Posttraumatic stress disorder (PTSD) is a debilitating psychiatric condition that is common in veterans returning from combat operations. While the symptoms of PTSD have been extensively characterized, the neural mechanisms that underlie PTSD are only vaguely understood. In this study, we examined the neurophysiology of PTSD using magnetoencephalography (MEG) in a sample of veterans with and without PTSD. Our primary hypothesis was that veterans with PTSD would exhibit aberrant activity across multiple brain networks, especially those involving medial temporal and frontal regions. To this end, we examined a total of 51 USA combat veterans with a battery of clinical interviews and tests. Thirty-one of the combat veterans met diagnostic criteria for PTSD and the remaining 20 did not have PTSD. All participants then underwent high-density MEG during an eyes-closed resting-state task, and the resulting data were analyzed using a Bayesian image reconstruction method. Our results indicated that veterans with PTSD had significantly stronger neural activity in prefrontal, sensorimotor and temporal areas compared to those without PTSD. Veterans with PTSD also exhibited significantly stronger activity in the bilateral amygdalae, parahippocampal and hippocampal regions. Conversely, healthy veterans had stronger neural activity in the bilateral occipital cortices relative to veterans with PTSD. In conclusion, these data suggest that veterans with PTSD exhibit aberrant neural activation in multiple cortical areas, as well as medial temporal structures implicated in affective processing. Posttraumatic stress disorder (PTSD) is a psychiatric disorder involving re-experiencing, avoidance, negative mood and cognition and arousal symptoms , and whether aberrant processing would have been detected beyond fronto-limbic regions in patients with PTSD remains unknown. One preliminary MEG study of traumatic memory recall in women with PTSD found activation largely outside of fronto-limbic regions  using television commercials, flyers, and social media. Following clinical assessment (see below), it was determined that 31 of these male combat veterans met the diagnostic criteria for PTSD and the remaining 20 male combat veterans did not meet criteria for PTSD or any other psychiatric or neurological condition. Participants without PTSD were matched to those with PTSD on age, education, ethnicity and handedness. General exclusionary criteria included any medical diagnosis affecting CNS function, known brain neoplasm or lesion, history of significant head injury and ferromagnetic implants. This study was carried out in accordance with the recommendations of the Creighton University Institutional Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Creighton University Institutional Review Board.PTSD was diagnosed using the Clinician Administered PTSD Scale . MEG data from each participant were individually corrected for head motion and subjected to noise reduction using the signal space separation method with a temporal extension . Once the participant was positioned for MEG recording, an electric current with a unique frequency label  was fed to each of the coils. This induced a measurable magnetic field and allowed each coil to be localized in reference to the sensors throughout the recording session. Since coil locations were also known in head coordinates, all MEG measurements could be transformed into a common coordinate system. With this coordinate system , each participant’s MEG data were coregistered with T1-weighted sMRI data using the Statistical Parametric Mapping software  approach implemented in the SPM12 software , which was calculated directly from the data according to the theory of Gaussian random fields. All statistical analyses for behavioral and clinical variables were conducted in SPSS (Release 21.0.0).The resulting 3D maps of functional brain activity were statistically evaluated in SPM12 using a mass univariate approach based on the general linear model. Briefly, the effects of group were examined using a mixed random effects model, whereas one-sample p = 0.78). Mean educational level was 14.71 (SD: 2.41) years in the PTSD group and 14.13 (SD: 2.70) years in the control group (p = 0.42). By design, veterans with PTSD had significantly higher PTSD scores than the no PTSD comparison group (p < 0.0001). As expected, veterans with PTSD also reported higher rates of depressive and anxious symptoms compared to those without PTSD  doses of psychiatric medication , and the other 20 veterans with PTSD were not taking any psychiatric medication. One veteran in the non-PTSD group was taking a SSRI. Mean age was 32.13 (SD: 5.98) years in the PTSD group and 31.60 (SD: 7.18) years in the control group . Significant activity was also detected in bilateral medial temporal structures, including amygdalae, hippocampi and parahippocampal regions of each group . In veterans with PTSD, bilateral prefrontal activity was also found . In contrast, veterans without PTSD had stronger activity in the left and right lateral occipital cortices compared to veterans with PTSD . To this end, we filtered MEG signals into conventional frequency bands  and imaged each band separately. Our results indicated that theta (4–7 Hz) was the critical frequency for our medial temporal findings , while alpha (8–14 Hz) activity was central to our occipital, temporal and sensorimotor findings. Our findings in the DLPFC extended across theta and alpha . Beta and gamma differences were much weaker, non-significant and likely made a negligible contribution to our primary findings.We used high-density MEG and advanced image reconstruction methods to quantify resting-state  activity in combat veterans with and without PTSD. Our main findings were that veterans with PTSD exhibited significantly stronger activity relative those without PTSD in bilateral motor regions, lateral temporal areas, the DLPFC and multiple medial temporal areas including the hippocampi and amygdala. The opposite pattern of stronger activity in the non-PTSD group was observed in the lateral occipital cortices. Major strengths of this study included the thorough clinical assessments, state-of-the-art neurophysiological imaging, and the close matching of veterans with PTSD to a psychiatrically-healthy combat-exposed comparison group. Lifetime traumatic events were similar between groups, and although combat exposure was greater in the PTSD group, the comparison sample had also experienced significant combat-related traumas. Given that the experience of trauma in the absence of PTSD symptoms appears to have a lasting effect on the brain , both groups exhibited very similar patterns of resting-state neural activity. Group comparisons of the patients with PTSD and matched combat-exposed controls revealed regionally-specific patterns of increased and decreased neuronal activity during the resting-state. Specifically, we found stronger spontaneous neural activity in the bilateral sensorimotor cortices , paracentral lobule and DLPFC in veterans with PTSD as compared to non-PTSD controls. The sensorimotor and prefrontal findings are consistent with evidence of aberrant neurodynamics in veterans with PTSD in these same brain regions in two recent MEG task-based studies , as our findings suggest that spontaneous neural activity may be altered in PTSD. Future studies should also evaluate women and PTSD resulting from different types of trauma to determine whether the neurophysiological effects differ from what we observed in male combat veterans.In conclusion, we examined the neural basis of PTSD in a sample of USA combat veterans using advanced MEG methods and clinical assessments. We focused on the amplitude of resting-state  neural activity using a voxel-based whole brain approach, as previous studies had largely restricted their analyses to Finally, our study sample was not without limitations, including our focus on men and combat-related PTSD, possible medication or anxiety effects in the veterans with PTSD, and expected differences in the severity of combat exposure between the two groups. In addition, we examined only the resting state using MEG , and some of our findings in the medial temporal lobe will need to be confirmed by future studies given the reduced sensitivity of MEG to neural sources distant from the sensors. That said, we used both magnetometers and gradiometers for source reconstruction, and magnetometers are inherently more sensitive to neural activity in distant brain regions. We also observed anticipated group differences in these medial temporal structures, providing converging evidence on the veracity of these findings, as MEG recordings would be equally sensitive (or insensitive) to such activity across both groups. MEG studies reporting neural activity in deeper brain structures such as the cerebellum, amygdala and other medial temporal regions are becoming more common across the field , grant R01-MH103220 from the National Institutes of Health (TWW), and grant #1539067 from the National Science Foundation (TWW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, p16 has been included in the TNM guideline for oropharyngeal carcinomas. The role of HPV and p16 in hypopharyngeal and laryngeal carcinomas has not yet been established sufficiently.Hundred and thirty-four patients with hypopharyngeal and laryngeal carcinomas were included in this retrospective analysis. Only patients with known HPV status were eligible for the investigation. Survival probabilities were estimated for different risk factors.Eighty-five patients presented with laryngeal carcinoma and 49 patients with hypopharyngeal carcinoma. 