OBJECTIVES/SPECIFIC AIMS: Improving human papillomavirus (HPV) vaccination rates ultimately decreases the morbidity and mortality of HPV-associated diseases. A school-based program was piloted in the Rio Grande City Consolidated Independent School District (RGCCISD) to increase HPV vaccination. METHODS/STUDY POPULATION: We assessed baseline HPV vaccination; surveyed 622 parents of eligible children aged ≥9 years; and piloted and developed a school-based HPV education and vaccination program in 1 middle school in 2017 and 4 additional middle schools in 2018. The parent survey included (1) demographic information, (2) an assessment of parental knowledge about the HPV vaccine, and (3) information about their children and HPV vaccine experience. Results of the parent survey and pilot program are in progress. RESULTS/ANTICIPATED RESULTS: As of 9/1/2016, 20.4% of the 7527 RGCCISD eligible students (≥9 years) had completed the HPV vaccine. Baseline completion rates were higher for RGCCISD students aged 12–14 years compared with students aged 9–11 and ≥15 years (28.4% vs. 16.5%). Baseline completion rates for RGCCISD adolescents were substantially lower than those reported in NIS-Teen and for Texas . DISCUSSION/SIGNIFICANCE OF IMPACT: Initial results show that engagement with key stakeholders is important and schools are a great venue for delivering and increasing HPV vaccination.


TTN (OMIM 188840), encoding the largest human protein, are known to cause dilated cardiomyopathy and several forms of skeletal myopathy. The clinical interpretation of TTN variants is challenging, however, due to the frequency of missense changes, variable testing and reporting practices in commercial laboratories, and incomplete understanding of the spectrum of TTN‐related disease.Pathogenic variants in TTN deletion segregating in a family with an unusual skeletal myopathy phenotype associated with facial weakness, gait abnormality, and dilated cardiomyopathy.We report a heterozygous TTN was identified in the proband and his symptomatic son, as well as in an additional son whose symptoms were identified on clinical evaluation. The deletion was found to be de novo in the proband.A novel 16.430 kb heterozygous deletion spanning part of the A‐ and M‐bands of TTN may be an unrecognized cause of skeletal myopathy phenotypes, particularly when accompanied by dilated cardiomyopathy.Pathogenic variants in  His early motor milestones included crawling at 12 months and walking at 18 months. He reported progressive difficulty with running and lifting. Manual muscle testing identified both proximal (predominant) and distal weakness with asymmetry (L>R), as well as mild facial weakness. He has a high‐arched palate, and ambulates with a bilateral uncompensated gluteus medius gait and is unable to walk on his heels or toes. Echocardiogram at age 15 was normal.TTN (exons 346‐362), interpreted as likely pathogenic. All first‐degree relatives were subsequently tested for this variant. Sons III‐1, III‐3, and III‐4 also shared this deletion. No other family members (including both parents of the proband) were found to have the deletion. Clinical evaluation of son III‐3 revealed facial weakness, neck flexor weakness, decreased muscle bulk, as well as proximal and distal weakness with asymmetry (L>R) , validated coverage‐based CNV detection algorithms designed to flag possible split‐read signals were applied. Once verified, the variant call format was updated and interpreted.Peripheral blood or saliva were evaluated by Next Generation Sequencing , centronuclear myopathy, and TTN‐associated distal myopathy  and hereditary myopathy with early respiratory failure (caused by dominant missense mutations in exon 344); neither causes cardiac disease  has a frequency of 0.11% in Europeans (Lek et al., TTN variants may produce both skeletal and cardiac muscle disease in a dominant fashion (Ceyhan‐Birsoy et al., Here we report an unusual skeletal myopathy phenotype with facial weakness, gait abnormality and DCM, associated with a novel de novoTTN sequencing and yielded inconsistent results. It is important that clinicians understand the variability between laboratories in detecting and reporting deletions, that the same variant may be interpreted or reported differently between laboratories, and reporting practices within the same laboratory may change over time. This issue is particularly acute in TTN and may lead to under‐recognition of clinically significant TTN variants and missed diagnoses.The proband's diagnostic odyssey illustrates the challenges associated with NGS technology and variant interpretation. Two different NGS panels were applied; both included TTN, which may impact the approach to genetic evaluation for both DCM and skeletal myopathy. We suggest that TTN be considered in the genetic differential diagnosis in any patient with both skeletal muscle weakness and DCM.Further data are needed regarding the frequency and pathogenicity of structural variants in C. Tan, M. Vatta and T. Winder are employees of Invitae Corporation. B. Elsheikh is a medical consultant f or Biogen Inc. J. Roggenbuck, K. Rich, A. Morales, D. Eck, W. King, R. Hershberger, and J. Kissel report no disclosures.A signed Patient Consent‐to‐Disclose Form has been obtained for photos/videos of any recognizable patient. Click here for additional data file.


P < 0.0001). Elimination of HPV16/18 by vaccination is predicted to prevent 42% (95% CI 37.0‐46.7) of CIN2, 57% (95% CI 53.8‐60.5) of CIN3 and 64% (95% CI 60.3‐67.7) of cervical cancer. Prevention of the five additional HPV types HPV31/33/45/52/58 would increase the protection to 68% (95% CI 63.0‐72.2) in CIN2, 85% (95% CI 82.4‐87.2) in CIN3 and 80% (95% CI 77.0‐83.2) in cervical cancer. This study provides large‐scale and representative baselines for assessing and evaluating the population‐based preventive impact of HPV vaccination.It is valuable to establish a population‐based prevaccination baseline distribution of human papillomavirus (HPV) types among women with high‐grade cervical intraepithelial neoplasia (CIN) grade 2 or 3 and cervical cancer in order to assess the potential impact of HPV vaccination. In four countries , we collected consecutive series of cervical cancers (n = 639) and high‐grade precancerous cervical lesions (n = 1240) during 2004‐2006 before implementation of HPV vaccination and subjected the specimens to standardized HPV genotyping. The HPV prevalence was 82.7%  in CIN2, 91.6% (95% CI 89.7‐93.5) in CIN3, and 86.4% (95% CI 83.7‐89.1) in cervical cancer. The most common HPV types in CIN2/3 were HPV16  and HPV31 , while HPV16 and HPV18 were the most frequent types in cervical cancer . The prevalence of HPV16/18 decreased with increasing age at diagnosis in both CIN2/3 and cervical cancer ( Denmark and Norway initiated HPV vaccination in 2009, followed by Iceland 2011) and Sweden (2012), with all HPV vaccination programs targeting 10‐12 years old girls. In Denmark, a catch‐up program aimed at girls aged 13‐15 years was included from 2009 and was in 2012 expanded to women up to 27 years of age. In Sweden, girls aged 13‐17 years had access to subsidized HPV vaccination during 2007‐2011, while the national vaccination program in 2012 included a catch‐up program with free vaccination of girls aged 13‐18 years. and SwedThe aim of this study was to assess the prevalence and age‐specific distribution of HPV types in different grades of severe CIN (grades 2 and 3) and cervical cancer among women from Denmark, Iceland, Norway, and Sweden, during 2004‐2006. This will enable establishment of a baseline for surveillance of the effect of HPV vaccination as well as an estimation of the proportion of CIN2, CIN3, and cervical cancer that could potentially be prevented when women are vaccinated either against two (16/18) or seven (16/18/31/33/45/52/58) oncogenic HPV types.22.1This is a multicountry, cross‐sectional study including four countries . We aimed to collect formalin‐fixed paraffin‐embedded cervical histological specimens of 200 cervical cancers and 300 CIN2 or CIN3, randomly selected from each country, with a smaller number of cancers from Iceland due to the limited size of the female population. Women with a histologically confirmed diagnosis of CIN2, CIN3, or cervical cancer, during 2004‐2006, were eligible for the study . The study was approved in each country by the relevant authorities; the Scientific Ethics Committee and the Data Protection Board. In total, cervical tissue samples from 1964 women were collected . All cervical specimens were classified based on the histological diagnosis as CIN2, CIN3 or cervical cancer. Cancers were further classified into squamous cell carcinoma (SCC), adenocarcinoma (AC), and other/cervical carcinoma not otherwise specified (NOS). In total, we excluded 85 