing de-identified data submitted by nine participating pediatric clinics, HPV vaccination rates were assessed in girls and boys, aged 11 to 16, from March 2013 through December 2019. The 16- month time period of interest was September 2018 to December 2019, which is the time period after the systems-based intervention went into effect.The FDOH was interested in developing partnerships with FQHCs to support systems-based interventions to increase HPV vaccinations. Solicitations for intervention studies to implement systems-based interventions were released in Fall 2017 with a FQHC in SW Florida selected for this intervention in February 2018.In July 2018, this SW Florida FQHC’s Quality Assurance and Quality Improvement (QA/QI) Team for the nine participating pediatric clinics implemented policy and procedural changes to improve the use of the organization’s IIS. The changes included the use of patients’ EHR and Florida State Health Online Tracking System (SHOTS). Florida SHOTS is an IIS in which immunizations administered by participating providers in Florida are tracked. It is a free, statewide, centralized immunization registry that can be accessed by enrolled healthcare providers, schools, and parents [The policy and procedural changes included: (1) provider reminders to alert staff through automated prompts on patient forms and within the EHR system; (2) patient reminders to alert parents/caregivers of patients due for vaccination on their 11th and 12th birthdays through postcard mailings; and (3) provider assessment and feedback through verbal and written notification of individual physician vaccination rates for overall patient population at monthly clinical meetings. By integrating Florida SHOTS into the organization’s policies and procedures, patients who have received vaccinations at clinics outside the FQHC system can be updated into the patient’s EHR. Also, the FQHC implemented an Incentive Program for Adolescent Immunization compliance for pediatric providers and staff starting in January 2018, seven months prior to the implementation of this systems-based intervention.In August 2018, the FQHC’s QA/QI Team implemented policy and procedural changes. The training was developed and presented by the QA/QI Director. Thirty-five training sessions were held and 199 medical assistants and office staff members were trained on best practices and new policies and procedures for increasing HPV vaccination rates during August 2018. Separate training sessions were held for the 46 providers  at their monthly meetings from August 2018 to December 2019. Each month (September 2018–December 2019) the QA/QI Team went to each of the separate clinic offices to address barriers the clinics were facing, review HPV vaccination data, and train any new staff.The HPV vaccination protocol was implemented in nine pediatric clinics and involved the use of FQHC’s EHR and the FDOH statewide immunization registry, Florida SHOTS. Each month, clinic site managers reviewed patients using Florida SHOTS to determine patients who were due for vaccinations including HPV. Figure Prior to a clinic appointment, the clinic site manager would print patients’ HPV immunization records from Florida SHOTS, highlighting which patients were due for their HPV vaccination and give the record to the provider for review. Patient reminders were sent to the patient/parent/caregiver via postcard and/or phone call, depending on patients’ preference. Additionally, all pediatric clinics would offer “vaccine only” appointments to decrease wait times for patients.During the patient appointment, HPV vaccination education was provided to the patient/parent/caregiver using the “announcement approach”  where thAt post-visit, records in Florida SHOTS were updated and printed, highlighting upcoming vaccines and information was added to the recall list. Each month, the organization's Management Information System (MIS) generated monthly reports on HPV compliance that included non-compliant patients (11–15 years old), where the youth were in the HPV vaccination series , follow-up attempts on the type of reminder sent, and date of first and second follow-up calls. Data from monthly reports were shared with pediatric clinic sites.Using a non-randomized design, de-identified individual-level HPV vaccination data were collected from the pediatric clinics using an approved HPV Intervention Database. Total observations were 8,960. Patients who received the first dose within 12 months of the study’s end date but had not received the second dose yet were not considered non-compliant. The data were then analyzed for descriptive and inferential statistics using Microsoft Excel and SAS 9.4 for Windows.The descriptive data of patients ages 11 to 16 years receiving HPV vaccination first and/or second dose was calculated at baseline prior to the intervention and again during the 16-month period after the intervention. A logistic regression model was analyzed with first dose completion as the dependent variable. The independent variables were age and sex. Ethnicity was not considered since this variable was not in the HPV Intervention Database. Pearson’s chi-squared tests were performed on the baseline versus intervention group for first dose completion as well as second dose completion. For the chi-squared test on first dose completion, the baseline group included all patients in the study population who had turned 11 years old before September 1, 2018. Patients who received the first dose of the HPV vaccination were then removed from the dataset and the remaining patients were considered the intervention group. For the chi-squared test on second dose completion, all patients who had not received the first dose of the HPV vaccination were removed from the dataset. Patients were included in the baseline group if they had received their first dose prior to September 1, 2017 and, therefore, were eligible to receive their second dose by September 1, 2018. Patients were included in the intervention group if they had not received their second dose before September 1, 2018 and received their first dose before December 31, 2018.There were 8960 patients (11–16 years old) followed during the nearly 7 year study period with 48.8% girls (n = 4370) and 51.2% boys (n = 4590). For this study period, 80.5% of FQHC patients received the first HPV vaccine dose and 47% received the second dose. For the first dose, 55.5% of 11 year old girls and 54.3% of 11 year old boys were vaccinated. From age 12 to age 16, first dose vaccination rates ranged from the lowest rate of 84.5% for 14 yo girls up to the highest rate of 90.5% for 13 yo boys  with compliance rates increasing with age Table  showed a2  = 64.94, p < 0.00001  = 140.39, p < 0.00001.Counts of youth who received the second dose of the HPV vaccine rose after the intervention (1470 before and 2629 after). However, counts for youth who did not receive the second dose also rose (111 before versus 670 after) leading to a 92.98% rate for patients receiving the second dose before and a 79.69% rate for patients receiving the second dose after the intervention , the second dose rate decreased after the intervention. There are limited studies that provide insight into decreased adherence to the second dose ; howeverThis study also supports the importance of FQHCs’ role in providing access to the HPV vaccinations for the uninsured with only 5.5% of the organization’s patients having private insurance. To effectively increase HPV vaccination rates, theory-informed interventions and implementation strategies are urgently needed . The FQHDuring the FQHC intervention, the practice level was responsible for collecting HPV vaccination data from Florida SHOTS and integrating them into the EHR system, sending reminders to providers when patients were due for vaccination, and conducting staff training sessions on policies and procedures to increase HPV vaccination. The provider level was responsible for sending vaccination reminders to patients and setting follow-up appointments, establishing a standard recommendation for HPV vaccination, providing examples of language to be used to answer frequently asked questions, and ensuring the ability to serve all patient populations (e.g. Spanish-speaking staff). The patient level was responsible for taking advantage of community and clinic materials  to become educated about HPV vaccination benefits and protocols and to initiate and complete the HPV vaccination series. Using this model in future interventions could provide a framework for program implementation and evaluation.There are limitations to this study. The intervention was limited to one FQHC in SW Florida which may not be representative of other primary care pediatricians in other FQHCs or in private practice. Additionally, this study used a non-randomized design so there was no control group for comparison. We were also not able to address the impact of ethnicity on initiation and completion of HPV vaccination series since this information was not captured in the database. Finally, there are unknowns related to the impact of the organization’s Vaccination Incentive Program, staff training related to new employees hired after August 2018, and accuracy of contact information for patients to initiate HPV vaccination and follow-up on receiving the second HPV vaccination.