8% were HPV positive . Median follow-up time was 58 months. We observed a significantly better overall survival for patients with an early tumor stage compared to advanced carcinoma. One of the hypopharyngeal HPV positive carcinomas was also p16 positive and one was p16 negative. Of the nine HPV positive laryngeal carcinomas, four were p16 positive and five p16 negative. Neither patients who were HPV positive nor patients positive for p16 showed a significantly better outcome than HPV or p16 negative patients. In contrast, nicotine pack-years showed a highly significant correlation with survival in our patient collective.The data suggest that tumor stage and nicotine exposure seem to have the highest impact on survival in hypopharyngeal and laryngeal squamous cell carcinoma patients. There is no evidence for a better survival for p16 positive or HPV positive patients with hypopharyngeal or laryngeal squamous cell carcinoma. HPV seems to play a minor role in these entities of head and neck carcinoma. More than 30 years ago, a link between human papillomavirus (HPV) and head and neck cancer (HNC) was found. Since then numerous studies have been performed on this topic and showed a better prognosis for HPV positive oropharyngeal squamous cell carcinomas (OPSCC) than for HPV negative OPSCCs. This fact is underlined by the inclusion of p16 in the updated TNM classification .The HPV prevalence is described by the percentage of HPV positive patients [with an exact 95% confidence interval (CI)]. The inverse Kaplan–Meier method  was usedIn total, 134 patients were included. Of all 134 patients, the following characteristics were known: patient age at time of diagnosis, TNM classification, treatment modality, HPV status, and tumour localization. P16 status, nicotine and alcohol consumption was not known in all patients. Median follow-up time was 58 months (range 0–104 months). Patients were treated with surgery, surgery and (chemo-/immuno-) radiotherapy or primary (chemo-/immuno-) radiotherapy. Two patients refused any treatment.Patient characteristics are summarized in Tables Of the 134 patients included in the study, 85 patients had laryngeal carcinoma and 49 patients hypopharyngeal carcinoma. Nine patients were HPV high-risk positive (type 16) and two low-risk positive (type 6). In total, 11 of 134 patients were HPV positive .Nine patients with laryngeal carcinoma and two patients with hypopharyngeal carcinoma were HPV positive (10.6 vs. 4.1%).Of the nine HPV positive laryngeal carcinoma patients, seven were HPV high-risk positive and two low-risk positive. Of the two hypopharyngeal HPV positive carcinomas both were HPV high-risk positive.In 81 cases p16 immunohistochemistry was available, 57 were p16 negative and 24 were p16 positive (70 vs. 30%). Of the 24 patients with p16 positive carcinomas 17 were laryngeal carcinomas and 7 hypopharyngeal carcinomas, respectively.One of the hypopharyngeal HPV positive carcinomas was also p16 positive and one was p16 negative. Of the nine HPV positive laryngeal carcinomas four (44%) were p16 positive and five (56%) p16 negative. Both HPV low-risk positive carcinomas were p16 negative.There was no significant difference between the two groups of patients (HPV positive and negative carcinomas). There was a similar distribution of gender, smoking as well as drinking habits and age Table .p < 0.001).All patients were included in survival analysis. In total, median survival for laryngeal carcinomas was 81 months and for hypopharyngeal carcinomas 25 months with a significantly better survival for laryngeal carcinomas (log-rank test: p = 0.956).Comparing overall survival for 81 patients with known p16 status, 24 p16 positive and 57 p16 negative, there was no significant impact on survival as shown in Fig. p = 0.175; HR = 0.53, 95% CI (0.14–1.35)] . Patients with an advanced tumour stage were four times more likely to die than patients with a lower tumour stage .Overall survival of patients with a tumour stage I or II (according to UICC guidelines)—summarized to an early tumour stage—showed a highly significant better overall survival than patients with a tumour stage III–IVc (according to UICC guidelines)—summarized to an advanced tumour stage (p = 0.077) for overall survival of patients .Treatment options were divided into three subgroups. Group 1 consisting of patients undergoing surgery alone, group 2 included patients undergoing surgery followed by radiotherapy, chemoradiotherapy or immunoradiotherapy and finally group 3 included patients treated by primary radiotherapy, chemoradiotherapy or immunoradiotherapy. Patients in group 1 had a better outcome than patients in group 2 and both had a better outcome than patients in group 3. Comparison of all three groups reached a significant difference , p = 0.087].Multivariable analysis revealed the prognostic factor tumour stage as the only statistically significant parameter on overall survival [HR = 4.32, 95% CI (2.11–9.95), Tobacco and alcohol have been the most important risk factors for the development of head and neck carcinomas for the past decades. After studies including those of Fahkry et al., which could demonstrate that patients with HPV positive oropharyngeal carcinomas have at least half the risk of death than HPV negative patients , HPV hasp = 0.0185). Joo et al. found a rate of 10.9% HPV positive hypopharyngeal carcinomas in 64 patients. In these patients there was a significant correlation of HPV high-risk positive carcinomas, younger age and non-smoking status. HPV-positive patients had a significantly better disease-free survival (p = 0.026) and disease-specific survival (p = 0.047) [p = 0.031) could be found [The role of p16 and HPV in hypopharyngeal and laryngeal carcinomas has not sufficiently been established yet. Especially in laryngeal and hypopharyngeal cancers HPV expression vary enormously. The prevalence of HPV DNA in laryngeal squamous cell carcinomas varies between 0 and 75% . Survivabe found .The percentage of HPV positive carcinomas in our patient cohort  is similar to other studies , 10, 12.Another reason for variable HPV prevalence is the method of detection, where p16 plays an important role, as it is often determined as a surrogate marker for HPV status. P16 is a cyclin-dependent kinase inhibitor, which becomes up-regulated in HPV infected cells when E7 inactivates the tumour suppressor Retinoblastoma protein . In oropThis hypothesis is supported by the results of studies by Meshman et al. or Chung et al. who had similar results to our study with a missing correlation of p16 and HPV , 16. Of P16 might not be a good marker for HPV in non-oropharyngeal head and neck carcinomas.Additionally, our results show that p16 expression and presence of HPV in laryngeal and hypopharyngeal carcinomas have no correlation to overall and disease-free survival. These results can also be compared to Wendt et al. , and MesFor normal laryngeal mucosa an incidence rate of HPV of up to 19% has been reported . HPV canAnother important factor when analysing HPV and p16 positive tumours is the method of detection.The golden standard for HPV detection has been discussed widely. At present, PCR is the most sensitive technique used in the search for viral genomes . A positIn our study, HPV was detected routinely by in situ hybridization (ISH) as shown in Fig. Not only the presence and detection of HPV play an important role, but also the type of HPV has an important impact. In total there are more than 100 known HPV subtypes. Kreimer et al. presented a systematic review of HPV types in HNSCCs, where HPV16 was the most common subtype in HPV positive HNSCCs. In oropharyngeal HNSCC HPV 18 was found less and more often in squamous cell carcinomas of the oral cavity and laryngeal carcinomas . Other tIn our study, nine patients were HPV high-risk positive (HPV type 16) and two were HPV low-risk positive (HPV type 6), which corresponds to the results mentioned before.Neither patients who were HPV positive nor patients positive for p16 showed a significantly better outcome than HPV or p16 negative patients.In contrast, nicotine pack-years and tumour stage showed a highly significant correlation with survival in our patient collective. Correlation of tumour stage and survival in all carcinomas of the head and neck has been shown in several previous studies. Patients with a lower tobacco exposure were more likely to have a better prognosis than heavy smokers. Eight out of 11 HPV positive tumour patients were smokers and 10 out of 11 were also exposed to alcohol, which does not allow further analysis of HPV positive non-smokers and non-drinkers. In oropharyngeal carcinomas HPV positive patients often are non-smokers and do not or rarely consume alcohol. This fact cannot be transferred to laryngeal and hypopharyngeal carcinomas, whether in the present study or in our personal experience.The survival results in this study depending on treatment options show that patients treated by surgery alone have a better outcome than patients treated by multiple modalities and patients treated by primary (chemo- or immuno-) radiotherapy. These results can be attributed to the fact that tumours with less extension are more often and easier treated by surgery alone and no further treatment is required. Patients with larger and/or inoperable tumours are treated with primary conservative treatment. These results therefore reflect the difference between early and advanced tumour stages rather than therapy outcomes.In conclusion, the data presented suggest that tumour stage and nicotine exposure seem to have the highest impact on survival in hypopharyngeal and laryngeal squamous cell carcinoma patients. We could not observe any sufficient correlation of p16 and HPV. There is no evidence for a better survival for p16 positive or HPV positive patients with hypopharyngeal or laryngeal squamous cell carcinoma. Active HPV infection seems to play a minor role in these non-oropharyngeal entities of head and neck carcinoma.