specimens, due to inadequacy of the sample for HPV testing (n = 64), diagnosis other than CIN2, CIN3, or cervical cancer (n = 9), withdrawal of consent (n = 5), or lack of appropriate specimen identification (n = 7), thereby leaving 1879 samples for study. The age distribution of the included women in this study was comparable to the age distribution of all women with cervical high‐grade lesions and cancer in each country.2.22.2.1All formalin‐fixed paraffin‐embedded blocks were sectioned according to a standardized protocol (previously verified to not result in contaminations) at a commercial, accredited laboratory, HistoCenter, Inc in Gothenburg, Sweden. Every case‐block was sectioned with a new knife, using new gloves. In between each case‐block, a blank‐block containing only paraffin was sectioned as a control for contamination. The blank‐block was sectioned first. Four 5‐µm sections were transferred to a 1.5 mL Eppendorf screw cap tube, using a new toothpick. The case‐block was mounted on the microtome and six sections, 5 µm each, were made with the same knife as the blank‐block. The first and last section were stained with hematoxylin‐eosin for rereview by a senior pathologist to assess the histology on the actual tissue, and the four 5‐µm sections in‐between were transferred to a tube in the same way as the sections from the blank‐block. After each case‐block, the knife was “removed and the microtome cleaned with DNAZap .2.2.2g, for 10 minutes. The supernatant was removed with a sterile transfer pipette and the process repeated once. After the second removal of xylene, the samples were washed with pure ethanol twice and air‐dried. Dried pellets were incubated in 100 µL Digestion buffer  with Proteinase K (50 mg/mL) in 37°C for about 24 hours and then boiled at 100°C for 10 minutes, to inactivate Proteinase K. After extraction, case‐blocks and blank‐blocks were subjected to polymerase chain reaction (PCR) with modified general primers (MGP) targeting L1, and HPV genotyped using Luminex, as previously described.All samples, case‐blocks and blank‐blocks, were extracted with Xylene as previously described.Betaglobin real‐time PCR was performedFor case‐blocks with a valid result positive for a universal probe, but not for any specific HPV type, the MGP PCR product was separated with 2% agarose gel electrophoresis. Fragments of length 170 bp were cut out and purified using QIAquick Gel Extraction Kit , 30 µL elution buffer. After purification, the product was sent for Sanger sequencing at Eurofins MWG Operon, Ebersberg, Germany.2.3We assessed the HPV prevalence anddistribution of HPV types in the CIN2, CIN3, and cervical cancer specimens. The prevalences are shown both among the whole study population and among HPV‐positive women. The following HPV types were defined as oncogenic: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, or probably oncogenic: HPV68, HPV types.3P = 0.079).In the present study, we included specimens from 1240 high‐grade CIN lesions (404 CIN2 and 836 CIN3) and 639 cervical cancer specimens . Infection with single HPV types was the commonest among cervical cancer overall , both in SCC  and AC . HPV16 and HPV18 were the most frequent HPV types in cervical cancer overall . Among all SCC specimens, the prevalence of HPV16 was 53.3% (95% CI 48.9‐57.8), followed by HPV18 , HPV33, 45, and 31. HPV16 was also the most prevalent HPV type among all AC specimens , while HPV18 accounted for almost all the remaining infections within this group . The prevalence of nononcogenic HPV types was very low in cervical cancer, ranging from 0% to 0.6%.The distribution of HPV types among women with cervical cancer is displayed in Table P < 0.0001 for both). In contrast, the prevalence of HPV31/33/45/52/58 remained relatively constant in all age groups, and the prevalence of HPV‐negative samples increased with increasing age (P < 0.0001 for both). Among the women with SCC and AC, the prevalence of HPV16/18 decreased with increasing age at diagnosis (P < 0.0001 for both), whereas the prevalence of HPV31/33/45/52/58 increased slightly with increasing age among women with SSC (P = 0.055). The age pattern in the prevalence of HPV‐negative samples showed an increase with increasing age at diagnosis .Figure Figure 4In this large multicountry study on cervical specimens obtained in the period 2004‐2006 from almost 2000 women with high‐grade precancerous cervical lesions or cervical cancer, we found that HPV16 and 31 were the most common types in CIN2 and CIN3, while HPV16 and 18 were the most common types in cervical cancer, with HPV18 being far more common in AC than in SCC. In CIN2 and CIN3 as well as cancer specimens, the prevalence of HPV‐negative samples increased with increasing age at diagnosis, while the prevalence of HPV types 16/18 was found to decrease. The preventive potential of HPV16/18 vaccination was 57% and 64% in CIN3 and cervical cancer, respectively, while inclusion of HPV31/33/45/52/58 in a vaccine increased the protection to more than 80% in both the groups.Other studies also report HPV16 as the most prevalent type in women with CIN2, CIN3, and cervical cancer, followed by HPV31 in CIN2 and CIN3 and HPV18 in cancer.With increasing age at diagnosis of the women, the specimens were less likely to harbor HPV16/18. A decrease in the prevalence of HPV16/18 with respect to woman’s age at diagnosis has been reported in previous studies.Infection with HPV is recognized as a necessary cause of cervical cancer, so our finding of around 10% HPV‐negative specimens in women with cervical cancer is relatively high, although consistent with several other studies.A small number of CIN3 and cervical cancer cases harbored nononcogenic HPV types only, as reported in other studies.Since the introduction of HPV vaccination programs, ecological studies have reported declines of genital warts and CIN of different grades.In the present study, we found a substantial effect of including five additional oncogenic HPV types compared to an HPV16/18 vaccine among women with high‐grade precancerous cervical lesions, where the potential prevention increased from 42% to 68% in CIN2 and from 57% to 85% in CIN3. This illustrates the high prevalence of especially HPV31, 33, and 52 in CIN2 and CIN3. Furthermore, this underlines the potential impact of a vaccine including HPV16/18/31/33/45/52/58 or a vaccine with substantial cross‐protection against these types on the future incidence of high‐grade CIN among Nordic women. When comparing the effect of an HPV16/18 vaccine to an HPV16/18/31/33/45/52/58 vaccine on cervical cancer, the estimated prevention increased from 64% to 80% (among HPV positive: 74%‐93%), corresponding to results from a recent population‐based US study and a meta‐analysis of two worldwide HPV distribution studies.Strengths of our study include the population‐based design, the large sample size, and inclusion of women from four Nordic countries with a similar demographic composition, thus making our results generalizable to the Nordic female population. Our HPV results were from histologically confirmed diagnostic categories , which increases the likelihood of a true causal association. HPV genotyping was performed by use of a highly sensitive PCR method at a central reference laboratory to ensure standardized testing. In addition, we tested for a wide spectrum of HPV types. Previous studies have reported results for CIN or cervical cancer overall, or in some cases for combined CIN2+ or CIN3+ groups, whereas we provide results for CIN2 and CIN3 separately, as well as histological subgroups  of cervical cancer, to fully report the differences in specific genotypes between groups.In conclusion, we present here a baseline assessment of the overall and type‐specific HPV prevalence and age‐specific distribution in both CIN2 and CIN3 as well as cervical cancer among women from four Nordic countries before implementation of HPV vaccination. These results form the basis for future monitoring of the effect of HPV vaccination. In addition, they contribute with estimates of the potential preventive impact of both HPV16/18 and HPV16/18/31/33/45/52/58 vaccination in the Nordic countries.SKK received lecture fee from Merck and Sanofi Pasteur MSD, scientific advisory board fee from Merck, and unrestricted research grants through her institution from Merck. JD reports having received research grants to his institution for the funding of the study. CM received lecture fees and travel grants from Sanofi Pasteur MSD. CL and MH report that their institution received a grant from Merck. KLL is a full‐time employee of Merck & Co. Inc and owns stocks and options of Merck. MN received research grants from MSD Norway/Merck through the affiliating institute. CDC, SG, LT, KS, and BTH report no conflicts of interest.