There is an increasing number of oral squamous cell carcinoma (OSCC) associated with HPV-16. However, p16 expression by immunohistochemistry as the current gold standard for a surrogate marker for virus infection reveals unsatisfying diagnostic accuracy. The aim of this study was to investigate a new rapid test for L1 antibody detection (Prevocheck®) and to validate its diagnostic performance.In a prospective study, the HPV 16 association of all consecutive patients with an OSCC treated between 2015 and 2019 were analyzed by L1 seropositivity (via PrevoCheck®), p16 immunostaining, and partly multiplex PCR for subtype analysis.n = 107), p16 expression was positive in 17 cases (15.9%), and L1 antibody seropositivity in 7 cases (6.5%). In PCR analysis, two cases of HPV35 and 50 were found. Total HPV prevalence was 8.4% overall and 6.5% for HPV-16. An inferior diagnostic accuracy for HPV-16-associated OSCC in comparison to PrevoCheck® was revealed.Overall (The rapid test for L1 antibodies showed an optimal sensitivity and specificity, positive and negative predictive value, and an overall diagnostic accuracy of 100%. However, HPV prevalence seems low in OSCC.L1 rapid test may represent an additional diagnostic staging method to detect HPV-16 association rather than p16 immunohistochemistry. In head and neck oncology, oral squamous cell carcinoma (OSCC) is still an urgent health threat with a growing morbidity in various countries , 2. Whilof different profiles of HPV 16-positive and HPV 16-negative OSCC, and other studies confirmed that each profile should be considered as a distinct cancer entity ) is now available. While the test is easy to perform chair-side and delivers immediate results without any laboratory effort, its clinical benefit for the diagnosis of HPV status in OSCC should be further analyzed. Therefore, the aim of this study was to evaluate PrevoCheck® as a potentially reliable diagnostic tool for HPV status of OSCC in comparison with the p16 expression as the commonly used surrogate marker.While the early viral genes E1, E2, E3, E4, E5, E6, and E7 are necessary to control viral transcription, replication, and cellular transformation, the late genes L1 and L2 encode for the correspondent proteins for the capsid formation . Recent n = 107) included in the study were treated at the Department of Oral- and Maxillofacial Surgery, University Medical Centre Mainz, Germany, from November 2015 to August 2019. The inclusion criteria to participate in the study were the histopathological diagnosis of OSCC located between the lips and palatoglossal arch, including tongue, mandible, maxilla, and planum buccale. OSCC relapse, any other SCC localization, or other malignant tumor entities were defined as exclusion criteria. For all patients, HPV status, age, gender, tumor localization, TNM classification, adjuvant therapy, and clinical outcome were evaluated. All procedures performed within this study were in accordance with the ethical standards of the institutional research committee ”) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was given by each participant in this study.All subsequent patients  according to the manufacturer’s protocol. For this, a venous blood sample was taken. All supplements and reagents were used at room temperature (15–30 °C). Twenty-five microliters of venous blood were added to the falcon tube with HPV-reagent, included in the kit, mixed by pipetting, and incubated for 10 min. Then 100 μl of the incubated blood sample were pipetted in the test-cartridge. After 10–15 min, the result (displayed by color changing or no color changing at the test line) was recorded for further analysis.After surgical removal of the OSCC, the resection specimens were analyzed by the Department of Pathology, University Medical Centre Mainz. HPV status was investigated by immunohistochemical staining against p16 as previously described . Brieflyn = 10), an additional reference analysis was used for further HPV typing at the Institute for Pathology, University Medical Center Halle, Germany, as described previously [In the event of p16-positivity by IHC but L1 seronegativity . 2 × 2 cross-tabulation was used for specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV). For multivariate analysis, ANOVA testing was performed to detect possible risk factors for tumor relapse and overall survival. For graphic display, Kaplan–Meier plots were chosen in combination with log-rank Mantel–Cox regression. The Spearman correlation coefficient was taken to identify correlations of a positive test result and an HPV infection status. n = 66) and 38% female (n = 41). Sixty-five percent of the malignancies were detected at an early tumor stage . Twenty-eight percent (n = 30) initially showed one or more cervical lymph node metastasis. The most affected anatomical sites were the tongue  and the alveolar ridge/gum of the mandible , followed by the floor of the mouth . Tumors of the alveolar ridge/gum of the maxilla , and the cheek  were sparse. No distant metastases could be detected. Histopathologically, the majority of tumors were categorized as well differentiated  ; 62% of the patients were male (n = 58). Due to advanced T-status and/or positive lymph node metastasis, 27 patients received radiation therapy (25%), in 15% in combination with cisplatin as radiosensitizer (n = 16). In the median follow-up interval of 36.6 ± 13.6 months, 10 patients developed a local recurrence (9%) and 8 patients a subsequent lymph node metastasis (7.5%). A total of 14 patients died (13%) due to their tumor manifestation  did significantly worsen overall survival. There was no significant association of L1 seropositivity (p = 0.186), a negative p16 expression (p = 0.360), tumor recurrence (p = 0.586), differentiation grad (p = 0.157), or lymph node metastasis (p = 0.119) on overall survival. In multivariate analysis, the age of the patients had a significant influence on overall survival (p = 0.019), while gender did not (p = 0.291).Advanced tumor size . L1 antibody seropositivity was found in seven cases (6.5%). The additional PCR analysis in ten cases where p16 expression was evaluated as positive and L1 serum antibodies were negative revealed an HPV infection with HPV subtypes 35 and 50 in two cases. For the remaining eight OSCC of this subanalysis, no HPV DNA could be detected by PCR. Therefore, the total HPV prevalence was found to be low at 8.4%, and an even lower HPV-16-associated infection rate of 6.5%.p < 0.005).For p16 expression by IHC to detect HPV 16 association in OSCC, a sensitivity of 100% : 59.04–100%), specificity of 90% (95%CI: 82.38–95.10%) and an overall diagnostic accuracy of 90.65% (95%CI: 83.48–95.43%) were demonstrated. Furthermore, a positive predictive value (PPV) of 41.18% (95%CI: 28–55.76%) and a negative predictive value (NPV) of 100% were found. PrevoCheck® revealed a sensitivity of 100%, a specificity of 100%, a PPV of 100%, and a NPV of 100% as well as overall diagnostic accuracy of 100%. A positive result in both methods was statistically significantly correlated with HPV-16 infection (n = 107) by L1 seropositivity (via PrevoCheck®), p16 immunostaining, and partly multiplex PCR. As a major result of this study, a low overall prevalence of HPV infection in patients with OSCC of 8.4% (6.5% for HPV 16) was revealed. Furthermore, it was shown that the immunohistochemical expression of surrogate