In Australia, screening for colorectal cancer (CRC) with colonoscopy is meant to be reserved for people at increased risk, however, currently there is a mismatch between individuals’ risk of CRC and the type of CRC screening they receive.This paper describes the development and optimisation of a Colorectal cancer RISk Prediction tool (‘CRISP’) for use in primary care. The aim of the CRISP tool is to increase risk-appropriate CRC screening.CRISP development was informed by previous experience with developing risk tools for use in primary care and a systematic review of the evidence. A CRISP prototype was used in simulated consultations by general practitioners (GPs) with actors as patients. GPs were interviewed to explore their experience of using CRISP, and practice nurses (PNs) and practice managers (PMs) were interviewed after a demonstration of CRISP. Transcribed interviews and video footage of the ‘consultations’ were qualitatively analyzed. Themes arising from the data were mapped onto Normalization Process Theory (NPT).Coherence: Clinicians understood the rationale behind CRISP, particularly since they were familiar with using risk tools for other conditions; 2) Cognitive participation: GPs welcomed the opportunity CRISP provided to discuss healthy and unhealthy behaviors with their patients, but many GPs challenged the screening recommendation generated by CRISP; 3) Collective Action: CRISP disrupted clinician-patient flow if the GP was less comfortable with computers. GP consultation time was a major implementation barrier and overall consensus was that PNs have more capacity and time to use CRISP effectively; 4) Reflexive monitoring: Limited systematic monitoring of new interventions is a potential barrier to the sustainable embedding of CRISP.Fourteen GPs, nine PNs and six PMs were recruited from 12 clinics. Results were described using the four constructs of NPT: 1) CRISP has the potential to improve risk-appropriate CRC screening in primary care but was considered more likely to be successfully implemented as a nurse-led intervention. Australia and New Zealand have the highest rates of colorectal cancer (CRC) globally, with estimated age standardized rates of 44.8 and 32.2 per 100,000 in men and women respectively , and onlRisk-stratified screening requires valid risk prediction models that accurately discriminate people at increased risk from those who are not. These risk models require easy-to-use risk assessment tools for implementation into clinical practice . A recenMedical Research Council Framework for the Development and Evaluation of Complex Interventions to improve healthcare ‘Coherence’ refers to how the participants make sense of and value an intervention. Clinic staff agreed CRC screening was relevant to their practice and were familiar with using risk assessment tools to support clinical decision making, particularly for assessing cardiovascular or diabetes risk, or when screening for depression.…but don’t forget that little person; okay let’s make sure that’s not you. GPs used the icon array to explain CRC risk, either to emphasize the need to be screened,there’s a whole lot of people and this is your chance out of them of cancer… …or to reassure the patient that their risk was low.The graph was more challenging to explain. In the first iteration of CRISP the y axis (CRC risk) ranged from 0% to 20% which generated a steep curve at times, and caused concern that this might mislead patients about their risk Fig. . All sta…that’s just the tricky bit of saying [to the patient] that this is what’s [sic] the recommendation but we don’t always follow what recommendations are… Screening advice was challenged by some GPs who did not agree with or were not familiar with current NHMRC guidelines.Because once you go beyond that first degree relative, really the risk diminishes so significantly that getting all that extra data isn’t going to be that significant. Many GPs also challenged the necessity for collecting second degree relative information.Anything that gets people to have a think about their health is a good thing, anything at all. ‘Cognitive participation’ explores how users engage with and understand an intervention in the context of their everyday work. There were specific aspects of CRISP that everyone liked, including the opportunity to reinforce healthy behaviours or discuss strategies with patients for changing unhealthy behaviours .Do you smoke?Patient: No.GP: Good man! GP: The GPs responded easily and almost instinctively to patients’ responses to these questions.On the other hand, there was a general distrust of some of the recommendations, particularly for FOBT. Colonoscopy was commonly referred to as the ‘gold standard’ for CRC screening. GPs were more likely to recommend colonoscopy if the patient wanted one or they had previously been recommended one by a gastroenterologist, even if the patient was at average risk.… nurses … see the patient first a lot of the time and so they could go through these sorts of things… ‘Collective action’ refers to the capacity for the intervention to fit within existing organizational practices. GPs, PNs and PMs all agreed that lack of GP consultation time would limit the use of CRISP by GPs. There was a consensus that nurses have the capacity, time and expertise to complete the risk assessment as part of routine preventive health consultations.The existing health checks for 45 – 49 year olds  which arI’ve got a 26-year-old patient that has …metastases everywhere …of course, everybody said it was her endometriosis… … Opinions about who would take responsibility for the final decision about screening advice was split between GPs and PNs; many GPs had a fear of missing a diagnosis and they often relayed anecdotes of cases of a missed or late CRC diagnosis.…Yeah. I just don’t think it will work in the waiting room…We might start getting asked questions at the front desk… That’s the thing, how do we answer? You know what I mean? Using CRISP as a self-completed tool in the waiting room was explored but everyone felt that administrative staff would not have the time or expertise to manage patients using CRISP in the waiting room.…the doctors are very open to suggestion. If the practice nurses, management, or another doctor feels that this could be a really good idea for the doctors, it’s taken to the doctors’ meeting and overall feeling of shall we do this, shall we not is taken place…Implementation of new interventions into a practice was generally the GPs’ decision although PMs and PNs felt they could propose new ideas to the practice.So I’m in a really big group practice… and … people would be really excited… [But it] would vary because some people don’t change their practice much at all… Despite this, even if a decision was made at a practice level, GPs still acted independently when incorporating new technology into their clinical work.…this is a labyrinth!… especially having 3 pages for the 2nd degree relatives…Many GPs found certain aspects of using the CRISP prototype laborious, especially the method for collecting family history information.GPs wanted CRISP integrated into existing clinical software so data could be auto-populated and then updated into the patient record but acknowledged that the variety of software used in different practices would make it difficult to integrate CRISP universally.There were challenges using CRISP within a GP consultation. GPs felt uncomfortable taking their attention away from the patient by looking at the computer. Despite this, GPs often re-engaged with the patient when explaining the risk output and screening recommendations. Younger GPs, particularly those who could touch type, were able to move more seamlessly between the computer and the patient. When GPs asked questions of their patients prior to opening CRISP, which was not uncommon, they felt the tool was unnecessarily repetitive.the capacity’s there. Yeah, and we certainly have things that we can do like that, but often we do nothing. … - it’s like ships in the night, so we can be here and not even say hi to each other all day sometimes…… ‘Reflexive monitoring’ refers to how new interventions are monitored, evaluated and appraised . ClinicsConsequently, there were limited systems within the practices to monitor or evaluate the effectiveness of CRISP over time.As a preliminary qualitative study in a program of research to develop and trial a novel CRC risk prediction tool, we identified clear barriers and facilitators to the implementation of our prototype tool (CRISP) into general practice. We found that GPs were capable of using CRISP in simulated consultations, and were generally used to applying risk tools as part of their clinical decision making. Despite this, GPs did not always adhere to the CRISP screening advice, with many favouring colonoscopic screening even for people at average risk as it was considered a ‘better’ screening method. Barriers to using CRISP included limited consultation time, long-winded family history collection, and for some GPs, lack of confidence with computers. All clinic staff thought the questions about lifestyle were very helpful for facilitating a discussion about how to change unhealthy behaviors and could see how CRISP might fit into a health-check conducted by the PN. For CRISP to be implemented into practice, it was obvious that it would need to be more intuitive and shorter and would be easier if it was integrated into the patient clinical medical record.development of an interactive family history collection page on a single web-page to collect family history; this was more intuitive and user friendly, and substantially reduced the time to complete CRISP Fig. ;Fig. 4Thmodifications to the natural frequency icon array to include a larger denominator population and therefore present smaller risks more clearly Fig. ;Fig. 5Anmodifications to the graph to reduce potential overestimation of risk over time Fig. ; andFig.as a result of our findings on GPs’ perceptions about the relative benefits and harms of FOBT and colonoscopy, we developed additional risk communication output in CRISP to highlight the potential harms of colonoscopy and the sensitivity of FOBT to detect CRC using ‘expected frequency trees’ Fig.  16].Fig.Fig16].FTo increase the useability of CRISP, we reduced the data entry pages from up to 27 pages to a maximum of eight pages. Other modifications included:Family Healthware Impact Trial, health promotion within a risk tool increased clinician ‘buy in’ [Our results are consistent with findings about the few available cancer risk assessment tools used in primary care . The use‘buy in’ . In comp‘buy in’ , 25.The CRISP research team previously conducted an exploratory study of a diagnostic risk assessment tool (QCancer) using similar methods, but the CRISP study involved GPs, PNs and PMs. CRISP was more acceptable to GPs than QCancer, although both were difficult to introduce into a GP consultation. The advantage of interviewing all staff in the CRISP study was the discovery that clinic staff were in agreement that CRISP would be better suited to use in a nurse consultation. Another significant difference between QCancer and CRISP was the user interface. A major limitation to the accuracy of QCancer was input error . This isOur study demonstrated that primary care in Australia has the capacity to implement risk assessment tools using PNs and potentially contribute to more risk-appropriate CRC screening. Practice staff felt CRISP would fit into the context of a preventive health consultation conducted by a PN, in particular as part of ‘45 – 49 year old’ health checks already funded by the Australian government to increase screening for chronic diseases . This prAlthough some of the participants were recruited from a practice-based research network, we recruited a range of GPs, PNs and PMs including: 1) rural and metropolitan practice staff, 2) solo and group practice staff, and 3) staff from a range of ages and experience level in general practice. The sample of PNs was mainly female and of similar ages, however, this was not atypical for this population and is likely to be representative . This apWe attempted to recruit GPs, PNs and PMs from the same clinics, to assess congruency of ideas between staff at the same clinic. However, this was not always possible with some clinic staff unavailable for an interview and one clinic did not have a PN.Another limitation was the potential for social desirability bias as participants might not have felt comfortable criticizing CRISP to the researchers during the interviews. This was offset by the data gained from the simulated consultations which have been demonstrated to predict actual clinical performance .This study has provided important contextual information on some of the potential barriers and facilitators to implementing CRISP and risk-stratified CRC screening in primary care. It has informed further development of CRISP so that it is more likely to meet the needs of users and provide risk information that could support improved screening decision making. We have identified a preferred model of implementation in practice involving PNs. This approach will now be tested in an RCT to assess the effects of implementing CRISP on GP and patient CRC screening behaviours.