HPV-16–positive HNSCC and HPV-16–negative HNSCC have different clinical factors, representing distinct forms of cancers. The study aimed to identify patient-specific factors for HPV-16-positive HNSCC based on baseline clinical data.Factors associated with HPV-16-positive HNSCC were identified using the data from 210 patients diagnosed with HNSCC at University College of London Hospital between January 1, 2003, and April 30, 2015, inclusive. A series of models were developed using logistic regression methods, and the overall model fit was compared using Akaike Information Criterion. Survival analysis was carried with Cox proportional hazards model for survival-time outcomes. The survival time for individual patients was defined as the time from diagnosis of HNSCC to the date of death from any cause. For patients who did not die, they were censored at the end of study on April 30, 2015.Of the 210 patients, 151 (72%) were found to have HPV-16-positive HNSCC. The logistic regression model showed that the prevalence of developing HPV-16-positive HNSCC was 3.79 times higher in patients with Type 2 Diabetes Mellitus (T2DM)  than in those without T2DM, and 8.84 times higher in patients with history of primary HNSCC  than in those without a history of primary HNSCC. HPV-16–positive HNSCC was also observed more in tonsils  and less in non-alcohol drinker’s oral cavity . Furthermore, individual patients were followed-up for 1 to 13 years (median of 1 year). Patients with HPV-positive HNSCC had a median survival of 5 years . Among HPV-16–positive HNSCC cohort, T2DM was a risk for poorer prognosis , and had lower median survival of 3 years , as compared to 6 years  in non-T2DM.Patient-specific factors for HPV-positive HNSCC are T2DM, history of primary HNSCC and tonsillar site. T2DM is associated with poorer prognosis. These findings suggest that it might be beneficial if routine HPV-16 screening is carried out in T2DM patients which can provide better therapeutic and management strategies. Over the past 3 decades, an increase in the incidence of human papillomavirus (HPV)-positive head and neck cancer (HNSCC), particularly oropharyngeal cancer, has been observed in the United Kingdom, most notably among men under the age of 60 . PreviouFew studies developed risk predictive models of tumor HPV-16–status using tumor biomarkers  or histoThe study protocol was approved by the Institutional Review Board of the UCLH. The ethic number for the study is 04/Q0505/59 by National Research and Ethics Committee (NRES) London-Harrow through University College of London Hospitals NHS Foundation Trust. Patients’ records were fully anonymized prior to access.The study cohort was derived from the head and neck cancer department medical records at University College of London Hospital (UCLH). Patients diagnosed with HNSCC between January 1, 2003, and April 30, 2015, at UCLH were included, inclusive. Patients were eligible for inclusion if they were older than 18 years and were newly diagnosed with a histological confirmed squamous cell carcinoma of the oral cavity, oropharynx, tonsils, hypopharynx, larynx, nasopharynx, or multiple HNSCC tumor sites. Multiple HNSCC tumor sites was defined as the presence of two or more tumors synchronously in head and neck region. Anatomic site of the tumor was determined by a physical examination that was performed by the treating head and neck surgeon. SPSS file consisting of all patients’ parameters used in model prediction for the study is provided in in situ hybridization approach for biotinylated probes [The outcome of interest was tumor HPV-16 status. Patients were classified as having a diagnosis of either HPV-16-positive or HPV-16-negative HNSCC based upon HPV-16 DNA detection in formalin-fixed and paraffin-embedded (FFPE) tumors using the d probes . Tissue Patient independent variables consisted of demographic variables, including age and sex; co-morbid conditions, including type 2 diabetes mellitus (T2DM), hypertension, vascular diseases, previous primary HNSCC, history of current or former smoking, or alcohol use; treatments history, including use of anti-hypertensives, hypoglycemic agents, or anti-infective for systemic use; and HNSCC tumor anatomic sites. A patient’s co-morbid conditions and treatment history were categorized as present or absent at the time of HNSCC diagnosis. The anatomic sites of head and neck included in the analysis were oropharynx, tonsils, oral cavity, larynx, hypopharynx, paranasal sinus, and nasopharynx. In this study, a large number of our patients (36%) were diagnosed with HPV-positive tonsillar carcinoma; therefore, tonsils which is a part of the oropharynx anatomic site were analyzed separately. Oropharynx included tumor in soft palate and uvula.All selected variables were tested for multi co-linearity to avoid any strong correlation between the variables. The presence of co-linearity was examined by the evaluation of variance inflation factors and magnitude of standard errors. Variables with more than 30% missing values were not included in the analysis. All other missing data were imputed using the multiple imputation technique using SPSS .P >0.10) were excluded from further analyses. Variables were then entered in each model in accordance with the chronology in which patient demographic and clinical data were available at the time of HNSCC diagnosis. Model with more variables or with greater complexity was successively compared with the simple ones. Using this approach, 4 Models were generated from the data provided.The first aim of this study was to identify patient- specific factors associated with HPV-16–positive HNSCC based on baseline demographic and clinical data. Therefore, we developed a sequential series of logistic regression models using HPV-16–positive HNSCC as the dependent variable . We usedP < 0.05) was identified between the oral cavity tumor site and alcohol use; therefore, both variables were added to the final model (Model 4).Model 1, was developed by adding patient demographics . Model 2, was developed by adding patient co-morbidities  and compared with model 1. Model 3, was developed by adding patient HNSCC tumor anatomic sites  and compared with model 2. We then tested for interaction effects between predictors or variables retained in model 3. A significant interaction (Improvement in model performance through the addition of new variables in multivariate logistic regression models was tested using measures of discrimination and goodness of fit. The discriminatory power of each model was assessed using concordance statistics (C statistics). Discrimination refers to the ability of a model to clearly distinguish between 2 groups of outcomes (discriminate between the HPV-16–positive HNSCC and HPV-16–negative HNSCC patient groups) and can range from 0.5 (no discrimination) to 1.0 (perfect discrimination) , 11. TheThe second aim of this study was to compare the median survival time between patients with HPV-positive and HPV-negative tumors during the follow-up period of 1 to 13 years (median of 1 year). We also compared the median survival time between T2DM and non-T2DM subgroups within the HPV-positive HNSCC cohort. The survival time for individual patients was defined as the time from diagnosis of HNSCC to the date of death from any cause. For patients who did not die, they were censored at the end of study on April 30, 2015. Survival probability was computed with a Cox proportional hazards model and the resultant cumulative survival probability curve was plotted. Cox proportional hazards model was adjusted for age, male sex, alcohol use, smoking, and American Society of Anesthesiologist (ASA) co-morbidity score .2) test or Fisher exact test. A finding of P < 0.05 was considered indicative of a statistically significant difference for all tests.All analyses were performed using SPSS 21.0. Categorical variables are presented herein as percentages, and the continuous variable (age) are presented as means (SDs). The age variable was assessed for normality using the Kolmogorov-Smirnov test. Comparisons of categorical variables were performed using the chi-square . Consistent with recent epidemiological studies , almost P = 0.100). Compared to HPV-16–negative patient groups, those with HPV-16–positive HNSCC had significantly higher number of co-morbidities such as T2DM , history of primary HNSCC , or alcohol use history ; and had significantly higher and lower rates of HNSCC in tonsils  and oral cavity , respectively.The patient demographic and clinical characteristics captured in the database are listed in The odd ratios for the variables and statistics for discrimination and goodness of fit for successive models are shown in Individual patients were followed-up for 1 to 13 years (median of 1 year), during which time 47 deaths occurred. Patients with HPV-positive HNSCC had a median survival of 5 years . HPV-positive HNSCC cohort with T2DM and a history of previous HNSCC, or with diagnosis of tonsillar or oral cavity were further stratified. A multivariate Cox proportional hazards regression analysis, adjusted for sex, age, alcohol use, smoking, and ASA co-morbidity score, revealed that among above-mentioned HPV-positive HNSCC specific factors, T2DM were a risk for poorer prognosis , and had lower median survival of 3 years , as compared to 6 years  in non-T2DM. .In this study, we developed a series of risk prediction models in a cohort of HNSCC to identify patient specific factors associated with tumor HPV-16 status. A comprehensive clinical predictive model would serve to help clinicians decide which patient-factor is associated with higher prevalence of HPV-16–positive HNSCC. As there is a different factors associated with tumor HPV-16–status, we also followed-up individual patients for 1 to 13 years (median of 1 year), and found that there is also heterogeneity in the prognostic factors associated with tumor HPV-16-status.P = .01) and oropharyngeal subsite (P = 0.02). Similarly, D’Souza et al [Several prior studies have developed prediction models from several likely patient-related factors in patients with HNSCC , 4.Byrd e P = 0.0. Similarza et al .In this study, we found that patients who had a history of primary HNSCC were significantly at higher risk of recurrent second primary HPV-16–positive HNSCC. Our finding correlates with previous studies that have reported a trend between previous HNSCC and second primary HNSCC in an era of oncogenic HPV , 16. DiaA noteworthy finding in our study was that patients with T2DM were significantly associated with higher prevalence of HPV-16–positive HNSCC and were associated with poorer prognosis in HPV-16-positive HNSCC subgroups. Numerous epidemiological studies have reported a higher risk of oral precancerous lesions  and oropIn our patients, HPV-16–associated HNSCC predominantly affected the tonsillar region, compared to the small percentage of tumors at the oral cavity. Our results are consistent with the previous findings that showed a higher prevalence of oncogenic HPV-16-DNA in palatine tonsils (50%) , compareSimilar to any observational study, this investigation has a number of limitations. Although, through development of a series multivariate logistic regression models, the effect of observed confounders were adjusted, there might be a number of unobservable factors that could only be controlled with a randomized controlled trial. In addition, the limited sample size of this study could have resulted in some bias in the results produced. We also cannot exclude the possibility of associations between patient sexual behavior and HPV-16–positive HNSCC since we did not have access to this factor. Finally, this study was performed in one hospital, which may also limit the generalization of the results. Nonetheless, the data presented in this study are the first, to our knowledge, to suggest that T2DM is a prognostic factor for HPV-16 positive HNSCC, thus, these findings will need to be confirmed in a larger database, and the impact of preventive measures such as routine HPV-screening, testing for HPV, and even the prophylactic effects of HPV-vaccination on these high-risk groups be evaluated. Our findings could lead to further research about the impact of T2DM for promoting viral carcinogenesis and in deciphering the molecular mechanisms behind T2DM lower responsiveness to chemotherapy agents.In conclusion, our data show that history of primary HNSCC and occurrence of HNSCC in the tonsillar regions are patient-specific factors for HPV-positive HNSCC. We also were able to identify T2DM as a prognostic factor for HPV-positive HNSCC suggesting potential preventive strategy through HPV-16 screening of T2DM patients.S1 File(SAV)Click here for additional data file.