marker p16 reveals a poor diagnostic accuracy for HPV 16-associated OSCC (90.65%). For the first time, a rapid test to analyze antibodies to the major capsid protein HPV 16 L1 was introduced. Here, an optimal sensitivity and specificity could be evaluated.This study is the first to exclusively investigate the HPV status of OSCC  controls were analyzed. This could have helped determine test results in context of a coincidental infection. Furthermore, a HPV vaccination that may lead to false positive serological findings was not applied as an exclusion criterion. Finally, the multiplex PCR analysis only analyzed the subgroup that differed in p16 IHC and L1 seroprevalence. For an ideal verification of HPV association, the PCR analysis should have been run in addition to the p16 staining for all obtained samples.With an optimal diagnostic accuracy, the L1 antibody rapid test (PrevoCheck®) was superior to the commonly used p16 expression by IHC as a surrogate marker for HPV 16 association with OSCC. In addition, the eligibility of PrevoCheck® as a possible screening tool for HPV infection or for the early detection of relapse in HPV 16-positive HNSCCs cannot be evaluated yet. In accordance with previous studies, the present study showed a low prevalence  of HPV infection in SCC of the oral cavity. Therefore, in contrast to OPSCC, HPV association does not seem to play a major role in the carcinogenesis of OSCC. To date, it can be concluded that the test is not suitable as a screening or therapy monitoring tool for OSCC.


This study evaluates the pre-vaccination prevalence of HPV infection in women from Vojvodina, Serbia, according to age and cytological status. A total of 1,495 women, ranging from 18 to 65 years of age, with different cytological results were enrolled. The HPV genotyping assay was performed using the EUROArray HPV test in order to detect thirty genitally relevant HPV subtypes. In our study, the most prevalent genotypeswere HPV 16, 31, 51, and 53. Among these, HPV 16 was consistently present in all cytological subgroups. Twelve HPV genotypes classified as carcinogenic to humans (Group 1) were detected in 77.8.0% of HSIL/ASCH and 55.0% of NILM with abnormal colposcopy findings. Six possible carcinogens—HRs (group 2B) were often found in women with normal cytology (14.8%) and mild abnormalities (ASCUS and LSIL), but with lower frequence in HSIL/ASCH lesions (7.1%). HPVs 6 and 11(Group 3) were not found in the cases of HSIL/ASCH. Unclassified HPV types were equally distributed in all cytology groups: 20.7%, 19.1%, 16.3% and 13% of NILM, ASCUS, LSIL and HSIL/ASCH, respectively. Our findings highlight that majority of abnormal Pap test results are caused by Group 1 HPVs among women from our region. Low frequency HPVs of group 2A/2B, especially HSIL/ASCH, supports the conclusion that individual genotypes require consideration of each type as an individual agent. We expect a positive impact of HPV vaccine in reducing HPV-associated cervical lesions among women from Vojvodina province, after establishing vaccination programs in our country. More than 200 human papillomaviruses (HPVs) genotypes have been molecularly characterized to this day, while only a small fraction of them has the potential to induce malignant transformation of human cells. International Agency for Research on Cancer (IARC) has classified twelve HPV types  from phylogenetic five alpha papillomavirus species α5, α6, α7 and α9 in Group 1 “carcinogenic to humans“, because there is sufficient evidence of carcinogenicity in human population [th most frequent cancer among women in Serbia and the 2nd most frequent cancer among women between 15 and 44 years of age [The human papillomavirus infection is considered to be the main risk factor for cervical cancer development, due to well documented involvement of HPV in malignant transformation of cells –4. GlobaNumerous studies have reported that HPV 16 is the most prevalent HPV type in HPV-related cancers worldwide –4. Also,In large epidemiological studies, the prevalence of HPVs genotypes from Group 1 has been of the prime interest, while the distribution of probably 2A) and possibly carcinogenic (2B) types among women’s populations in the world is less known A and pos. The WorOnly a few studies examined HPV prevalence among women in Serbia, usually women from Belgrade with surrounding areas and from the province of Vojvodina –13. ThesThe primary objective of this study was to determine the prevalence of different HPV genotypes, including Group 1, 2A, 2B and unclassified types in cervical samples taken from unvaccinated women from Vojvodina province, Serbia. The secondary objectives of this study were to describe and evaluate demographic and clinical parameters according to HPV genotypes and to establish a scientific rationale for implementation of HPV vaccination in Serbia.This study was conducted within the ongoing project: “Significance of HPV DNA analysis in the prevention of cervical cancer” and the results shown in this paper present the outcome of the analysis conducted from 2016 to 2018. This was a cross-sectional study with convenience type of sampling. Cervical cancer screening was performed by the clinician at the time of pelvic examination, after visualization of cervix. Conventional Pap smears were obtained with the use of cervical Ayer spatulas. Slides were evaluated at the Cytology Laboratory of Department of Gynecology, Community Health Centre Novi Sad, Vojvodina, Serbia and classified according to the Bethesda Classification Diagnosis . The DepSamples for HPV DNA analysis were collected using ThinPrep Pap (Hologic Inc.) or eNAT  specimen transport medium, according to the manufacturer’s original instructions. The study group consisted of 1,495 women, where 567 women had normal cytology , 751 women ASCUS , 136 women LSIL  and 41 women had either HSIL  or ASCH . Women enrolled in the study were informed about the research objectives and they all signed an informed consent form. The study protocol was reviewed and approved by the Medical Ethics Committee of the Institute of Public Health of Vojvodina, Novi Sad, Serbia .DNA was extracted from 200 μl specimen by automatic nucleic acid extraction system . In each EUROArray HPV test 5 μl of the extracted DNA was used. If amplification was not performed on the same day as extraction, the processed samples were stored at 2–8°C for a maximum of five days or frozen at -20/- 80° C for longer periods. The HPV genotyping assay was performed using the EUROArray HPV test  according to the manufacturer’s specifications. This test uses an extensive panel of specific primers and probes, to detect thirty genitally relevant HPV types in one reaction. Among them, 18 HPV types  included in IARC Group 1, 2A and 2B were regarded to be HR HPV and 12 HPV types  included in IARC Group 3 and unclassified types were regarded to be low risk HPVs (LR HPV). Amplification and hybridization steps allowed identification of both the target viral genetic material (E6 /E7 gen) and a fragment of the human Hsp90 gene used as endogenous controls for valid sample extraction and amplification.Beside positive and negative controls, cross-contamination control (CC-I and CC-II) was incorporated into the test ensure high result security. Chip uses two orientation points (OS I and OS II) for automatic detection of position by EUROArray Scan. The evaluation, interpretation