This educative scientific meeting will take place in Rio de Janeiro from 4–7 May 2009under the theme: Brain Aging and Quality of Life. We are convening an internationallyrenowned group of scientists who are experts in the field of research and clinicalpractice related to neurology, psychiatry and geriatric medicine to address issues inaging, neuroscience and mental health in old age. The International Meeting will providea forum to meet and network with other clinicians, educators and researchers from allover the world.http://www.abnpg.com/contatos.htm, call 55 31 3201 7905 or send ane-mail to exsecretaria@abnpg.comWe are looking forward to your participation. For more information, please visitJoão Carlos Barbosa MachadoJerson Laks


Cytological and histological cervical screening methods for human papillomavirus may be subjective. Current guidelines recommend the use of direct human papillomavirus screening by molecular methods in conjunction with cytology for the detection of high-risk human papillomavirus types with carcinogenic potential. In this study, we compared the performance of the molecular Cepheid Xpert HPV test to the FDA-approved HC2 High-Risk HPV DNA Test on samples from patients presenting for cervical screening, regardless of the cytology results, in which cervical cell samples were originally collected for Papanicolaou (Pap) smear specimens in Becton Dickinson (BD) SurePath preservative fluid.Cervical cells were obtained for Pap smear specimens from 343 women attending Qatif Central Hospital in Saudi Arabia for cervical cancer screening using a Cytobrush Plus GT and immersed in BD SurePath preservative fluid in BD SurePath collection vials. The study was carried out between December 2015 and July 2016.n=15) or in combination with HPV16 (n=3). The overall concordance rate between the tests was 98.5 %. The positive concordance was 84.4 %.The Xpert HPV test was positive in 27 (7.9 %) of the samples. The HC2 High-Risk HPV DNA Test was positive in 32 (9.3 %) of the samples. The most common HPV types according to the Xpert HPV test were HPV other types, either alone (The Xpert HPV test is convenient to use on cervical cell samples collected for Pap smear specimens in BD SurePath preservative fluid within an hour and is a viable alternative to the HC2 High-Risk HPV DNA Test for HPV testing. A numbeaviridae .Chlamydia trachomatis or bacterial vaginosis [The International Agency for Research on Cancer (IARC) divides HPV types into groups according to their carcinogenic potential . Group 1aginosis . Women aRegular cervical cancer screening is important for early detection and treatment. Currently, the most common screening method involves the collection of cervical cells for staining with Papanicolaou (Pap) stain and microscopic examination . This caHowever, there are some concerns that the cytological and histological screening methods may be subjective and that there is considerable variation in interpretation between laboratories . Thus thIn this study we compared the performance of the Cepheid Xpert HPV test to the HC2 High-Risk HPV DNA Test using samples from all patients presenting for cervical screening, in which cervical cell samples were originally collected for Pap smear specimens in Becton Dickinson (BD) SurePath preservative fluid. This is the first time, to the best of our knowledge, that these tests have been performed directly on cell samples routinely collected for a standard Pap stain in this reagent.sd) was 40.6±10.7 years and the age range was 20–80 years. The distribution by age group was 20–29 years, n=54; 30–39 years, n=109; 40–49 years, n=115; 50–59 years, n=53; ≥60 years, n=12.The study population included all women attending Qatif Central Hospital for cervical cancer screening, regardless of the cytology results. The study was approved by Qatif Central Hospital Institutional Review Board (IRB). Samples were taken from 343 women, of whom 338 (98.5 %) were Saudi nationals. The mean age±standard deviation  and the HC2 High-Risk HPV DNA Test was carried out according to the manufacturer’s instructions, as in Rabaan et al. .et al. [Cell solution taken directly from the SurePath collection vials was homegenized and transferred to BD centrifuge tubes (c-tubes) containing pre-aliquoted density reagent, using the BD PrepMate Automated Accessory, according to the manufacturer’s instructions (Becton Dickinson). Two different centrifugation steps were then carried out to produce a concentrated and enriched cell pellet, as normally used for SlidePrep and cytological screening. In this study, 1 ml of enriched pellet suspension was placed in an Xpert HPV cartridge  and run and analysed on the GeneXpert Dx system for second-generation real-time PCR analysis, according to the manufacturer’s instructions, as in Rabaan et al. .P<0.05 was accepted as significant. We also calculated the percentage agreement between the two methods using the Kappa statistic. The results were interpreted based on the guidelines that negative Cohen’s kappa means no agreement between methods, between 0 and 0.20 indicates slight agreement, 0.21–0.40 indicates fair agreement, 0.41–0.60 indicates moderate agreement, 0.61–0.80 indicates substantial agreement and 0.81–1 indicates almost perfect agreement [We calculated the overall concordance between the HC2 High-Risk HPV DNA Test and the Cepheid Xpert HPV test, and the positive concordance between the tests. The McNemar chi-squared test was applied to test the statistical significance of the discordance between the tests; greement . The pron=15) or in combination with HPV16 (n=3)  .P value was 0.07, indicating that there was no significant degree of discordance. Cohen’s kappa was 0.907 , indicating almost perfect agreement. The positive concordance was 84.4 %.P=0.678).When the 338 samples for which the two tests gave the same result were considered by age group , there was no significant difference in the proportion of positive versus negative results , vaginitis (n=6), post-coital bleeding (n=3), uterine fibroid/polyp (n=3), menorrhagia (n=2), vaginal discharge (n=2), abdominal pain (n=1), Bartholin cyst (n=1) and infertility (n=1), in addition to attendance for routine screening (n=2). For 26 of these 27 samples, pathology results were available. For 21 samples there was nil pathology, for 1 nil with atrophy and for 1 nil with severe inflammation. Three samples were identified as ASC-H , and two were identified by histology as carcinomas. The pathology was nil for the five cases that were positive by HC2 but negative by Cepheid.There were a range of clinical signs for the 27 samples that were positive for both tests, including irregular menstruation  SurePath preservative fluid. The overall concordance between the tests, and a comparison with the prevalence results from other studies, suggests that this did not interfere with the tests and that they worked efficiently under these conditions . This woAlthough the HC2 High-Risk HPV DNA Test was positive in five samples that gave a negative result with the Cepheid Xpert HPV test, it has been previously noted that the HC2 High-Risk HPV DNA Test has a false positive rate of 5 % in tests where PCR does not detect HPV DNA . The proThe Cepheid Xpert HPV test has the advantage of providing information on the presence of individual HPV types rather than a pooled result. It is also more rapid to perform, giving results within an hour as compared to taking several hours, as with the HC2 High-Risk HPV DNA Test, meaning that the patient can be referred for colposcopy immediately. Other studies have also indicated substantial agreement between the HC2 High-Risk HPV DNA Test test and the Cepheid Xpert HPV test. For example, in a recent assessment of anal hrHPV prevalence in 199 HIV-positive women in South Africa there was 86.7 % agreement between the tests , while iIn conclusion, in this study we have shown that the Cepheid Xpert HPV test can be used on cervical cell samples collected for Pap smear specimens in BD SurePath preservative fluid and shows good agreement with the FDA-approved HC2 High-Risk HPV DNA Test test. Thus the Cepheid Xpert HPV test is a viable alternative to the HC2 High-Risk HPV DNA Test test and can be conveniently performed on samples taken routinely for Pap smear to generate a result within an hour.