Four novel human gammapapillomaviruses were characterized from penile specimens using genome amplification, cloning, and sequencing. The HPV-219 L1 gene showed 87% nucleotide identity to that of HPV-213 of species gamma-13, HPV-220 had 72% identity to L1 of HPV-212 (gamma-17), HPV-221 had 80% identity to L1 of HPV-142 (gamma-10), and HPV-222 had 73% nucleotide identity to L1 of HPV-162 (gamma-19). Four novel human gammapapillomaviruses were characterized from penile specimens using genome amplification, cloning, and sequencing. The HPV-219 L1 gene showed 87% nucleotide identity to that of HPV-213 of species gamma-13, HPV-220 had 72% identity to L1 of HPV-212 (gamma-17), HPV-221 had 80% identity to L1 of HPV-142 (gamma-10), and HPV-222 had 73% nucleotide identity to L1 of HPV-162 (gamma-19). Papillomaviridae family that infect mucosal and cutaneous epithelia  are small nonenveloped DNA viruses of the pithelia , 2. The enotypes . Here, wenotypes .de novo assembly function in CLC Genomics Workbench (GW) version 8.5.1 . Splice site prediction was carried out as outlined by Van Doorslaer et al.  for confirmation and assignment of type numbers.The penile swab collection and DNA extraction procedures have been described previously . The fulr et al. . ReferenHPV-219 is phylogenetically most closely related to HPV-213 of the gamma-13 species, sharing 87% identity in the L1 gene, HPV-220 is most closely related to HPV-212 (72% L1 identity) of gamma-17, HPV-221 is most closely related to HPV-142 (80% L1 identity) of gamma-10, and HPV-222 is most closely related to HPV-162 (73% L1 identity) of gamma-19. These HPV genotypes share <90% identity in the L1 gene (6-GGT) in their long control regions (4GK(T/S) (29CxxC] (All four viruses had a TATA box (TATAAA)  and pali regions . An ATP 4GK(T/S) , 11, was(29CxxC]  were ide(29CxxC] , in the Gammapapillomavirus genus. This knowledge expands the heterogeneity of the ever-growing members of the gammapapillomaviruses. The prevalence and clinical importance of these novel gammapapillomaviruses warrant further investigation.To conclude, we discovered four novel HPV genotypes of the MH172376, MH172377, MH172378, and MH172379, respectively.The GenBank accession numbers for the HPV-219, HPV-220, HPV-221, and HPV-222 genome sequences are


Cervical cancer ranks as the fourth most common malignancy in women affecting 500,000 women each year with an estimated 266,000 deaths. Uganda has one of the highest cervical cancer incidence rates globally with an age-standardised incidence rate per 100,000 of 47.5. This study assessed the level and the factors associated with uptake of HPV vaccine by female adolescents in Lira district, Uganda.a mixed methods approach was employed using a survey among 460 female adolescents. We collected data using an interviewer-administered questionnaire. We interviewed five key informants and conducted ten in-depth interviews. Uptake was defined as completing three doses of the vaccine as per the recommended schedule. Prevalence risk ratios were used as measures of association and were computed using modified poison regression. Content analysis was used for qualitative data.the mean age of the respondents was 13.97 (SD=1.24). Uptake was at 17.61% (81/460). The factors associated with uptake of HPV vaccine were: attaining ordinary level of education , positive attitude towards the vaccine , receiving vaccine doses from different vaccination sites  and encouragement from a health worker  or Village Health Team  to go for the vaccine. Other factors associated with uptake of HPV vaccine included; the existence of community outreaches , availability of vaccines at vaccination sites  and receiving full information about the vaccine at the vaccination site .HPV vaccine uptake was low in Lira district. Efforts to improve uptake of HPV vaccine should focus on ensuring a consistent supply of vaccines at the vaccination sites, health education aimed at creating a positive attitude towards the vaccine, sensitisation of the adolescents about the vaccine and conducting community outreaches. A woman's lifetime risk of acquiring HPV infection is higher than 80%, and most infections occur within 3-4 years of sexual debut . The priStudy area and design: we employed a mixed methods approach in which quantitative and qualitative data were collected using a concurrent triangulation design. By combining quantitative and qualitative data, we sought convergence and corroboration among the different data sources. The multiple perspectives provided an opportunity to develop a complete understanding of the factors associated with uptake of HPV vaccination. This study was conducted in Lira district, Northern Uganda in May 2016. Lira district is one of the 12 districts where the HPV vaccination program was first implemented (piloted) in Uganda in 2012, so the program was four years old at the time of this study.Study population: the study enrolled female adolescents aged 12-17 years because they were expected to have completed the vaccination schedule.Quantitative study: quantitative data were collected in a multistage cross-sectional design. The required sample size of 460 was determined using the formula by Bennett et al. (1991)  fo, (2011) , 10 housMeasurements: the dependent variable was the uptake of HPV vaccine which was categorised into two; fully vaccinated and not fully vaccinated. We determined HPV uptake using respondents' recall or vaccination cards if were present. An adolescent was considered fully vaccinated if they had received all the three doses of the HPV vaccine. Any adolescent who had received one, two or no dose of HPV vaccine was considered not fully vaccinated. The Independent variables included: individual factors, community factors and health service factors. Individual factors included; sociodemographic characteristics, knowledge about the HPV vaccine and attitudes towards HPV vaccine. We measured knowledge of respondents about the HPV vaccine as a composite variable by asking respondents six questions about the HPV vaccine. The same approach has been used in other studies to measure knowledge levels [e levels  Table 1.e levels . We usede levels .Data management and analysis: we reviewed the data for completeness, coded it, and analysed the data using STATA. We computed summary statistics like frequencies, proportions and means of different variables. We then conducted univariable, bivariable and multivariable analysis. At bivariable analysis, we computed prevalence ratios (PR) using a generalised linear model with Poisson family and a log link with robust standard errors as a measure of association because the prevalence of the outcome was above 10%. At multivariable analysis, we included all independent factors with p < 0.10 at bivariable analysis in the multivariable model to obtain adjusted prevalence risk ratios. We used the backward forward elimination with the logical model building approach of considering factors known from literature that affect HPV vaccine uptake to ascertain the best fitting model.Qualitative study: for the qualitative approach, we used purposive sampling. We conducted 20 in-depth interviews, five from each category of adolescents who had not received, received one, two and three doses respectively. We also conducted five key informant interviews with the district health team members who had an expert opinion about the health service factors that influence uptake of HPV vaccination in Lira district. The district team members included the following: the assistant district health officer for maternal and child health, the district vaccines focal person, district health educator and two sub-county health facility In-charges Qualitative data were transcribed from audio recordings for content analysis. We read the transcripts several times and then coded them to identify recurring themes. We then identified key quotations that represented the central themes on factors influencing uptake of HPV vaccine and presented them in the results as text.Ethical considerations: the study was approved by Makerere University School of Public Health Higher Degree Research and Ethics Committee. We sought permission to conduct the study from the Lira district and community gatekeepers. Voluntary participation in the study was sought using written informed consent.Social demographic characteristics of respondents: a total of 460 respondents participated in the study with a response rate of 100%. The age of respondents ranged from 12 to 17 years with a mean age of 14 years (SD =1.24). The majority of respondents 82.6% (380/460) were living with their parents, and 94% (432/460) were in school. Most of the respondents 79.8% (367/460) had both their parents still alive  had not received any dose of HPV vaccine, 18.0% (83/460) had received one dose, 14.8% (68/460) had received two doses, and 17.6% (81/460) had completed all the three doses. 0ut of the 232 respondents who had initiated on the vaccine, 180 (77.6%) had received it from school. The level of uptake was 17.6%  of the respondents had heard about the HPV vaccine. Half  of the respondents knew at least one benefit of receiving the HPV vaccine. More than half of the respondents  did not know the recommended doses of the HPV vaccine and the majority  of respondents did not know the recommended intervals between the HPV vaccine doses. More than half  of the respondents did not know the targeted age group for the HPV vaccine, and only 51.74% (238/460) of the respondents knew where to access the HPV vaccines. Overall, more than half of the respondents  had poor knowledge about the HPV vaccine ( vaccine .Attitudes towards HPV vaccination: the majority 70% (322/460) of the respondents agreed that HPV infections cause cervical cancer. Most of the respondents  agreed that cervical cancer is a deadly disease. A large proportion  of the respondents agreed that it is important for young adolescents to receive the HPV vaccine. Most  of the respondents agreed that the HPV vaccine is effective in protecting against cervical cancer. More than half  of the respondents agreed that the HPV vaccine has no side effects. The respondents who disagreed with the statement of HPV vaccine having no side effects gave side effects like; the injection is painful and it causes pain in the upper arms. Overall, the majority  of respondents had positive attitudes towards the HPV vaccineIndividual factors independently associated with uptake of HPV vaccine: the proportion of adolescent fully vaccinated with the HPV vaccine was 48% more among adolescents who had attained a secondary ordinary level of education compared to those who had attained an education level of primary and below  1.48, 95%CI 1.11-1.97). Having a positive towards the HPV vaccine was a strong predictor of HPV vaccine uptake. The prevalence of uptake of HPV vaccine was 3 times higher among adolescents with positive attitudes towards the vaccine compared to those with negative attitudes  adolescents who had received the HPV vaccine doses from more than one vaccination site had a 59% higher prevalence of HPV vaccine uptake compared to those who had received all the HPV vaccine doses from one vaccination site . This is also revealed in a statement by one of the interviewed adolescents; "At school, I got only two doses, but my mother took me to the hospital where I got the third dose" (In-depth interview 13). The qualitative results from the in-depth interviews revealed that knowledge about HPV vaccination was a predictor of HPV vaccine uptake. Adolescents who had not taken any vaccination dose were not aware of the HPV vaccine and did not know the benefits. For example, most of them commonly said that; "I am not aware of the HPV vaccine, and I don't know what it protects against" (In-depth interview 1). However, most of the adolescents who had received one dose of the HPV vaccine were aware of the HPV vaccine but did not know the right doses and intervals between the doses. "The teachers told us that the HPV vaccine is important and protects against cervical cancer but did not explain to us well the intervals and doses" (In-depth interview 7) (rview 7) .Community factors independently associated with uptake of HPV vaccine: uptake of HPV vaccine was two times higher among adolescents who had been recommended or encouraged to go for the HPV vaccine by health worker compared to those who had not been recommended or encouraged by a health worker to go for the HPV vaccine . The prevalence of uptake of HPV vaccine was three times higher among