and archiving of results has been fully automated by EUROArrayScan software.All statistical analyses were performed with software package STATISTICA for Windows 13.0 . The preA total of 1495 non-vaccinated women  were enrolled in the study. A summary of variables that describe sociodemographic characteristics, lifestyle and sexual habits of participants is provided in In the first step, samples were analyzed for the presence of all of 30 genital HPVs where 760 had HPV positive result, so the overall prevalence of HPV in the study population was 50.8%. All of genotypes covered by EUROArray HPV test were identified in study population and the five most prevalent HPV genotypes were HPV 16 (30.6%), HPV 31 (15.0%), HPV 42 (13.0%), HPV 53(11.7%) and HPV 51 (9.1%). Regarding cytological results and age groups, analyzed women were monitored for distribution of high risk and two low risk HPV types, while unclassified HPVs were excluded. HPV prevalence and distribution of high risk and low risk HPVs types among different age groups are presented in In the next step, HPV DNA positive samples were analyzed according to classification of the IARC. HPVs 6 and 11 which are not classifiable as carcinogenic (Group 3) were found in 7.4% of NILM, but in no cases in HSIL/ASCH. Six possible carcinogens—HRs (group 2B) were often found in women with normal cytology (14.8%) and mild abnormalities (ASCUS and LSIL), but in lower frequency in HSIL/ASCH lesions (7.4%). Among 2B, HPV 53 showed the highest frequency of 11.7%, regardless of cytological results. Unclassified HPV types were found in similar percentage in all cytology groups: 20.7%, 19.1%, 16.3% and 13.0% of NILM, ASCUS, LSIL and HSIL/ASCH, respectively , where HCo-infection with two or more HPV types was observed in 45.6% (347 out of 760) taking into account all HPV DNA positive samples. Women demonstrating positivity for a single HPV genotype accounted for 54.3% (413 out of 760), whereas 25.9% (197 out of 760) of women were positive for dual infections, 11.6% (88 out of 760) for triple infections and 8.2% (62 out of 760) had four or more infections. The percentage of single infection increased significantly from 49.6% in the normal cytological subgroup to 53.5% in the LSIL group and to 61.1% in the HSIL/ASCH group (p<0.05). Dual and triple HPV infections were equally distributed in all of cytology groups, while co-infections with four or more HPV types were not found in the HSIL/ASCH group .Five most prevalent HPV genotypes  were analyzed regarding their presence in multiple infections. Among them, HPV 53 was the significantly more common genotype detected in multiple infections (80.9%), followed by HPV 31 (66.7%), HPV 51 (66%), HPV 42 (65.7%) and HPV 16 (61.8%) .This study provides useful information about the rate of HPV infection and HPV types’ distribution, according to the cytology and age in a cohort of participants from the Province of Vojvodina, Serbia. To the best of our knowledge, this is the first work that examines such a wide spectrum of HPV types in this population. According to EUROArray HPV test of the entire group of 1495 unvaccinated women, the prevalence of HPV DNA was 50.8%. The observed prevalence is not surprising because women with different cytological findings were included in study. These results are similar with our previous studies ,13 and aClarification of the contributions of, particularly novel, HPV genotypes causing cervical carcinogenesis will be important to provide an estimate of the potential impact of prophylactic vaccines and cancer screening programs. For this reason, among women in our region, we have analyzed the frequency of each HPV type which IARC marked as carcinogenic, probably carcinogenic and possibly carcinogenic to humans. The specific genotype prevalence with only 12 cancerogenic (Group 1) genotypes, where observed 55.0%, 57.6%, 61.4% and 77.8% in NILM, ASCUS, LSIL and HSIL/ASCH, respectively. The most common 12 HPV types worldwide were consistent with the 12 HPV types classified as Group 1. They were also very frequently present in women of Vojvodina. Among them, HPV 16 was the most prevalent one and HPV 31, 33 51, and 58 were consistently identified, but in lower frequencies. These results correspond with our previous published data –13. HoweUsing the EUROArray HPV test, for the first time in the territory of Vojvodina, it has been possible to estimate the prevalence of six types  from group 2A/2B. All of six HPVs were detected within the group of women with normal cytology and mild abnormalities (ASCUS and LSIL). However, in women with high grade lesions as HSIL/ASCH we found only three types: HPV 53 (2.7%), 73 (5.5%) and 82 (2.7%). These results are in concordance with studies conducted by other authors who found that HPVs from group 2A/2B circulate among population women worldwide in different percentage ,24–26. HInterestingly, we have identified nine unclassified genotypes  with total prevalence of 20.7%, 19,1%, 16.3% and 13.0% in NILM, ASCUS, LSIL and HSIL/ASCH, respectively. It should be noted that HPV 42 is among the top five HPV types detected in present study. This type was consistently frequent in all cytologic subgroups, often in combination with others HPV types. Several studies have reported the presence of this type in various cervical lesions. However, exact role of HPV42 is not well understood ,25,28. DIn this study, multiple infections were found in 45.6%. Such a high percentage of multiple infections raise the issue of cross-contamination. However, EuroArray HPV test use integrated controls such as DNA positive control and cross-contamination control, which ensure the reliability of test results. In addition, EUROArray HPV compared with Anyplex II HPV28 showed high concordance ratio for genotype-specific detection . ObserveOne of the objectives of this study was to establish a scientific basis for implementation of HPV vaccination in Serbia. Our results emphasize importance of implementation of HPV vaccination in our region, which would offer direct protection against HPV types that are most commonly associated with cancerogenic alterations and found to circulate most frequently within the population in Vojvodina. According to the present data, the potential impact of the quadrivalent HPV vaccines would be about 50% while the usage of the nonavalent vaccine could prevent more than 84% of cervical precancerous lesions identified in this study.Although this study does not reflect all women from province Vojvodina Serbia, the results of this research provide evidence of the high percentage of oncogenic HPV genotypes within infected women group. This data adds support to introduction of HPV vaccine into the national immunization program and integration of HPV DNA tests into in cervical cancer screening programs. Also, data of certain HPV types could not be obtained from previously used methods, since EUROArray HPV test allowed the record of the frequency of “possibly carcinogenic types“, such as 53, 73 and 82, for the first time in our region. It would be of significance to further investigate rare and uncommon HPV genotypes and their possible association with cervical lesions. The data obtained in this study could be of great use in evaluation of the behavior of these infections and associated lesions, as well as for planning of relevant tools for HPV screening and future vaccine implementation.