Human papillomaviruses (HPVs) represent a large collection of viral typesassociated with significant clinical disease of cutaneous and mucosalepithelium. HPV-associated cancers are found in anogenital and oral mucosa, andat various cutaneous sites. Papillomaviruses are highly species and tissuerestricted, and these viruses display both mucosotropic, cutaneotropic or dualtropism for epithelial tissues. A subset of HPV types, predominantly mucosal,are also oncogenic and cancers with these HPV types account for more than200,000 deaths world-wide. Host control of HPV infections requires both innateand adaptive immunity, but the viruses have developed strategies to escapeimmune detection. Viral proteins can disrupt both innate pathogen-sensingpathways and T-cell based recognition and subsequent destruction of infectedtissues. Current treatments to manage HPV infections include mostly ablativestrategies in which recurrences are common and only active disease is treated.Although much is known about the papillomavirus life cycle, viral proteinfunctions, and immune responsiveness, we still lack knowledge in a number of keyareas of PV biology including tissue tropism, site-specific cancer progression,codon usage profiles, and what are the best strategies to mount an effectiveimmune response to the carcinogenic stages of PV disease. In this review,disease transmission, protection and control are discussed together withquestions related to areas in PV biology that will continue to provideproductive opportunities of discovery and to further our understanding of thisdiverse set of human viral pathogens. Papillomaviruses are an ancient group of viruses exquisitely adapted to their hostsin a tissue and species-restricted manner .c. A region containing two late genes coding for the capsid proteins.d. A non-coding region containing regulatory elements and a replication origin.insitu.Current control of HPV infections is focused on preventive strategies via inductionof neutralizing antibodies; by various ablative strategies for lesion removal, andattempts to activate antigen-specific cell-mediated immunity  and late (L) proteins. ORFs have been identified from the coding sequencefor E1, E2, E4, E5, E6 and E7 early proteins and L1 and L2 late proteins . Confirmation as to whether additional viral proteins are present  are challengingexperiments to conduct yet several potential reading frames remain unstudied and therefore are assumed to be non-functional and/ornon-essential. Spliced viral RNA species have been mapped and “new” proteins (e.g.E8^E2) have been confirmed by studies in an animal papillomavirus model HPV infections are believed to occur following wounding of epithelium and subsequentinfectious virion access to basal epithelial cells and basement membrane componentsof the epithelium  andtumor necrosis factor (TNF)-α cytokine-producing T cells Questions relevant to this section:1. Why do infections with some HPV types manifest only as asymptomaticdisease whereas others show active clinical disease?2. Why do infections by different HPV types progress to cancer whereasothers do not?3. Do HPV16-associated cancers at different anatomical locations have asimilar pathology, etiology, and progression rate?4. Are HPV-associated cancers less immunogenic (or have differences inimmune escape mechanisms) than precancerous or primary benign HPV infections ofthe same HPV type? 5. Does the detection of HPV viral DNA correlate 100% with active clinicaldisease?HPV infections present as epithelial lesions that are localized to cutaneous ormucosal sites. All cutaneous tissues are susceptible to HPV infections althoughmucosal infections are mostly confined to the anogenital and oral cavities as wellas laryngeal epithelium. Other less common mucosal sites include bladder,conjunctiva and lung epithelium. The infections range from asymptomatic infections(particularly from the beta- gammapapillomaviruses) to small epithelial lesions tovery large cutaneous lesions to large cancerous lesions. In general, most infectionsare small in size, and present with minimal evidence of infection .in situ analyses. Those HPV types that produceasymptomatic disease are challenging to locate in situ. That theseasymptomatic infections are true infections that complete the virus life cycle andrelease infectious virions is absolutely confirmed by the existence and persistenceof these many HPV types in patient populations.Many HPV infections are confirmed by sensitive DNA detection methods rather than viahistological or One HPV type (HPV16) stands out as highly associated with cancers at severaldifferent anatomical locations  areassociated with a potential differential susceptibility to HPV infections.3. Given the commensal nature of some skin and mucosotropic HPV and theirprevalence, do these commensal infections provide a potential localized immunetolerance for subsequent infection by the more pathogenic HPVs thereby reducingthe initial host immune response to these latter types?4. Is there competition or synergistic interactions between different HPVtypes during a co-infection?5. Do asymptomatic HPV infections trigger protective homeostatic hostimmunity to these and other common skin microbes? Do other microbes at skin andmucosal sites provide immune “cover” for many asymptomatic HPVinfections?6. Is there a correlation between codon usage profiles and tissue tropismfor HPV types?HPVs are one of the most ubiquitous viral infections with over 300 viral types thatinfect most cutaneous and many mucosal tissues. Most types are associated withminimal disease and can be described as part of the commensal fauna of thesetissues. Numerous epidemiological and molecular studies on cervical cancerdemonstrate that hrHPV are associated with up to 99% with cervical cancers 3410911011111311411511611811204HPV of the betapapillomavirus group such as HPV5 and HPV8 are associated with rareincidences of skin cancers, and these and other members of this family areconsidered to also play a role in non-melanoma skin cancers 30109103There is an exquisite interplay between HPV persistence, tissue tropism and thehost innate and adaptive immune response. The keratinocyte is the exclusive hostcell of HPVs, and has significant potential to mount both anti-viral responsesto PV infections as well as activate adaptive immunity. The tissue tropismintroduces the concept of micro-environments in which the host keratinocytes andlocal immune monitoring are not necessarily identical at different anatomicalsites. Possible differences in immune monitoring as well as differences in thenatural microbiome in oral and anogenital mucosa also provide mechanisticexplanations for HPV tissue tropism 4545125126insitu. This phenomenon represents another fruitful area of researchfor the papillomavirus community. Note too that some HPV types show littletissue restriction and can be found at both mucosal and cutaneous sites  become preferentially infected despite the observationthat most oral mucosa is susceptible to this virus . These studiesmimic to some extent, the pre-selection of oral HPV-associated cancers that areconfined to the base of tongue, tonsil and oropharynx 45A new mouse PV model has recently been described that infects cutaneous Questions relevant to this section:1. Why do some patients clear their HPV infections and others do not? Arethere genetic, epigenetic and/or environmental components to these differentoutcomes?2. Why do many patients clear infections against some HPV types but notothers?3. Do pre-existing co-infections with other infectious agents increase ordecrease susceptibility to and persistence of HPV infection?4. Does immune clearance of HPV infections lead to sterilization of theinfection or immune monitoring of a subclinical persistent infection?5. Why are there so many treatment failures when clinical “cures” appear tohave been achieved with various different strategies?6. Can we develop successful T-cell based immunity to existing HPV diseasethat can control/clear persistent disease and cancer? Are there additionalneo-antigens  such as host-viral fusionproteins that could serve as potential CD8 T-cell epitopes?ad hocbasis of treatment of visible lesions seen by colposcopy or acid-white staining.Asymptomatic HPV disease remains untreated as a default. Active clinical disease isdiagnosed by both sensitive viral DNA detection methods, and histology or cytology.Monitoring programs (e.g. PAP test) are used to determine the timing and severity ofintervention. The PAP smear test examines cells collected by lavage from vaginalsites and cells are examined for histological abnormalities, for viral DNA or forother diagnostic indicators such as increased p16INK4a immunostaining, acidphosphatase positivity, p53, p63, various host microRNA and other additional novelmarkers 8285118121126140141142143144145Three prophylactic vaccines are currently approved for control of severalhigh-risk HPV types, and two low-risk types commonly found in genital warts.These include Gardasil , Cervarix  and Gardasil9 147148150151154Therapeutic vaccines based on T-cell responses to virus-infected tissues havebeen extensively tested in preclinical models, but have enjoyed much lesssuccess in clinical trials. Nevertheless, there have been recent encouragingresults using long peptide vaccines from HPV16 E6 and E7 for therapeutic T-cellbased regression of vaginal intraepithelial infections 156in situ in HPV-associated cancers that are highly localizeda. We need to better understand the various immune suppressive events that occurb. We need to design improved strategies to overcome localized T-cell exhaustionand other functional deficiencies that are increasingly being defined in otherchronic viral infections c. We need to better understand and then counteract HPV-induced innate andadaptive immune escape mechanisms to improve T-cell recognition and eliminationof HPV infected cells.d. We need to improve our understanding of T-cell homing to non-immunologicaltissues  in order to improve thefrequency and effector functions of CD8 T-cells e. We need to improve therapeutic activation of HPV-specific T-cells and assessother potential non-HPV epitopes arising from mutations in cervical cancer tobetter design vaccines that develop long-lasting immunity 162Finally, there may be productive opportunities to explore possible sources ofneo-antigens in HPV-containing cancer cells that are represented as novelmutations in host cell proteins that provide new CD4 and CD8 epitopes 164165167168Questions relevant to this section:1. Is wounding essential for successful infection of tissues by infectiousvirions?2. Can HPV infections be established from virions that enter sites ofepithelial “conflict” as found in transition zones, hair follicles and tonsillarcrypts, without the need for environmental wounding?3. Do all HPV types use the same molecular entry pathway?4. Do different HPV types interact with different host factors duringdisease persistence and progression?5. Is the mechanism of virion entry different for cutaneous versus mucosalepithelium?6. What are the molecular, genetic and immunological criteria that definetissue-tropism of PVs?Papillomaviruses are predominantly epitheliotropic. Only a small number of PV typesconfined to the deltapapillomavirus group induce fibropapillomas in which bothepithelial and fibroblast cells are infected in vitroMolecular events of entry have been studied extensively in cell culture systems(reviewed in 36170171A series of different host proteins are involved in the various early stages ofbinding and entry. These include surface heparin-sulfate proteoglycans (e.g.syndecans) 35170182in situ onto skin and mucosal sites. These studiesinclude mouse cervicovaginal 42Several animal preclinical models have been used to assess some features of viralentry Do we have sufficient knowledge about the complex interplay between immunemechanisms of escape, viral carcinogenesis, host innate sensors, transcriptionalregulation and codon usage profiles to determine the species and tissue tropismof papillomaviruses?In summing up, the PV research community has built an impressive body of knowledge onthe biological, clinical and pathological activities of papillomaviruses.Significant challenges remain in the arena of improved treatments for persistent andlatent HPV infections and associated cancers. Preclinical PV models will continue toprovide opportunities for more mechanistic studies where the virus and host can bemanipulated both genetically and pharmacologically to tease apart contributions ofthe various components of the virus life cycle. Already, these viruses have enrichedour knowledge of viral evolution, tumor suppressor function, host restrictionfactors, innate immunity, codon usage profiles, viral vaccines, immunotherapy, DNAdamage response mechanisms, keratinocyte biology and viral tissue tropism. Futurestudies will allow us to assess the impact of the current prophylactic vaccines on aselect set of HPV types and the selection pressures these vaccines impose on viralevolution. Papillomaviruses have provided a resource for a variety of expertisespanning the fields of virology, immunology, pathology, gynecology, evolutionarybiology, vaccine manufacture, carcinogenesis and molecular virology. Questionsremaining will continue to challenge future researchers as we continue to studythese fascinating viruses.