adolescents who had been encouraged by a village health team member(VHT) to go for HPV vaccination compared to those who had not been encouraged a VHT . There was a 47% higher prevalence of uptake of HPV vaccine among adolescents who reported that HPV vaccine community outreaches were conducted in their residences compared to those who reported that the outreaches were not conducted in their residences .Health service factors independently associated with uptake of HPV vaccine: the prevalence of uptake of the HPV vaccine was five times higher among adolescents who reported that the vaccines were available at HPV vaccination site at all their visits compared to those who reported that the vaccines were not available at all visits . This result is corroborated by key informants as one of them stated that; "At first the vaccines were available but during the last lot, the vaccines became unavailable at the different vaccination sites because of inconsistent supply from the national medical store" (Key informant interview 1). Qualitative results further revealed that there was also a challenge of lack of sufficient funds to facilitate the transport/delivery of vaccines at the different vaccination sites and the health workers responsible for vaccinating the girls "Vaccines were sent without financial support so we had to divert some primary health care funds to facilitate those health workers who vaccinated in schools and community outreaches" (Key informant interview 2). The prevalence of uptake of HPV vaccine was 90% higher among adolescents who received full information about the HPV vaccine at the vaccination site compared to those who did not receive full information .The study estimated the level of uptake of HPV vaccination at 18% which is below the targeted coverage of 80% required to eliminate common serotypes i.e., 16 and 18) from targeted populations . Our res and 18 fA study by Ladner 2012) in the lowest income countries to assess eight HPV vaccination programs that used different modes of HPV vaccination showed that mixed models comprising school and health facility-based vaccination had better overall performance compared to models using just one of the methods . In Lira12 in theFindings from our study revealed that availability of HPV vaccines at vaccination sites on all adolescents' visits is associated with higher prevalence. This result is consistent with a study done by Small (2012)  which evWe found out that the uptake of HPV vaccine was low. Factors found to be associated with HPV vaccine uptake included: attaining an education of secondary ordinary level, positive attitude towards the vaccine, receiving vaccine doses from more than one site and encouragement from Health workers and Village Health Teams. Other factors associated with uptake of HPV vaccine included; participating in community outreaches, availability of vaccines and receiving full information about the vaccine.Recommendations: we recommended the ministry of health, Uganda to; conduct massive HPV vaccination community sensitisations to build positive attitudes, conduct HPV vaccination community outreaches and to ensure regular availability of vaccines at the vaccination sites. Further studies should also be conducted about the timeliness of HPV vaccinations among female adolescents to see the factors associated with untimely uptake of HPV vaccine doses and the implications of not receiving the doses in the correct intervals.Operational definitions: human papillomavirus vaccine is the vaccine that protects against human papillomavirus which is known to prevent cervical cancer and other HPV infections in women. HPV vaccine uptake was defined as being fully vaccinated with all the three doses of HPV vaccine. Knowledge is the familiarity with information, facts, descriptions, or skills acquired through experience or education about cervical cancer and HPV vaccination. Attitude is a set of health beliefs that determine or guide individual behaviours/conduct. A household consisted of a person or group of persons, related or unrelated, who lived together in the same dwelling unit, who acknowledged one adult male or female as the head of household, who shared the same meals and living arrangements and were considered as one unit.The study assessed different factors that hinder timely uptake of the HPV vaccine for instance poor attitudes towards the vaccine some of which were already known.The study adds an in-depth understanding of the importance of timely vaccination with HPV vaccine and the implications of not receiving the vaccine in time.The authors declare no competing interests.


Some veterans face multiple barriers to VA mental healthcare service use. However, there is limited understanding of how veterans’ experiences and meaning systems shape their perceptions of barriers to VA mental health service use. In 2015, a participatory, mixed-methods project was initiated to elicit veteran-centered barriers to using mental healthcare services among a diverse sample of US rural and urban veterans. We sought to identify veteran-centric barriers to mental healthcare to increase initial engagement and continuation with VA mental healthcare services.Cultural Domain Analysis, incorporated in a mixed methods approach, generated a cognitive map of veterans’ barriers to care. The method involved: 1) free lists of barriers categorized through participant pile sorting; 2) multi-dimensional scaling and cluster analysis for item clusters in spatial dimensions; and 3) participant review, explanation, and interpretation for dimensions of the cultural domain. Item relations were synthesized within and across domain dimensions to contextualize mental health help-seeking behavior.Participants determined five dimensions of barriers to VA mental healthcare services: concern about what others think; financial, personal, and physical obstacles; confidence in the VA healthcare system; navigating VA benefits and healthcare services; and privacy, security, and abuse of services.These findings demonstrate the value of participatory methods in eliciting meaningful cultural insight into barriers of mental health utilization informed by military veteran culture. They also reinforce the importance of collaborations between the VA and Department of Defense to address the role of military institutional norms and stigmatizing attitudes in veterans’ mental health-seeking behaviors.The online version of this article (10.1186/s12913-018-3346-9) contains supplementary material, which is available to authorized users. Some veterans using healthcare services at the Department of Veterans Affairs (VA), face multiple potential barriers to VA healthcare services, particularly mental healthcare , 2. PrevPatients, physicians, and researchers who have different life experiences and backgrounds may use different paradigms and language—that is, different cultural frameworks when thinking about healthcare or engaging with healthcare systems –12. For In this era of greater emphasis on patient-centered care approaches, where researchers are encouraged to meaningfully collaborate with populations they study , one criIn this paper, we report on the use of cultural domain analysis (CDA), a method common in ethnographic research , to elicWe used standard CDA procedures to elicit veteran-centered barriers to mental healthcare and to create a cognitive map , 26. We s): VISN 1 included one urban and two rural CBOCs in Maine serving primarily white veterans; VISN 16 included one urban health clinic and two rural CBOCs in Arkansas serving a largely white and African American veteran population; and VISN 21 included one urban and three rural CBOCs in Northern California serving a racially and ethnically diverse group of veterans. Table This study was conducted at ten sites in three geographically distinct VA administrations areas called Veterans Integrated Service Networks  to recruit, ultimately diversifying our sample. In wave 1, we recruited veterans of differing service eras obtaining representation of older and younger veterans ; in waves 2 and 3 we recruited veterans based on race and ethnicity. For all waves, we recruited veterans from both rural and urban contexts and over-recruited women veterans, an underrepresented veteran subpopulation.n = 52) and into the domain interpretation task until saturation was reached (n = 14).Of the 80 veterans in the sample, 66 veterans participated in the cultural domain analysis and interpretation interview. As described below, this method requires a relatively small sample size. Therefore, we recruited participants into the CDA phase until agreement was reached  examines how members of a group who share a culture characterize aspects of that culture through a cognitive domain, referred to as a cultural domain in CDA, defined by a set of words, phrases, or concepts that collectively symbolize a single idea . The CDAFree list exercise. We started with a free list exercise, an unstructured, open-ended task used to elicit the words and brief phrases that inductively characterize a domain. During data collection, each participant was asked to “list all the things that you think can make it more difficult for veterans to get help for stress-related and emotional health problems , white (n = 40), and had prior specialty mental health visits (n = 40); 24 screened positive for PTSD, 28 for depression, and 40 for at-risk drinking. All had experience using the VA healthcare system; consequently, their understanding of barriers to care was grounded in their experiences.Sixty-six unique veterans between the ages of 20 to 70 (average 44.0) years participated in one of the three CDA phases  Figure The CDA procedures resulted in five dimensions of barriers to care: worry and concerns about what others think; personal, financial, and physical obstacles; confidence in the VA healthcare system; Veterans’ discussion of this dimension grouped around stigma, vulnerability, and trust as barriers to VA mental health services use. Veterans expressed concern over stigmatizing labels such as “crazy” and “mental health patients” as well as military attitudes  fostering feelings of weakness and failure. Such attitudes contributed to veterans’ anxiety and alcohol and drug use and impeded mental healthcare services use. Veterans also indicated a lack of trust in the clinical encounter  decreased their motivation to remain in care.Discussions centered on personal life struggles  constraining on veterans’ daily lives and ability to meet responsibilities—including healthcare services use. Travel distance to VA facilities and lack of transportation or gas money also represented barriers to VA care within this dimension.Veterans’ discussions of this dimension indicated system-wide problems with VA processes of care and expressed their lack of confidence . Appointment problems, staffing issues, limited follow-up from VA healthcare providers and staff, and prioritization of some veterans over others impeded access to care. Long wait times between appointments, slow clinic response, and challenges to scheduling appointments  created barriers to timely and appropriate care. Staff issues, especially lack of available providers and high provider turnover, limited veterans’ access to care and provider continuity over time. Veterans also indicated that limited or no access to specialized care resulted in veterans’ disengagement with care. Furthermore, the appearance that services are prioritized to address the needs of younger veterans limits available services for older veterans and diminishes the importance of older veterans’ health and wellbeing.Veterans’ discussions highlighted the challenges of navigating VA benefits and healthcare services, which are connected to lack of understanding or misunderstanding of VA benefits and mental healthcare services. Veterans reported being unaware of VA benefits prior to leaving military service, having limited knowledge of the processes required to obtain benefits  once eligible, and struggling to understand the use of their benefits as veterans. This knowledge gap impeded initial enrollment into the VA healthcare system. Once veterans entered the system, they still struggled with a lack of awareness of available mental healthcare services. Veterans stressed the need for both the military and the VA to disseminate benefit material more effectively to service members prior to military exit.Veterans’ discussions of this dimension revealed concerns regarding healthcare privacy and misuse of the VA healthcare system by other