Self-collected HPV screening may improve cervical cancer screening coverage in low resource countries, yet data guiding implementation and follow-up of abnormal results are sparse.This is a prospective cohort implementation study of HPV self-testing program in Mbarara, Uganda with mobile phones to facilitate result notification and referral for treatment at a regional hospital. The effectiveness of the interventions was analyzed using Proctor’s model of implementation. Women were interviewed following screening and at 6 months to assess acceptability and barriers to follow-up. Data were analyzed using descriptive statistics.P = 0.001).159 of 194 (82%) of eligible women underwent HPV self-sampling; of these, 27 (17%) returned positive for high-risk HPV subtypes. We sent SMS messages providing test results and follow-up instructions to all participants. Seventeen (63%) hrHPV-positive participants reported receiving SMS text instructions for follow-up, of whom 6 (35%) presented for follow-up. The most common reasons for not returning were: lack of transportation (n = 11), disbelief of results (n = 5), lack of childcare (n = 4), and lack of symptoms (n = 3). Confidence in test results was higher for self-screening compared to VIA (Likert score 4.8 vs 4.4, Despite the use of SMS text-based referrals, only one-third of women presented for clinical follow-up after abnormal HPV testing. Cervical cancer is a leading cause of preventable cancer morbidity and mortality, contributing approximately 250 000 deaths in low- and middle-income countries (LMICs) annually . The 90-Recognition of the strong causal relationship between persistent cervical infection with high-risk human papillomavirus (hrHPV) subtypes and cervical cancer has led to a proliferation of HPV molecular assays. Compared with VIA and cytology, hrHPV DNA testing has a higher sensitivity and negative predictive value for precancerous lesions, less inter-observer variability, a more favorable cost-effectiveness profile, and remains efficient in settings with high HPV vaccine penetration -10. ImpoWhere cultural and program barriers limit women’s access to clinician-based cervical cancer screening, home- or community-based self-sampling methods can increase participation among women who are difficult to reach through conventional means . HoweverThe parent study is a population-based sociocentric social network cohort in a rural administrative sub-unit of Rwampara District in southwestern Uganda, whose primary objective is to characterize the social network context of HIV stigma and understand its impacts on HIV care. Community feedback on study procedures is both formally and informally sought on a regular basis, through a community advisory board and multiple community sensitization meetings . In respWomen aged 25- 65 years of age who were enrolled in the parent study were recruited to participate in the hrHPV pilot study. Attendees were recruited with a coordinated series of media and community outreach campaigns, including announcements delivered over the radio and from a mobile truck, banners displayed in public places, and leaflets distributed at church services. Community-wide sensitization meetings were held to provide information about the VIA screening and embedded hrHPV study and to answer questions. Eligible women were identified during these community meetings or at the time of the health fair and were referred to trained research assistants for a detailed explanation of the study and to obtain written informed consent. VIA was offered to eligible attendees independent of study participation. Women were excluded if they were (1) pregnant; (2) had a known gynecologic tract malignancy; or (3) had a prior hysterectomy  clinic at Mbarara Regional Referral Hospital (MRRH). Women who screened positive or suspicious for cancer with VIA, or who had received cryotherapy, were instructed to present to the CCP for clinical follow-up.All samples were transported to a central laboratory at the MRRH. All HPV samples were then analyzed using GeneXpert Xpert® HPV , which reports results as HPV 16, HPV 18/45, other hrHPV , HPV negative, or invalid. Tests returning with invalid results were tested in triplicate.Test results were delivered to women by SMS using an automated web-based algorithm developed by Innovation Streams Limited  .During the study period and following the health fair, nurses at the CCP kept a log of all returning participants. Participants who returned for follow-up underwent colposcopy, biopsy, and recommended treatment under the discretion of the clinic physician and nurses, and per WHO and Uganda cervical prevention guidelines . ClinicaPenetration was defined as the percentage of women meeting eligibility criteria for screening who consented to participate and had sample collection performed. Appropriateness was defined as the percentage of women with the literacy skills to read results via SMS and who had a phone to receive SMS results. Acceptability was measured using mean scores from the post-screen survey. Feasibility was defined as the percentage of women obtaining samples, having valid test results, and receiving SMS results. Effectiveness was defined as the number of women with positive results returning for follow-up in the CCP. Stata statistical software  was used for analyses.We aimed to understand the effectiveness of this intervention to achieve screening goals as well as to understand implementation outcomes for each component of the intervention. Thus we evaluated this pilot on both implementation outcomes  and service outcomes (effectiveness) chosen from Proctor’s model of implementation research . PenetraThis study was approved by the Institutional Review Boards at Mbarara University of Science and Technology, Mbarara Uganda, and Massachusetts General Hospital, Boston, MA. Study data were collected and managed using REDCap electronic data capture tools hosted at Partners Healthcare .Over a 5-day period, 280 women were screened. Of these, 194 (69%) met inclusion criteria, of whom 172/194 (89%) were enrolled and completed the baseline survey and 159/194 (82%) collected HPV self-samples, achieving penetrance of 79% of the intended population  women had undergone previous screening. VIA was the most commonly reported modality (37/56 [66%]), and most women reported that their prior results (51/56 [91%]) were reported to them as normal.  