The role of HPV type distribution on the disparity of cervical cancer (CC) incidence between human populations remains unknown. The incidence of CC in the Amazonas State of Venezuela is higher than the national average. In this study, we determined the diversity of known HPV types  in Amerindian and mestizo women living in the Venezuelan Amazonas State. Understanding the ecological diversity of HPV in populations undergoing lifestyle transformations has important implication on public health measures for cervical cancer prevention. Human papillomavirus (HPV), an etiological agent of cervical cancer (CC), has infected humans since ancient times. Amerindians are the furthest migrants out of Africa, and they reached the Americas more than 14,000 years ago. Some groups still remain isolated, and some migrate to towns, forming a gradient spanning urbanization. We hypothesized that, by virtue of their history, lifestyle, and isolation from the global society, remote Amerindian women have lower HPV diversity than do urban women (Amerindian or mestizo). Here we determined the diversity of the 25 most relevant cervical HPV types in 82 Amerindians spanning urbanization , and in 29 urban mestizos from the town. Cervical, anal, oral, and introitus samples were taken, and HPVs were typed using reverse DNA hybridization. A total of 23 HPV types were detected, including 11 oncogenic or high-risk types, most associated with CC. Cervical HPV prevalence was 75%, with no differences by group, but Amerindians from low and medium urbanization level had significantly lower HPV diversity than mestizos did. In Amerindians, but not in mestizos, infections by only high-risk HPVs were higher than coinfections or by exclusively low-risk HPVs. Cervical abnormalities only were observed in Amerindians (9/82), consistent with their high HPV infection. The lower cervical HPV diversity in more isolated Amerindians is consistent with their lower exposure to the global pool, and transculturation to urban lifestyles could have implications on HPV ecology, infection, and virulence.IMPORTANCE The role of HPV type distribution on the disparity of cervical cancer (CC) incidence between human populations remains unknown. The incidence of CC in the Amazonas State of Venezuela is higher than the national average. In this study, we determined the diversity of known HPV types  in Amerindian and mestizo women living in the Venezuelan Amazonas State. Understanding the ecological diversity of HPV in populations undergoing lifestyle transformations has important implication on public health measures for CC prevention. The typnih.gov) .Cervical cancer is one of the five deadliest types of cancer among women. As high as 80% of CC cases occur in developing countries , 10, witN = 238 and N = 409, respectively)  , with sectively) . Among Vctively) . One of ctively) .The evolution of HPV diversity is not well-known, but HPV has infected humans since times that preceded the human migrations out of Africa , 17. Amehttps://doi.org/10.6084/m9.figshare.5579299.v1). Principal-component analysis (PCA) showed better segregation of subject-based groups .We determined the prevalence and diversity of HPV types in 111 sexually active women in the northern part of the Venezuelan Amazonas State in the Orinoco River basin . The stu10.1128/mSphere.00176-18.1FIG S1 95%) ellipses, while low urbanization Amerindians cluster apart from mestizo women (ID refers to Identification Document possession). Subject-based medium urbanization and high urbanization Amerindians groups showed less overlap. The area of each ellipse represents each group distribution with the CI95%. (d and e) Urban index boxplots for community-based groups (d) and subject-based groups (e). Mean urban index comparison indicated significant increase from low to high urbanization Amerindian groups and with mestizos being the highest for both classification approaches . Different letters over the boxplots indicate significant differences. P values shown were still significant after Holm correction. (f) Distribution of women by their subject-based urban group (color legend), in each of the community-based urban groups (x axis). The dominant color in each community-based group was concordant with the subject-based urban group, the discordant cases showed heterogeneity within communities reflecting an urbanization transition that occurs at the subject level. (g and h) Linear regression of community versus subject urbanization indices including only Amerindians (g) and for all subjects (66 Amerindians and 24 mestizos) (h). A positive correlation was observed for both cases . Download FIG S1, PDF file, 0.9 MB.Urban score assignment based on community or subject attributes. (a) Principal-component analysis (PCA) depicting villages based on urban scores. Five villages with low urbanization scores (green), one village with an intermediate score (orange), and two villages with high scores (blue) are shown. Principal component 1 (PC1) separated by urbanization indicator , in a gradient from low to high urban level. Urban attribute vectors show directions of community location in the space, with high urban communities placed in the direction of the vectors. The length of the arrow is proportional to the contribution of each urbanization indicator explaining the spatial distribution of women. (b and c) PCA depicting individual urban scores colored by urban groups based on their village (community-based groups) (b) or subject-based groups (c). Community-based medium urbanization and high urbanization groups overlapped their 95% confidence interval , 77% of the women in the study had never had a cytological screening before. There were no age differences by urbanization level , use of hormonal contraceptives , or lifetime sexual partners , and the differences in the frequency of single or multiple HPV infections were not significant between groups  and did not differ between urban groups (10.1128/mSphere.00176-18.2FIG S2 P = 0.540 by χ2 test) or between Amerindians from the high urbanization group and mestizos (P = 0.570 by χ2 test). Unlike mestizos, Amerindian women showed higher prevalence of having only high-risk HPV types in relation to low-risk HPV types or both types . Dots represent prevalence, and bars are 95% confidence interval (CI95%). (c) Sample size-based Shannon diversity of cervical HPV by urban groups on an extrapolated 32 women. Amerindians for low and high urbanization groups were significantly less diverse than the mestizo group. There was a nonsignificant tendency of increasing HPV diversity with urbanization . The interpolation (solid line) curve fraction corresponds to the HPV diversity of the actual number of women sampled. The extrapolation fraction (dashed lines) corresponds to the estimated diversity. Curved shaded areas represent the CI95% estimated from the bootstrap (50 replications). Different letters indicate significant differences, reached when CI95% do not overlap. (d) Beta diversity analysis by urban groups. Median distance to centroid, using Sorensen dissimilarity index. No significant difference in variability between or within groups was observed (P > 0.05 by PERMANOVA and permutation test for homogeneity of multivariate dispersions). (e) Heat map of the prevalence of cervical HPV types. HPV18 and HPV39 of the α7 family show the highest relative proportions. HPV L1 region sequences were used to generate a maximum likelihood tree rooted with theta HPV type (not shown). HPV families and their relative proportions  are shown on the right. HPV68 and HPV73 were excluded from the tree, since the LiPA25 kit does not discriminate between these two types. Download FIG S2, PDF file, 0.8 MB.Prevalence and diversity of cervical HPV by community-based urban groups. (a) HPV general prevalence. (b) Risk type prevalence. No general differences were found in prevalence by Amerindian groups  at a rarefied/extrapolated sample size of 32 women or 83 women for all populations (gamma diversity) or at a coverage-based level of 0.937 and 0.977, respectively. Shannon and Simpson metrics show mestizo women with a significantly higher HPV diversity than low urbanization Amerindians. Mestizos had a significantly higher Simpson index than any other Amerindian group in the asymptotic estimation. There was a nonsignificant tendency of increasing diversity with urbanization in Amerindians. Download TABLE S3, PDF file, 0.1 MB.Cervical HPV alpha and gamma diversities among HPV-positive women by community-based urbanization groups. Sample size-based, coverage-based, and asymptotic diversity analyses were performed for observed richness and Shannon and Simpson diversity .A total of 23 HPV types were detected, of which 22 were from the cervix . Alpha d10.1128/mSphere.00176-18.7TABLE S4 q = 1, 2, and 3, respectively). Coverage-based analyses for urban groups and mestizos were performed at a coverage level of 0.906, while for the total population, the coverage level was 0.971. Mestizos showed significant higher diversity than Amerindian low urbanization group. At the asymptotic estimation, mestizos showed a significant higher HPV diversity than the Amerindian low urbanization group. There was a nonsignificant tendency of increasing diversity with urbanization in Amerindians. Download TABLE S4, PDF file, 0.1 MB.Cervical HPV alpha, beta, and gamma diversities among positive women by subject-based urbanization groups. Coverage-based and asymptotic diversity analyses were performed for observed richness and Shannon and Simpson diversity  at a rarefied/extrapolated sample size level of 12 women or at a coverage level of 0.854. The cervix had the highest observed richness. Beta diversity with Sorensen dissimilarity index did differ significantly between groups. Download TABLE S5, PDF file, 0.1 MB.HPV prevalence and alpha and beta diversity by body site in 18 women, of which 16 are HPV infected in at least one body site. Sample-size-based, coverage-based, and asymptotic diversity analyses were performed for observed richness and Shannon and Simpson diversity . The presence of cervical lesions in these women did not significantly change HPV diversity.Nine women presented cervical abnormalities, and they were all Amerindians with mostly high-risk HPV infections . There Understanding the causes underlying the high incidence of CC in Amerindians is of crucial importance for decisions in public health interventions. While the same virulent types circulate among Amerindian and