veterans. Veterans discussed military policy denying security clearances for service members known to seek mental healthcare and this fear influencing veterans’ decisions to seek specialty care within the VA healthcare system. Some also expressed a fear of their confidential information being shared and sensitive information  disclosed, which could have profound negative consequences on veterans’ lives . Both older (Vietnam era) and younger (Iraq and Afghanistan era) veterans’ expressed distrust of the VA healthcare system as a government institution and fear of digital communications . Last, misuse of VA services by some veterans for disability payments and pain medication, while perceived by veterans as rare, had a negative effect on the VA care experience of other veterans. Specifically, misuse of the system influences how society and some providers view veterans who use VA healthcare for certain treatments .This mixed-methods CDA analysis generated veteran-centered perspectives of barriers to VA mental healthcare services use grounded in veterans’ lives and during clinical interactions that add depth, breadth, and complexity to understanding barriers to care. This approach contributes to better understanding of veteran-derived perspectives on barriers to mental healthcare services use. The findings are intended to inform patient-centered care  and concthat historical, socio-cultural, and psychological factors inform veterans’ decisions to engage with VA mental healthcare services. Their perceptions highlight how military socialization, command structure influences, and institutional attitudes  reinforce attitudes around help-seeking as weakness, which significantly informs veterans’ post-military service healthcare experiences [Our interpretation of veterans’ perceptions of barriers to VA service use demonstrates eriences , 53. Theeriences  found coIn addition, our findings show how institutional betrayal, or as veterans explained, distrust of the VA as a government institution, influences their VA mental healthcare services use. Desai et al.  found nehttps://www.va.gov/COMMUNITYCARE/about_us/POI/poi_faq.asp). Veterans’ abuse of VA healthcare was present in our study—in the case of a participant retelling how veterans scheme VA doctors to obtain pain medication only to misuse their prescription. Whereas fraud was present in the case of a participant explaining that some veterans manipulate the system to obtain a PTSD diagnosis. Though veterans described them as rare, these practices can shape veterans’ and providers’ attitudes and impede development of a trusting therapeutic alliance between providers and veterans utilizing VA healthcare services.This study also provides insight into ways veterans perceive misuse or abuse of the VA healthcare system. The VA is well aware of potential abuse of its healthcare services. For instance, the VA Office of Community Care has a webpage with frequently asked questions about fraud, waste, or abuse of VA healthcare services  facilitating service member and veteran care coordination is a step towards reducing barriers to care for younger veterans . SimilarFurthermore, our findings regarding veterans’ confidence in the VA healthcare system points to service members and veterans’ mistrust in government healthcare. Both VA and DoD should work together to address veterans’ mistrust of the VA healthcare system, which reflects both current public relation issues and the historical treatment of veterans of different service eras  within the VA healthcare system , 58. FurSeveral limitations should be considered when interpreting the findings. Our findings reflect insight from veterans enrolled in VA healthcare services and may not reflect the experiences of veterans who have not used VA benefits and services. While we had much success recruiting participants from both rural and urban settings across the three sites , we enrolled a relatively small number of racial/ethnic diverse and women veterans into the study. The general trend of racial/ethnic diversity in our sample, including representation of Asian identity in North California, Black and White identity in Arkansas, and White identity in Maine and Northern California, corresponds with the demographic characteristics of the veteran population in these geographic regions –61. Addin = 16) into the study. However, our analysis could have been qualitatively more nuanced and rich if more women had participated in the study and shared their experiences and perspectives. Historically, servicewomen and women veterans have been underrepresented in research examining VA or DoD healthcare use [We oversampled women veterans and enrolled a fair number of women


Plasmodium falciparum gametocytes that are responsible for transmission from human to mosquitoes and bring radical cure in Plasmodium vivax by clearing dormant liver stages. Deployment of primaquine is thus of relevance for malaria elimination efforts but challenged by the widespread prevalence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) in endemic countries since primaquine in G6PDd individuals may lead to acute haemolysis. In this study, the prevalence of G6PDd was investigated in different settings in Ethiopia using phenotyping and genotyping approaches.8-Aminoquinolines such as primaquine clear mature G6PD gene and its flanking regions.Community and school based cross-sectional surveys were conducted from October to December 2016 in four administrative regions  in Ethiopia. Finger prick blood samples were collected for G6PD enzyme activity using the CareStart™ G6PD screening test and genotyping of 36 selected single nucleotide polymorphisms (SNPs) located in the Overall, the prevalence of phenotypic G6PDd was 1.4% (22/1609). For the first time in the Ethiopian population, the African variant (A−) was detected in 3.5% (7/199) of the limited set of genotyped samples, which were all phenotypically normal. Interestingly, all of these individuals had a variation at the rs2515904 locus. Strong geographical variation was observed for both phenotypic and genotypic G6PDd; three-quarters of the phenotypically G6PDd individuals were detected in Gambela.G6PD mutants and the patchy distribution of G6PDd indicate that larger studies specifically designed to unravel the distribution of G6PDd at small geographical scale may be needed to tailor malaria elimination efforts in Ethiopia to the local context.A very low prevalence of G6PDd was detected in the present study populations. The presence of the A− variant alongside other The online version of this article (10.1186/s12936-018-2437-8) contains supplementary material, which is available to authorized users. Plasmodium falciparum gametocytes  that are responsible for human-to-mosquito transmission : 10–30); of whom 46.3% (745/1609) were females, participated in this study. The overall phenotypic G6PDd detected by the CareStart™ G6PDd screening test was 1.4% , with considerable variation between study sites. Of the 22 G6PDd individuals 16 were from Gambela Regional State: 6.6% (95% CI 3.5–11.0) of participants from Abobo and 1.5% (95% CI 0.3–4.4) of participants from Lare Woredas ; rs5030872 (542A>T); rs137852328 (680) and the Mediterranean variant (rs5030868)]. One SNP (rs12389569) had < 1% minor allele frequency (MAF) (Table G6PD gene (2 synonymous and 2 missense variants) and the other in flanking regions of the gene; i.e. 3 immediately downstream of G6PD gene and the remaining were intergenic (1), intron (5), non-coding exon (2) and 5′ UTR variants (1).A total of 14 SNPs that did not meet the quality control were excluded from further analysis  (Tables Linkage disequilibrium (LD) plot could be constructed only for the whole population. It was not stratified based on gender, ethnicity and study setting due to small samples genotyped. One LD block was constructed that consisted of rs28470352, rs73573478, rs5986990, rs1050829 (376), rs762515, rs113492957, rs111827785, and rs60030796  of phenotypically evident G6PDd was detected in this study. For the first time in published literature, the African variant (A−) was detected in an Ethiopian population. Of the genotyped samples 3.5% (7/199) were A− variants  and consisted of both the G202A and the A376G mutations. Three quarters of the phenotypically deficient individuals were from one Region, Gambela. None of the phenotypically deficient individuals were found to have the A− variant. Uniquely, all individuals with the A− variant were found carrying a variation at the rs2515904 locus that was previously found in perfect LD with the A− variant and was reported protective against clinical malaria –42.The high prevalence of G6PDd detected by the phenotypic assay in Gambela Region is in line with a recent finding of 7.3% phenotypic deficiency in the same region  and withG6PD A+, resulting in 80% residual enzyme activity compared to normal values [G6PD deficient genotype A− is typically defined by the possession of both the rs1050829 (A376G) allele and G to A substitution at the rs1050828 locus . Type A− mutations cause on average 12% residual G6PD activity [G6PD gene. There was one phenotypically deficient individual who had the A376G, together with variations at rs28470352, rs5986990, rs762515, rs111827785, and rs60030796 loci, but not the G202A. All other SNPs tested in this study were monomorphic for phenotypically deficient individuals, which might be explained by G6PD mutations in the Ethiopian population that were not tested in the present study [G6PD gene, as well as their associations with RDT performance with precision. The very small sample size, especially with the genotyping analysis, could also have contributed to the observed geographic clustering. The detection of the African variant of G6PDd in the present study and the predominant geographic pattern of patchy distribution of G6PDd underline the need for larger studies to have complete picture of the nationwide distribution of the deficiency to help build a resilient programmatic approach in the move towards eliminating malaria in Ethiopia.Phenotypically evident deficiencies detected in G6PDd individuals often have matched genetic background (often of point mutation or haplotypes). A to G substitution mutation at the rs1050829 locus  is associated with reduced production of G6PD enzyme that causes a milder variant, l values . The G6Pactivity . Type A−nt study . A relevG6PD variants [With the limited number of genotyped samples from different study sites, 3.5% (7/199) of genotyped samples were of the A− variant. This contrasts with, previous studies conducted in different settings in Southwest Ethiopia, South Omo  and Jimmvariants  and diffvariants , 54, 55 variants . The Medvariants , 52. Allvariants . AllelicG6PD can cause varying degrees of G6PDd and thus severity of haemolysis may vary. The patterns of oxidative haemolysis are well characterized in hemizygous males and homozygous females; whereas, heterozygous females may exhibit symptoms to varying degrees due to random inactivation of X-chromosomes [Mutations of omosomes . The expomosomes  and can omosomes , 59, theomosomes . Heterozomosomes . A quantomosomes , 61.P. falciparum malaria and a 14-day course for radical cure in P. vivax is recommended by the WHO and this is being incorporated into country treatment policies. Most P. vivax malaria-endemic countries recommend PQ treatment without prior G6PD testing [A single low dose PQ as a gametocytocide in  testing . The WHO testing . On top  testing , using iP. falciparum malaria would likely pose little risk to the population due to the very low genotypic and phenotypic prevalence of G6PDd. Despite the low prevalence, alternative strategies including DOT or at least better screening for G6PDd, particularly among females, who are likely to be phenotypically normal by Carestart G6PD screening test, but may experience substantial haemolysis if they were genotypically female heterozygous, need to be considered to prevent P. vivax relapse.Prevalence of G6PDd is generally very low and varies substantially between settings in Ethiopia. The country could potentially benefit from tailored deployment of 8-aminoquinolines like PQ. This study supports the evidence that the incorporation of PQ into national treatment guidelines for Additional file 1. Flow chart for genotyping and data analysis. Indicated in a) is the schematic presentation of the quality check for data analysis and in b) is the genotyping procedure.Additional file 2. KASP Primer sequences.Additional file 3. Genotyping overview.