were hrHPV positive, 120 (81%) were hrHPV negative, and 4 (3%) had an invalid result. Of the 27 women with hrHPV positive results, 6/27 (20%) were positive for HPV 16, 6/27 (20%) were positive for HPV 18 or 45, and 21/27 (78%) were positive for other high-risk HPV types. Co-infection with one or more high risk strain was present in five (19%) women.Among those with positive hrHPV results, 4 (17%) also had positive findings on VIA, although none were suspicious for cancer. Three (11%) underwent cryotherapy. HPV outcomes according to VIA results are included in Nearly all (153 [96%]) participants who obtained HPV self-samples provided a mobile phone number (15 of whom provided a smartphone). Most (126 [79%]) participants were able to demonstrate literacy on enrollment, by completing enrollment surveys and reading alound SMS text messages options. SMS text messages were confirmed as delivered to 147 (93%) participants  followed up at the CCP for further screening, following receipt of text messages. Three women (11%) received cryotherapy, 2 (4%) had colposcopy and 1 (4%) had a pap smear. All 27 women with hrHPV positive results were reached by phone at the six-month mark. The 21 women who had not yet presented for follow-up screening reported the following reasons: lack of transportation (n = 11), disbelief of results (n = 5), lack of childcare (n = 4), asymptomatic (n = 3), forgot (n = 3), avoidance of male provider and/or pelvic exam (n = 2), lack of trust in care providers (n = 1), perception of already receiving treatment at health fair (n = 1).Most women rated self-sampling highly and overall reported less embarrassment, less pain, higher confidence, and higher ease of screening with self-collected HPV compared with VIA screening . Most women were appropriate for the intervention, having demonstrated high rates of literacy (79%) and access to a mobile phone (96%). We demonstrated high feasibility in obtaining self-collected HPV samples, obtaining valid test results (98%), and confirming delivery of SMS texts (93%). However, result receipt by SMS was only confirmed as successful in about 50% of women who were reachable by phone at the 6-month endpoint, with only 22% of women with hrHPV positive results following up at the tertiary care facility (Our evaluation approach using the Proctor model enabled us to identify target areas in the cascade of cervical cancer screening for improvement of implementation. We demonstrated that self-sample collection can be performed with high penetrance, and that the context of the community is appropriate for an intervention based on SMS text result notification. However, there was high non-follow up rate in our participants. More work is needed to understand this finding, especially since SMS has been tested and demonstrated to successfully improve linkage to care among people living with HIV in the same catchment area as our study ,23. HowePrimary prevention of cervical cancer hinges on successful vaccination. Currently available vaccines target HPV 16 and -18 genotypes . In our Our findings should be interpreted in light of several limitations. Our cohort was limited to women who presented to the community health fair and who were willing to participate in cervical cancer screening,. Due to small sample size, we were unable to assess for predictors for follow-up. There was no control group to facilitate comparison in follow-up in those who did vs did not receive SMS follow-up and limited our ability to analyze the efficacy of text message follow-up. There was a 6-month interval from screening to result notification, which may have decreased the willingness of participants to engage in follow-up. This was done according to study protocol, given that the progression from infection to cancer occurs over decades, and given that all women who completed sampling had undergone a physical exam during VIA screening by experienced gynecologic oncology nurses and physicians; This was done according to study protocol, given that the progression from infection to cancer occurs over decades, and given that all women who completed sampling had undergone a physical exam during VIA screening by experienced gynecologic oncology nurses and physicians. It is also possible that women presented for follow-up care at health facilities other than MRRH, causing us to underestimate the rate of appropriate follow-up in this cohort. Finally, HPV self-sampling was performed concurrently with VIA screening at the time of the health fair. This was done to achieve a service delivery goal of cervical cancer screening using the current standard of care available. However, our simultaneous use of both methods of screening may have reduced willingness to follow-up among women receiving HPV positive results due to their perception of having already being screened.Despite these limitations, our study presents a novel approach to community-based primary screening for cervical cancer and identifies key areas in the cervical cancer screening cascade and linkage to care that can be targeted for improvement. Though small in number, the willingness of women and reliance on community health workers for result delivery, unprompted by the study, reveals another potential avenue to achieve community-based screening, which may be promising. Further research will be need needed to examine the acceptability and performance of a community health worker-based HPV self-sampling strategy for community-based cervical cancer screening. This, coupled with strategies to reduce or cover transportation costs, either by providing travel reimbursements, or enabling follow-up screening at health facilities closer to women’s homes, may improve success rates across the cascade of care.


Human papillomavirus (HPV) causes over 99% of all cervical cancer globally. In 2019, it was responsible for 3286 deaths in Kenya. Data on the epidemiological distribution of HPV genotypes by cervical dysplasia and HIV-infected women which is important in designing prevention strategy monitoring treatment and management of cervical cancer is lacking in Eastern Kenya.  To determine HPV genotype prevalence and their association with cervical dysplasia among HIV-infected (cases) and noninfected (control) women aged 18-48 years seeking reproductive healthcare.  