mestizo women, Amerindians showed higher prevalence of infections by the virulent types than infection by low-risk types or both. Amerindians in this study did show high HPV18 and HPV16, common virulent types in other human groups, but they also had high prevalence of a rare high-risk HPV type of the α7 family, HPV39, consistent with reports for Amerindians in the northern United States . Its preThat cervical abnormalities were found only in Amerindians, consistent with the epidemiological evidence of high CC incidence in this human group , suggestThis study included young adult, nonpregnant, healthy women from the Venezuelan Amazon. The women were from the following two groups: Piaroa Amerindian from villages in a spectrum of urbanization  or urban mestizo. All experimental protocols were approved by SA Centro Amazónico de Investigación y Control de Enfermedades Tropicales Simón, Bolívar, Venezuela , and University of Puerto Rico (IRB 1314-163).n </it>= 117) were mostly due to recent exposure to antibiotics or antiparasitic drugs (28%), menses (25%), postmenopausal (13%), pregnant (12%), urinary infections (8%), refusing to participate (4%), sexual contact in the last 24 h (3%), hysterectomy (2%), belonging to a different ethnicity (1%), diabetes (1%), and HIV (1%).Women included in the study belonged to eight different villages in northern Amazonas State, Venezuela: one urban town, Puerto Ayacucho , one village in the periurban area, and six villages at the Orinoco Basin on the Sipapo River, Autana River, and Cuao River . A totalhttps://doi.org/10.6084/m9.figshare.5579299.v1). Subject-based surveys included education, identification document (ID) possession, purchasing power, preservation of traditional practices, frequency of mobility to urbanized towns, level of environmental exposure , use and acceptance of Western medicine, and level of adoption of nontraditional diets (see data posted at https://doi.org/10.6084/m9.figshare.5579299.v1). Community-based urbanization survey included access to health, urban services , political representation, education, salaries, and language command (Spanish-Piaroa). This village survey was completed with the community captains, schoolteachers, or health workers (see data posted at https://doi.org/10.6084/m9.figshare.5579299.v1).Each woman received two urbanization indices, one based on her individual exposure to urban practices (subject-based index) and another on her community urban level (community-based index) (see data posted at n = 24 women), medium , and high . Subject-based urbanization groups were built first, sorting in ascending order individual women scores and then grouping them in tertiles: the first group corresponds to the low urban group , the second group corresponds to the medium urban group , and the third group corresponds to the high urban group . Mestizo women had a high urban level by both classification approaches  (scores of 0.70 to 0.93 for subject-based groups).Categorical variables of the urbanization surveys were transformed into numeric values ranked between 0 and 1, with 1 being the highest level of urbanization . Each indicator component was equally weighted, and its values were averaged using arithmetic means. Community-based groups included 111 women, but subject-based groups included only 91 women due to missing data in the surveys. Urban groups had similar sample sizes (https://doi.org/10.6084/m9.figshare.5579299.v1). Surveys were coded without personal identifiers.Clinical history, sexual behavior, contraceptive usage, and hygiene practices were also recorded in a separate clinical survey (see the data posted at N = 111), introitus (N = 18), anal (N = 18), and oral (N = 18) sites. DNA was extracted using Power Soil DNA kit (Mo Bio Laboratories Inc.) according to the manufacturer’s instructions. The main concern about HPV detection methods is obtaining false-negative results, usually after not being able to extract/detect viral DNA in an HPV-positive sample. The Power Soil method involves an aggressive bead beating step and allowing good extraction of the viral DNA. Cervical smears were performed by an obstetrician-gynecologist using an endocervical brush and spatula, and biopsy specimens were taken and treatment was provided if indicated. Papanicolaou’s stain was performed for the cytological analysis. Results were reported according to Bethesda 2001 classification system. A drop of blood was taken from fingers for in situ hemoglobin  to detect anemia according to the WHO limits    , was use primers , 30, repggfortify package  for the villages and for women based on their urbanization indicator values were performed with the  package  in R  with Yates’ continuity correction if needed or Fisher exact test for count data. P values were adjusted for multiple comparisons by the Holm method  counts all HPV types present in each group, Hill number of order 1 (q = 1) can be interpreted as the number of common HPV types per group, and Hill number of order 2 (q = 2) can be interpreted as the number of dominant HPV types. Alpha diversities were compared using the nonasymptotic and asymptotic analysis for incidence type data. For the nonasymptotic analysis, we compared groups at the same sample size (sample size based) or at the same level of sampling coverage (sample coverage based). The latter measures the proportion of the total number of individuals that belong to the HPV type detected in the sample and has been shown to better evaluate the magnitude of the diversity differences among groups than the traditional sample size-based comparison  comparison was performed with the permutation test for homogeneity of multivariate dispersions (betadisper function was used to reduce the distances to the principal coordinate. The method computes the F statistic to compare median distances-to-centroids of each group. P value was generated with the permutest function based on 999 permutations. The plot function was used for the principal-coordinate analysis visualization (Beta diversity was analyzed with the vegan  package vegan . Beta divegan  to compapersions . The betlization  and S4.reshape2 (ggplot2 (R 3.3.2 version functions (Statistics and graphics were also performed using reshape2 , ggplot2(ggplot2  and defaunctions . The mapunctions .https://doi.org/10.6084/m9.figshare.5579299.v1.Three data sets containing urbanization survey results and metadata and other data have been deposited in figshare at


Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression. All HPVs complete their life cycle in stratified squamous epithelium, either cutaneous or mucosal dependent on their tropism (reviewed in [Human papillomaviruses (HPVs) are small, non-envelope, double stranded DNA viruses, and there are more than 200 papillomavirus types identified to date (see curated list at Papillomavirus Episteme (PaVE); iewed in ,2). The iewed in . There iiewed in ,5. WhethE) viral genes are expressed. E1 and E2 support HPV DNA replication and the measured expression of E6 and E7 [In the basal layer, the HPV genome escapes its viral capsid and is maintained at 50–60 copies per cells (ranging from 10 to 200 copies) [6 and E7 ,9. EvadiL) gene expression is activated. L1 and L2 form the protein shell of HPV, and they incorporate the HPV episomal DNA into new infectious virions. Those released as epithelial cells are sloughed off at the top of stratified squamous epithelium, completing the viral life cycle.As cells leave the basal layer, they rise through the suprabasal and spinous layers and progress through differentiation. HPV requires this cellular differentiation program to complete its own viral life cycle. Without it, HPV has an abortive infection. Therefore, as HPV-infected host cells rise through the differentiating layers, HPV progresses through its own life cycle. In these differentiating layers, HPV DNA becomes amplified to several thousand copies per cell , and latE6 and E7 genes. E6 and E7 drive cells to continue to grow and divide when they otherwise would not, and, to that end, E6 and E7 are expressed in the differentiating layers of stratified squamous epithelium [Although HPV requires its host cell to differentiate in order to complete its life cycle, it also requires it host cell to continue to grow when normally it would not. HPV dysregulates the balance of growth and differentiation found in stratified squamous epithelium to do so. In low-risk HPV types, this is manifest as warts. In high-risk HPV types, this leads to dysplasias and carcinoma in situ . This dyithelium ,10. By EE6 and E7 genes, thus are considered to be HPV’s viral oncogenes. If HR E6 and E7 are introduced into normal diploid cells, they become immortalized [E6 and E7 genes required for oncogenesis, but they are also required for the maintenance of malignant phenotype. There are critical oncogenic pathways that HR E6 and E7 affect. HR E7 targets the retinoblastoma protein (Rb) for degradation in epithelial cells [PSD-Dlg-ZO-1/2 (PDZ) containing proteins for degradation, disrupting cellular apicobasal orientation and cell-to-cell adhesion in the epithelium, leading to hyperplasia [There are at least 15 HPV types that are defined as high-risk (HR) by their association with cervical cancer . HPV-assrtalized ,13. If Hrtalized ,15. Thisal cells ,17. Thisal cells ,19. It serplasia ,22,23,24erplasia ,13.In this review article, we will describe the roles E6 and E7 have in telomerase induction during HPV infection and in oncogenesis. We will first define telomeric DNA, its role in DNA protection, and the enzymatic function of telomerase. Then, we will highlight the multiple ways HR E6 and E7 derepress hTERT to activate and accelerate telomerase activity. Finally, we will discuss how E6, E7, and hTERT expression changes during oncogenesis.Telomeric DNA caps the ends of linear chromosomes, is repetitive, and is approximately 5000 to 15,000 nucleotides in length in humans ,26. No gWithout telomerase activity, the linear chromosomes of cellular DNA are serially shortened with every cell cycle and division by 100 to 200 nucleotides . This DNStudies in the late 1980s defined the roles HR E6 and E7 played in cellular immortalization, cancer development, and cancer progression ,13. In aRecent studies have confirmed that low-risk (LR) E6 does not activate telomerase  while HRThe E3 Ubiquitin Ligase E6 Associated Protein (E6AP) is important for the activation of hTERT expression and telomerase activity by HR E6 ,54,55. EhTERT gene is constitutively