In Mexico, HPV vaccines available immunize against genotypes 16/18 and 16/18/6/11; however, there is limited surveillance about carcinogenic subtypes in different states of the country that allow evaluating the effectiveness of vaccination and cervical cancer screening programs. Here, we report the regional and age-specific prevalence of 14 hr-HPV genotypes as well as their prevalence in abnormal cytology  among Mexican women which were undergoing from cervical cancer screening in the Salud Digna clinics in 20 states of the country. This study includes women with social security from the majority of public health institutions , and women without social security. For cervical cancer screening, we used the SurePath liquid-based cytology and the BD Onclarity HPV Assay. From December 1, 2016, to August 2, 2018, the hr-HPV prevalence among 60,135 women was 24.78%, the most prevalent types were HPV 16 (4.13%), HPV 31 (4.12%) and HPV 51 (3.39%), while HPV 18 (1.70%) was less prevalent among infected women. Interestingly, the genotypes not covered by current vaccines in Mexico were commonly found in precancerous lesions, evidencing their carcinogenic potential, so it is necessary to increase their surveillance and inclusion in cervical cancer screening triage. Carcinogenic types include predominantly, alpha 9 (HPV 16/31/33/35/52/58), alpha 7 (HPV 18/39/45/59/68), alpha 6 (HPV 56/66), and alpha 5 (HPV 51) genus4. These families differ in their mucotropisms, for example, members of alpha 7 genus have been associated with glandular lesions and adenocarcinoma of the cervix; meanwhile, alpha 9 genus has been found in squamous intraepithelial lesions and squamous cell carcinoma6.The human papillomavirus (HPV) is the leading cause of cervical cancer and recently, has increased the evidence that relates HPV infections with anogenital, head, and neck cancers7, as well as screening programs had contributed to the reduction of cervical cancer mortality9; it might expect that together vaccination and screening contributes to reducing most cervical cancers.Variability and prevalence of alpha members among populations would have an epidemiological impact in the incidence of some types of cancer. So that, screening programs, as well as preventive vaccination, have to be implemented in each population to reduce the burden of HPV-related cancers. Vaccination has reduced the abnormal cytology rate10. This differences in genotypes distribution may affect the effectiveness of the HPV vaccines in different populations. Regional data on the prevalence and distribution of HPV are essential for estimating the impact of vaccines and cervical cancer screening program (CCSP) in each country.The study of HPV genotypes allows identifying oncogenic strains present in human populations; since it has been reported that its prevalence and distribution vary according to countries and continents12; this information has contributed to changing cancer screening from cytology to HPV triage. Regarding HPV prevalence, different works had been carried out in some states of the country  in 60,135 women  from 20 states of Mexico. Also, we identified the most common genotypes present in precancerous lesions. This study, together with previous works can serve as a reference to guide cervical cancer screening and HPV vaccination programs in Mexico.Salud Digna is a private not for profit and non-government institution which provides diagnostic services in Mexico through outpatient diagnostic clinics located in 20 of 32 states of the country, in which could are attendant all people, no matter their socio-economic status, or if they have social security or not.15; in this case, for an expected HPV-infections prevalence of 10% based on the mean of prevalence reported by population-based studies in Mexico17.For this retrospective cross-sectional study, we analyzed anonymized electronic health records of cervical cancer screening from patients whose where attendant in the outpatient care clinics Salud Digna in 20 states of Mexico from December 01, 2016 and 02 August 2018. We include all available data collected during gynecological examinations, including data on demographics, gynecologic, and obstetric information. A unique standardized and validated survey was applied for data collection in all clinics. The cases included were from adult women (18 to 90 years), which were screened for cervical cancer. The exclusion criteria were the following: women younger than 18 years, pregnant women, and women with hysterectomy. For geographical analysis, we included states with 140 or more subjects. This threshold was considered from the sample size calculations for cross-sectional studiesThe consent for the use of information from health records was obtained according to the Mexican Federal Law on Personal Data Protection . People who are attendant in the Salud Digna clinics accept our privacy policy which includes the use of their information for scientific research purposes; by the above, we do not need specific informed consent from each people included in this work, because this study is a cross-sectional analysis of an electronic health registry. We handled data protection and privacy of its, according to national laws and guidelines in Mexico. Data obtained was anonymized by the assignation of a unique ID code to protect the identity of people and to prevent data duplication in subsequent analysis. Aggregation of information was used to enhance data protection.This study was approved by the Ethical Review and Research Board of Salud Digna. All methods were carried out by the approved guidelines and the Declaration of Helsinki.The procedures for the Pap test and HPV genotyping used to generate the information collected in the electronic health records used in this study are described below. Cervical cancer screening results from the registry were obtained from the samples collected in the BD SurePath liquid-based cytology collectors during gynecological examinations in each clinic, then, were processed in the Cytology unit, and Molecular biology laboratory at the National Reference Center of Salud Digna in Culiacan, Sinaloa. Cytology samples were processed into the BD Totalys MultiProcessor  for the cell enrichment of cervical samples  as well as to make aliquots for an HPV test.rd ed. of The Bethesda System for Reporting Cervical Cytology18.The slides preparation and staining for Pap smears were performed into the BD Totalys SlidePrep  according to the manufacturer´s recommendations. Slides were visually inspected in Carl Zeiss Primo Star HD microscopes  by 13 certified cytotechnologists; all abnormalities observed were confirmed for certified pathologists and 14% of total slides inspected by cytotechnologists were re-analyzed by independent pathologists (Pathologists were blinded for the commentaries of cytotechnologist). Pap test was performed according to the 319. This assay simultaneously detects 14 high-risk HPV types, in three different reactions. R1 detects individually HPV 16/18/45, R2 detects individually HPV 31 and two groups: G1 (HPV 33/58), and G2 (HPV 56/59/66); R3 detects individually HPV 51/52 and G3 (HPV 35/39/68); all reactions use the human β-globin gene as an internal control.The HPV DNA testing was performed using the automated workflow on the Viper LT system. HPV type was determined using the real-time PCR BD Onclarity HPV Assay  which detects and amplify HPV type-specific E6 and E7 genesDescriptive statistics were performed on all data sets. For categorical data, the Wilson score method without continuity correction was performed to calculate 95% CI. Genotypes detected in the G1, G2, and G3 had regarded as a single unit for results descriptions. We evaluated the associations between demographic, gynecobstetrics, and other factors to HPV infections using a stepwise multivariable logistic regression model. All variables significantly associated in the univariable analysis (p < 0.05) were selected for inclusion in the multivariate logistic regression model, which was used to control for potentially confounding factors. The adjusted odds ratio (OR) were calculated from age-adjusted multivariate logistic regression. To analyze the oncogenicity of HPV genotypes, we used registries from women undergoing both tests (HPV and Pap test); odds ratio (OR) of each genotype were calculated from the univariate logistic regression model. The analyses were done in SPSS V. 24 .Between December 01, 2016 and 02 August 2018, 60,328 women were attendant for cervical cancer screening in outpatient diagnostic clinics Salud Digna located in 20 of 32 states of the country. 