A cervical broom was softly rotated 360 degrees five times to exfoliate cells from the region of the transformation zone, squamocolumnar junction, and endocervical canal for HPV genotyping. Social-demographic and risk factors responsible for HPV acquisition were collected using a questionnaire. Laboratory outcome and questionnaire data statistical relationships were computed using Pearson chi-square test.  317 women (cases: 161 (50.8%), control 156 (49.2%), mean age: 34.3,SD ± 10.4, range 18-46 years) were recruited from Embu (85/317 (26.8%)), Isiolo (64/317 (20.2%)), Kirinyaga (56/317 (17.7%)), Meru (81/317 (25.6%)), and Tharaka-Nithi (31/317 (9.8%)). The frequency HPV genotypes detected by cervical dysplasia were CIN1 (cases: HPV81 (12/317 (3.8%)), HPV11 (2/317 (0.6%)); control: HPV53 and 66 coinfection (1/317 (0.3%)), CIN2  each), HPV81 (6/317 (1.9%)), and single case (1/317 (0.3%)) of HPV11 and 66, HPV81 and 44, HPV81 and 88, HPV9 and 53, and HPV16 and 58 coinfection; control: HPV81 (2/317 (0.6%)) and invasive cervical cancer (cases: HPV16 (1/317 (0.3%)) and HPV81 (3/317 (0.9%)); control: HPV16 and 66 (1/317 (0.3%))). There was a higher frequency of both high-risk and low-risk HPV genotypes associated with cervical dysplasia among HIV-infected than HIV-uninfected women seeking reproductive health care. This study provides epidemiological data on the existence of nonvaccine HPV types associated with cervical dysplasia in the region. Human papillomavirus (HPV) is primarily responsible for 99.7% of cervical cancers globally . It can Human papillomaviruses are grouped based on their oncogenicity as Group I: “Carcinogenic” and include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59; Group 2A: “probably-carcinogenic” which includes HPV26, 53, 66, 67, 70, 73, 82, 30, 34, 69, 85 and 97; Group 2B: “possibly-carcinogenic” which includes HPV6 and 11; and Group 3: “unclassifiable to carcinogenicity” [Oncogenicity of HPV is further increased by immunocompromised status following human immunodeficiency virus (HIV) infection, long-term exposure to hormonal-contraceptives, and autoimmune drugs which favor tumorigenic effects of HPV-genome and weaken the immune system responsible of clearing cancer cells , 4. OtheLiteratures review wide spectra of HPV genotypes associated with cervical dysplasia among HIV-infected women , 5–7. NoThis cross-sectional study involved 317 women aged 18-46 years who attended reproductive health clinics in referral hospitals of Isiolo, Kirinyaga, Meru, Tharaka-Nithi, and Embu Counties in Eastern Kenya from January to December 2019. A sample size was calculated by using an ICO HPV infection rate (1) of 2.8% . Samples were distributed according to reproductive health clinic's diagnosis 27 VIA/VILLI-positive cases starting July–December 2017 in the region. The stratified technique of sampling was used to obtain five strata based on county of residence then simple random sampling was then used to recruit the required number of a participant in each stratum.Inclusion criteria are as follows: consenting women aged 18-46 years from Eastern Kenya.Exclusion criteria are as follows: menstruating and pregnant women, mentally incompetent, and those with eroded cervix or history of ablative procedures or medical treatment for cervical disease in the last six months , 7.A two-step approach was applied where visual inspection with acetic acid solution (VIA) was performed followed by Lugol's iodine solution (VILLI) which are based on the colors taken up by the cervical transformation zone , 9. The Social-demographic data was collected from participants with precaution after consenting using a questionnaire. The questionnaire focused on data on the residence, age, education level, parity, sexual orientation, and choice of family planning method.Uni-Gold™ Recombigen® HIV-1/2 by Trinity Biotech was used as a tie breaker test [HIV serostatus was determined as per national algorithm. Alere Determine®HIV-1/2 test by Abbort Co. was used as baseline screening while the First Response® HIV 1-2-0 card test by Premier Medical Corporation was used as a confirmatory test in case of discrepancies. ker test .External genitalia were examined while the participant lays in a lithotomic position. A speculum was rinsed in warm water, lubricated, and used to locate the cervical opening (os) under direct light. The mucus plug in os was removed and wiped to ensure sufficient cells were collected. A cervical broom  was softly rotated 360 degrees five times to exfoliate cells from the region of the transformation zone, squamocolumnar junction, and endocervical canal . ExfoliaA standardized protocol for cytopathology request form , 6, Pap DNA extractionAll samples stored for HPV DNA detection were analyzed by the following procedure:μL Lysis-buffer, 8 μLRNA carrier, and 280 μL isopropanol sample/volume. Samples stored at 1-4°C were thawed, vortexed for 5 minutes, and then centrifuged at 10000 r/min for 5 minutes to extract cytological material from the brush into MEM. Samples were subjected to the kit protocol to obtain DNA extracts by magnetic bead technique The eluate was stored at -20°C [(b) HPV DNA PCRA 96-well format HighPrep™ Viral-DNA/RNA, MagBio Genomics, Inc. USA/Canada Lysis kit was used. Master Mix was prepared by mixing 240 at -20°C , 14. (b)μM working stock of each primer was prepared by adding 50 μL biotinylated PGMY09 100 μM to 350 μL nanopure water and 50 μL PGMY11 100 μM primers to 750 μL nanopure water. They were later distributed each 5 μM working stock in 45–90 μL aliquots and stored at -20°C. In the primary PCR, 5 μL of the extract was amplified in a Master Mix containing PCR buffer (1x), 2.0 mM MgCl2, 100 μM dNTPs, 0.13 parts Taq polymerase enzyme and 500 nM MY09, and 500 nM MY11 . First reaction: 4 minutes at 95°C  then 30 cycles of 20 seconds at 95°C, 40 seconds at 56°C, and 2 minutes at 72°C, and final extension for 7 minutes at 72°C. In nested PCR, 5 μL of the first PCR product, 2 mM MgCl2, 500 nM GP5+, and 500 nM GP6+ , 400 μM of dNTPs, and 0.13 units of Taq polymerase enzyme composed the Master Mix. Nested reaction: 4 minutes at 95°C  then 30 cycles of 20 seconds at 95°C, 40 seconds at 60°C, 40 minutes at 72°C, and final extension for 7 minutes at 72°C. Positive control of CIN2+ and negative control of distilled water were incorporated in all primer cycles [(c) Gel electrophoresis and UV visualizationHPV detection was achieved by amplifying an L1 portion of the HPV genome that is relatively conserved through L1 consensus nested PCR in the ABI-thermocycler Model 9600; Applied Biosystems®. HPV consensus primary primers PGMY09 (GCACAGGGACATAACAATGG) and PGMY11 (CGTCCCAAAGGAAACTGATC)  that tarr cycles . (c) GelμL PCR-product in 4% agarose was used in gel electrophoresis where samples that showed a band of 160 bp were considered positive. The positive PCR product was purified using the QIAquick DNA purification kit™  before sequencing [(d) HPV DNA sequencingTris-Borate-EDTA 10x was prepared by dissolving 162 g Tris-base, 50 g boric acid, and 9.5 g EDTA in nanopure water to 1.0 liter volume (pH 8.8). 5 quencing , 13. (d)μL positive PCR-product, 1 μL of 5 μM GP6+ primer, 1 μL BigDye® Terminator, 3.5 μL buffer (5x), and 13.5 mL nanopure water according to the protocol supplied by the manufacturer. Centri-Sep column  was used for dye-terminator cleanup; then, the reaction mixture was sequenced in an automated ABI 3130 four-capillary genetic analyzer [(e) HPV genotyping and phylogenetic analysisDNA sequencing was performed in ABI-thermocycler Model 9600 (Applied Biosystems) for 20 reaction cycles of 1 analyzer , 16. (e)http://blast.ncbi.nlm.gov/blast.cgi. Sequences with unique divergences from the same HPV type underwent phylogenic analysis, and references were obtained from GenBank. The input file of representative sequences and their references was made and subjected for multiple alignments with CLUSTAL W in MEGA X software [rd [CHROMAS software Version 2.4.3 was used to edit the sequences obtained. HPV sequences were blasted in NCBI software , 16. Theware [rd .This study was approved by the KEMRI Scientific Ethical Review Unit . Participants were consented orally where all steps and procedures for HIV and cervical exfoliated cell sample collection; analysis and collection of their results were explained. Laboratory and questionnaire data were confidentially stored by the principal investigator.p < 0.001, df = 12)  .p < .001, df = 1)  HIV-infected women were infected with single HPV type compared to (93.2%) HIV-uninfected women while 73 (23.03%) were infected by multiple HPV types compared with 13 (4.1%) HIV-uninfected women ( df = 1) .SD ± 10.4, range 18-46) women were recruited. Overall HPV prevalence was 27.1% ((86/317); cases: 23.2% (73/317) and control: 4.1% (13/317)). There was significant association between HPV infection and age, parity, education level, and reported number of sex partners , Isiolo (p < 0.001), Meru (p < 0.001), and Tharaka-Nithi (p = 0.048)), age (≤30 years (p < 0.001); <30 years (p = 0.017)), parity (≤35 (p = 0.031); >35 (p < 0.001)), family planning method ; nonhormonal contraceptives (p < 0.001) use), and number of sex partners ) (HPV type distribution among HIV-infected women was significantly associated by residence (Embu ( 0.001)) .p = 0.001)  HIV-positive women had normal cytology as compared to 143 (45.1%) HIV-negative, whereas 65 (20.5%) HIV-positive women had abnormal cytology results compared with 13 (4.1%) HIV-negative (= 0.001) .p < 0.001) ): HPV81 (12 (3.8%)), HPV11 (2 (0.6%)), CIN2 (16 (5.0%)): single case (0.3%) of HPV11, HPV16, and HPV66 and HPV81 (6 (1.9%)), CIN3 (6 (1.8%)): HPV11 (1 (0.3%)), HPV81 (4 (1.2%)) and invasive cancer (5 (1.5%)): HPV16 (1 (0.3%)) and HPV81 (3 (0.9%)), (< 0.001) .Phylogenetic tree of HPV samples marked in red aligned against the representation of the different HPV genotypes and subtypes distributed in different regions worldwide, constructed using MEGA6 neighbor-joining using 1000 bootstrap reference method. Most HPV81 clustered with those cases was detected in Bangkok Morocco and Thailand, HPV66 clustered with those from Kenya and Iran, while HPV66 clustered with cases reported in Tunisia, Morocco, Iran, and India .This study established an overall HPV infection rate of 27.12% (cases: 73 (23.03%); control: 13 (4.1%)). It agrees with the overall age-specific HPV infection rate of 27% in neighbouring Nairobi region  that couWomen aged below 35 years of age had a high rate of mixed HPV genotypes and a significant association between HIV infection and abnormal cytology outcome which agrees with published observations .A high infection rate was also noted among Christians who comprised the majority of participants than Muslims. This study concurs with other studies that have shown high HPV infection rate among secondary school educated women. This is attributed to a higher risky sexual behaviour and low knowledge on HPV and associated risk factors as reported among young women in South Africa , 21.HPV infection rate was high among HIV-infected women with high parity, married, and those using hormonal contraceptive than other categories. This could be associated with high persistence and subsequent low clearance of HPV upon infection among these groups as reported by other studies , 22.There was a significant association between having multiple sex partners and HPV infection. Most of the participants in this category were HPV infected. This could be because having multiple sex partners increased their chance of contracting HPV and HIV. The dominance of HPV infection in normal cytology was detailed high (28.8%) as published . YoungerAlphapapillomavirus 3 (α3) HPV species by both HIV serostatus and cytology outcomes. The predominance of HPV81 (CP8304) has moreover been detailed in women with anomalous cytology in Kenya [The foremost common HPV detected in the region was lrHPV81—in Kenya , 7 and Qin Kenya . It is ain Kenya , 25 as sα9) and HPV66 (α6) were most common high-risk types associated with invasive cancer by HIV serostatus. Their predominance in abnormal cytology among HIV-infected women has been established [α9). This study and others therefore recommend routine testing for HPV16, HPV52, and HPV66 [α7) and (α9) dominate neoplastic tissues. Though they were also detected in normal cytology, a study has demonstrated the genetic variability and frequent mutating L1, E6, and E7 genes mostly HPV52 and HPV53 (α7) resulting into intratypic variants with increased oncogenic potential [HPV16 , and HPV88 which do not feature in many studies as potential oncogenic types Here, their association with CIN+ could have been increased by HIV infection as reported in other studies [Single HPV type infection in CIN1+ showed diversity compared with multiple HPV type's infection in CIN1+ by HIV infection. Diverse infection was also seen among HIV-infected women with multiple HPV infections. Other common lrHPV types detected were HPV11, HPV44  species and HPV81 will cross-react with HPV61 [There were a significantly low number of participants infected with HPV types of the same alpha group in this study. There were also a reduced number of hrHPV52 and 58 and absence of most of other hrHPV types, notably HPV18. This could be attributed to antibodies' cross-reaction which is achieved within species unlike across species. Antibodies against HPV16 cross-react and protect against HPV52 and HPV58 of the same th HPV61 .This study and literature data in Kenya establish the predominance of mixed HPV types other than those included in the current vaccine . This puThe phylogenetic trees illustrated that single or multiple HPV types infected each participant as shown by 15 participants. Another reason is that HPV genes are replicated by the host replication machinery suggesting that a very low human autosomal–like mutation rate would be operating as established in another study .The study group is a minimal presentation, and data presented cannot be generalized as exact outcome if all women of reproductive age in Eastern Kenya were sampled. Responses to the questionnaire could have been altered by missing key words like ‘cervical cancer' in local direct during translation but caution was taken to give accurate translation and meaning.There was a higher frequency of both high-risk and low-risk HPV genotypes associated with cervical dysplasia among HIV-infected than HIV-uninfected women seeking reproductive health care. This study provides epidemiological data on the existence of nonvaccine HPV types associated with cervical dysplasia in Eastern Kenya. Genotypic HPV diversity established has potential impact on new vaccines design and need for increased cervical screening among HIV-infected women.