5 registries (belonging women younger than 18 years) were excluded from the dataset; also 188 registries were excluded because they belong from states with less than 140 women screened. For the HPV prevalence as well as geographical and age-specific analysis, 60,135 registries were included. For risk factors analysis and the relationship of HPV genotypes according to cytology results, 52,415 registries were included, and 7,721 registries with incomplete gynecological and clinical information were excluded .In this study, women from 18 to 88 years were screened for cervical cancer; the mean age was 38.76 years; 53.69% of women had their first sexual intercourse at 18 years or older. Also, the majority of women declared not to use tobacco (75.59%). Most of the women studied have been pregnant (71.64%), 51.34% declared to used contraceptive methods, from these, 21.74% corresponding to condom use; 70.72% of women previously have been undergoing to Pap test. It is important to note that most of the women declared not having social security (70.81%). A summary of the demographic characteristics of women included in this study is shown in Table The prevalence of hr-HPV in 60,135 women was 24.78% Fig. . In cont15. The expected HPV prevalence was 10% based on the mean of prevalence previously reported by population-based studies in Mexico17. Among states analyzed, Chihuahua (28.74%), and Nayarit (28.33%) had the highest prevalence of infections, while in Quintana Roo (21.17%) and Sinaloa (21.54%) we found the lowest prevalence  was at least 140. This threshold was considered from the sample size calculations for cross-sectional studiesWe found that the HPV 31 was more prevalent in San Luis Potosi (6.07%) and Aguascalientes 5.20%); while the lowest prevalence was observed in Quintana Roo (1.95%), and Sinaloa (3.01%). The HPV 51 was more frequent in Nayarit (4.78%), and Nuevo Leon (4.39%), in contrast, we observed the lower prevalence in Quintana Roo (2.43%), and Durango (2.45%). The HPV 52 was most prevalent in Chihuahua (4.68%) and Veracruz (4.63%); in contrast in Sinaloa (2.84%), and Nuevo León (2.93%) it was observed the lowest frequencies  and Nuevo Leon (2.66%) than in Quintana Roo (0.97%), and Durango (1.15%) in which was less prevalent. In Chihuahua (3.60%) and Nayarit (2.26%), HPV 45 was the most prevalent; meanwhile in Quintana Roo (0.73%), and Durango (1.15%) was less prevalent. The G2 (HPV 56/59/66) was more prevalent in Nuevo Leon (11.32%), and San Luis Potosi (10.90%), and less frequent in Quintana Roo (6.81%) and Sinaloa (7.44%). The G3 (HPV 35/39/68) was more prevalent in Veracruz (7.48%), and Chihuahua (6.85%), while in Guanajuato (4.83%), and Coahuila (4.86%) was less prevalent. Finally, the G1 (HPV 33/58) was more prevalent in Chiapas (4.86%), and Michoacan (4.37%), and less prevalent in Quintana Roo (2.19%), and Nuevo Leon (2.40%) . Similarly, sexual intercourse before 18 years was significantly associated . However, menarche age lower than 12 years did not show a significant association with HPV infections  Table .Table 3CFurthermore, we found that women that never had been pregnant showed increasing odds to be infected  than women with a history of pregnancies. In this way, women who have had more than one pregnancy showed an odds reduction to get infected. Regarding birth types, the cesarean surgeries reduced the odds of infections ; however, births were not related . Women with a history of abortions slightly increased their odds  , Candida  or Gardnerella vaginalis  showed significant odds  as well as sexually transmitted diseases (STD) were significantly associated to HPV infections . Of the STD’s, infections with We analyzed the prevalence of hr-HPV according to cytological results in women undergoing co-testing procedures (Pap test and HPV typing at the same time). We found that the prevalence of hr-HPV was 21.21% in women negative for intraepithelial lesions or malignancy (NILM), 73.04% in atypical squamous cells with undetermined significance (ASCUS), 82.14% in atypical squamous cells cannot exclude high-grade squamous intraepithelial lesions (ASC-H), 73.68% in atypical glandular cells (AGC), 84.09% in low-grade squamous intraepithelial lesions (LSIL), 92.22% in high-grade squamous intraepithelial lesions (HSIL), and 78.57% in cervical cancer cases , HPV 31 , HPV 51  and HPV 52 . In the cervical cancer cases, HPV 16 (42.86%) and HPV 31 (21.43%) were the most prevalent genotypes  were more prevalent in all cytological abnormalities including cervical cancer; while the alpha 7 genus  were less frequent among cytological abnormalities and cervical cancer. Despite that genotypes of the alpha 6 genus  were the most prevalent in all categories of the cytology test; these genotypes were not associated with precancerous lesions and cervical cancer. Similarly, HPV 51  was not significantly associated with precancerous lesions and was absent in cervical cancer cases , and others do not have social security Table .21; the above can be reviewed in detail in the Supplementary Table The prevalence of 14 hr-HPV in 60,135 women was 24.78%. Previous studies in Mexico in different health care institutions reported prevalences of 10–49.7%, which includes low and high-risk HPV types. There are some differences between previous works and this study that limits the comparison of the results, including the type and design of the study, and the intrinsic limitations of HPV test used in each work which has been further discussed22. This assay detects 13 carcinogenic HPV types and 1 possible carcinogenic (HPV 66)23. Six genotypes are detected individually  and the others are detected in groups: G1 (HPV 33/58), G2 (HPV 56/59/66), and G3 (HPV 35/39/68), reducing the possibility of knowing the real prevalence of each genotype, representing a limitation for HPV genotypes surveillance, but not for clinical purposes22. The G2 and G3 groups have a lower risk of cervical lesions (among carcinogenic types) compared to HPV 16 according to globally reports24.One of the main differences is that we used the real-time PCR Onclarity HPV assay test for HPV typing which has been recently approved by the Food and Drug Administration (FDA) agency in the US for HPV testE6 and E7 genes, this implies that those infections in which the L1 gene has been lost by viral integration can be detected because the E6 and E7 genes are not affected by this event21; in contrast, L1-based typing tests could miss HPV infections in cervical samples. Also, a comparison between consensus PCR targeting L1 primers (MY09/11) and type-specific E6/E7 HPV PCR showed that L1 PCR failed to detect 10.9% of HPV infections25, also MY09/11 showed lower sensitivity (87.9% vs 98.3%) and specificity (38.7% vs 76.14%) than type-specific E6/E7 primers25. Another primer set is GP5+/GP6+, which also targeting L1 and has similar sensitivity than MY09/1126. However, this primer set to fail in the amplification of DNA from multiple HPV present in a single sample26. The above might explain why we observed a high prevalence of HPV infections using E6/E7-based typing test than previous works in the country that used L1-based typing tests  than women with social security (22.48%) affiliated to the majority of public health institutions of the country . Also, the age-distribution of HPV infections observed in this study was similar to previous studies in Mexico30. Interestingly, multiple pregnancies were associated with a reduction in the odds of infections. Also, cesarean surgeries and abortions were weakly associated with HPV infections. The information about lifetime-sexual partners is an important risk factor for HPV infection; however, we could not get that information during interviews for cervical cancer screening, which could confuse some of the factors studied here.In this work, we found that women without social security increase their odds of getting HPV infections. The above shows the need to increase the accessibility to cervical cancer prevention programs to all women in Mexico. Additionally, sexually transmitted diseases were highly related to HPV infections which had been reported in other populations13; this program used vaccines against HPV 16/18 and HPV 6/11/16/18.Regarding the prevalence of HPV genotypes, we found that HPV 16, 31 and 51 were the most prevalent, also G2 (HPV 56/59/66), G3 (HPV 35/39/68) were the commonest, while HPV 18 was less prevalent, which differs with the majority of previous reports in the country . In consequence, we consider that the low prevalence of HPV 18 observed would be explained by different factors such as mucotropism of HPV genotypes36, this might explain the raising of cancer cases in old women in the country37. Furthermore, we found that the most prevalent genotypes from normal to abnormal cytology were HPV 16/31/51, which was similar to previous reports in women with normal cytology38 and cervical cancer cases39. However, HPV 18 and 45 were less frequent in this population. In contrast, genotypes detected in groups (56/59/66 and 35/39/68) were commonly found in normal cytology and LSIL, but lower in HSIL and CC cases, showing a lower risk for high-grade cervical lesion promoted by this genotypes; which is consistent with worldwide reports24.Additionally, we found a difference in the prevalence of hr-HPV genotypes between young and old women . Recently, a nine-valent vaccine (Gardasil9) which immunizes against HPV 6/11/16/18/31/33/45/52/58 has been tested to assess their safety and efficacy to reduce precancerous lesions43 and has been approved for use in Europe, Canada, Australia, and the US44. This vaccine immunizes against the most prevalent carcinogenic genotypes found in this work and other previous studies in the country, so that could be suitable for vaccination in Mexico.It is important to know that we found a high prevalence of HPV 31 and HPV 33/58 in normal and abnormal cytology; a previous work found that these genotypes had a comparable risk for HSIL development that of HPV 1845, including HPV 16/18 typing to refers women to colposcopy49. In Mexico, the HPV triage coupled to hr-HPV 16/18 genotyping shown a good cost-effectiveness relationship than hr-HPV plus pap test50. A recent analysis showed that HPV 31/33 have higher positive predictive values for CIN3 and CIN2 than HPV 1851. Taking into account the results shows here, and the previous works in the country, we consider that the inclusion of HPV 31 in the HPV typing triage in Mexico would improve cervical cancer screening due to the high prevalence of this genotype among cervical neoplasias.Moreover, with the development and validation of HPV tests, different screening algorithms for cervical cancer had been proposedThe information showed in this work does not represent the Mexican population entirely; however, this is one of the first approaches to known the circulating hr-HPV genotypes in Mexico. Our results show a growing prevalence of HPV genotypes not included in the current vaccines applied in the country. Together all work on HPV in Mexico in women from different regions of the country, with or without social security and with different population profiles provides valuable information that will help to guide the future cervical cancer screening programs and HPV vaccination in Mexico. Also, it is necessary to extend and continue the surveillance of circulating HPV genotypes to evaluate the effectiveness of vaccination and screening programs to reduce of cervical cancer and other HPV related cancers in Mexico in the coming years and long-term.Supplementary information