repressed in somatic epithelial cells; this repression occurs at its promoter. The hTERT promoter is approximately 1100 nucleotides in length, with its core promoter being only 200 to 300 nucleotides long [cis elements in its core promoter, blocking transcription [cis elements are E boxes, GC-rich sites, and X boxes.Most research on telomerase regulation has focused on the expression of its catalytic subunit, hTERT. The des long ,61,62. Ncription ,67,68,69cis elements flank the transcriptional start site of hTERT [cis E boxes within the hTERT promoter are required for its transcriptional activation with or without HR E6.Two E box of hTERT ,62,66, aof hTERT ,54. Thesof hTERT ,71,72. Uof hTERT ,73,74 Alof hTERT ,72,75. Eof hTERT ,54,74,75of hTERT . The reqof hTERT ,76,77, bcis elements in the hTERT promoter 5′ of the transcriptional start site [cis elements leads to loss of hTERT promoter-driven transcriptional activation [There are five GC-rich art site ,71,74. Sart site ,71. Maz tivation .Finally, there are two X boxes in the hTERT promoter . One is cis elements and the transcriptional proteins that bind those elements, epigenetic studies of the hTERT promoter demonstrate important structural chromatin changes that affect transcriptional activation of hTERT [cis element but also binds SIN3 transcription regulator family member A (mSin3A), a transcriptional co-repressor that recruits HDACs to promoters [Beyond studies of the hTERT promoter of hTERT . Severalof hTERT ,78. The romoters .DNA methylation patterns at the hTERT promoter also shift during an HPV infection and in tissue culture studies of HPV positive cells. Specific regions of the promoter become hypermethylated, while other regions become hypomethylated, during long-term tissue culture of cells with HR E6 and E7 ,80,81,82Post-transcriptional regulation of hTERT by alternative mRNA splicing and mRNA stabilization is important for telomerase activity ,85,86. INFX1 gene  [We found that hTERT and telomerase activity are upregulated post-transcriptionally by HR E6 through the host cellular protein NFX1-123 . NFX1-12variant) ,89. Greavariant) ,89. The variant) .NFX1-123 contains two protein motifs important for binding, stabilizing, and augmenting hTERT expression. The R3H domain of NFX1-123 has putative single-stranded nucleic acid binding capabilities ,89,90, aCytoplasmic poly(A) binding proteins themselves are important in hTERT expression and telomerase activity in HR E6 positive cells. When PABPC types 1 and 4 are knocked down, hTERT and telomerase activity are reduced . ConversCollectively, these research findings highlight multiple ways hTERT mRNA is post-transcriptionally regulated. Again, the duplicative mechanisms, from DNA, chromatin, and RNA regulation, that HR E6 uses to increase hTERT and telomerase emphasizes its importance to HPV and oncogenesis.E6 genes drive hTERT expression and telomerase activity.Most studies examining the regulation of telomerase by HPV have focused on HR HPVs from the α genus. More recent work has examined the role β genus HPVs play in nonmelanomatous squamous cell carcinoma, and how the beta E6 and E7 proteins may also activate oncogenic pathways, whether similar or disparate to α HR E6 and E7 proteins. Work by Galloway et al. has determined the oncogenic potential of β HPV types through direct analysis of their E6 and E7 protein functionality, and specifically how different E6 types activate hTERT expression, telomerase activity, and immortalization ,93. β E6During cervical cancer initiation and progression, the expression of hTERT and the activity of telomerase parallels worsening disease ,95,96,97E1 and E2 genes are often lost. Even in non-integrated HPV driven cancers, the binding sites for E2 often become methylated. These changes allow for greater expression of E6 and E7, as E2 moderates the expression level of these viral oncogenes [Interestingly, during the transition from HR HPV infection, to dysplasia, to frank cancer, HPV DNA typically no longer remains episomal. It becomes integrated in the cell’s chromosomes. This happens within the context of genomic instability, created and supported by the functions of HR E6 and E7 themselves. This, by definition, means HPV can no longer form infectious virions; it also means HPV gene expression itself is dysregulated. With HPV DNA integration, the HR E6 and E7 genes are universally preserved, but the regulatory ncogenes ,101. Altncogenes . During High-risk HPV infections are associated with cancers in other sites besides the cervix . These icis and trans elements, the chromatin structure, the mRNA product, and associated RNA regulatory proteins. There are still many unanswered questions in the dysregulation of telomerase activation by HPV during infection and oncogenesis. However, its universality implies it is critical to the core function of HR HPV types and to induction of cancers caused by HPV.High-risk E6 hijacks host cell proteins from their usual function  to activate hTERT and telomerase activity. This supports cellular immortalization. These viral-cellular protein partnerships primarily control the derepression of telomerase’s catalytic subunit, hTERT. They increase hTERT through the promoter’s


Cervical squamous cell carcinoma (SCC) continues to be a significant cause of cancer morbidity and is the third leading cause of cancer-related death in women worldwide. In sub-Saharan Africa, cervical cancer is not only the most common female cancer but also the leading cause of cancer-related deaths in women. Malawi, in particular, has the highest burden of cervical cancer. With the increasing use of human papillomavirus (HPV) vaccination, documenting the prevalent HPV types in those high-risk populations is necessary to both manage expectations of HPV vaccination and guide future vaccine development.In this study, we performed HPV typing on 474 cervical SCC samples and analyzed the potential impact of HPV vaccination in this population.Ninety-seven percent of tumors were positive for at least one HPV type, and 54% harbored more than one HPV type. HPV 16 was the most common type (82%), followed by HPV 18 (34%), HPV 35 (24%), and HPV 31 (12%). A vaccine against HPV 16 and 18 would ideally prevent 53% of cervical SCC, and the nonavalent HPV vaccine  would prevent 71% of cervical SCC in Malawi (assuming 100% vaccine efficacy). The main reason for a lack of coverage was high prevalence of HPV 35, which was also present as a single infection in a small subset of patients.Although any HPV vaccination in this population would likely prevent a significant proportion of cervical cancer, the nonavalent vaccine would provide better coverage. Furthermore, investigation of the role of HPV 35 in this population, including possible cross-protection with other HPV types, should be pursued. With the advent of prophylactic vaccination for specific HPVs , Gardasil , and Gardasil-9 , the HPV type-specific distribution in target populations becomes particularly germane to both expectations from large-scale vaccination and anticipated clinical burden. Bivalent human papillomavirus vaccine and recombinant human papillomavirus cover HPV 16 and 18 , and 9-valent human papillomavirus vaccine covers the low-risk HPV 6 and 11, in addition to seven high-risk HPVs .15 Although HPV 16 and 18 are the most common genotypes worldwide, including in SSA,17 studies have suggested that some HPV types that are relatively uncommon in Western countries  might have a much higher prevalence in resource-poor settings, especially SSA.24 Furthermore, an immunocompromised state, most notably, HIV infection, may also affect the number and distribution of HPV genotypes in a given population31 and confound the estimations of influence exerted by each HPV type in a mixed infection.Cervical squamous cell carcinoma (SCC) continues to be a significant cause of cancer morbidity and is the third leading cause of cancer-related death in women worldwideThe goals of this study were to determine the number and genotypes of HPV present in cervical SCC specimens from Malawian women, and determine the relative percentage of the cervical cancer population predicted to benefit from HPV 16 and 18 vaccination versus the more recent nonavalent vaccine .This study was performed with approval from the institutional review boards at the Malawi College of Medicine and Brigham and Women's Hospital. Patients were selected by reviewing pathology reports from the Malawi College of Medicine in Blantyre, Malawi, to identify invasive SCCs of the uterine cervix. Approximately 500 patients with cervical SCC diagnosed from 2009 to 2012 with available tumor blocks were retrieved from Blantyre. When the age and HIV status of the patient was provided in the pathology report, this information was recorded. Stage, clinical outcome, and survival data were not available.32A hematoxylin and eosin–stained slide was made from each retrieved block, and all slides were reviewed (B.E.H.) to confirm the diagnosis of invasive SCC and adequacy of the block for DNA extraction. Tumors were evaluated for grade  and whether they demonstrated keratinizing and/or basaloid features.Genomic DNA was extracted from 474 samples of formalin-fixed, paraffin-embedded tumor tissue using the NucleoSpin DNA FFPE XS kit  according to the manufacturer’s instructions.a,b, 70, 73, and 82) using the type-specific E7 polymerase chain reaction bead-based multiplex assay .34 This ultrasensitive HPV genotyping assay, allowing detection of fewer than 10 copies of the viral genome, was previously validated by a WHO LabNet proficiency panel.35 Two beta-globin primers were included to control for DNA quality. The 21 HPV genotypes included all of those previously reported in cervical cancers at a frequency of 1% or greater.36All samples were interrogated for 21 HPV genotypes  and the median age of patients per year  were similar.Patient age was reported for 395 patients (80%), with a median age of 45 years . Of the 15 patients for whom HIV status was documented on the pathology requisition form, all were reportedly positive for HIV; however, insufficient data were available to understand the complete role/relationship of HIV and cervical cancer in this population. On the basis of the prevalence of HIV in Malawi,SCC was confirmed in 474 patients. Eighteen samples (3.8%) were well differentiated, 250 (53.2%) were moderately differentiated, and 202 (43%) were poorly differentiated. In four samples, the tissue preservation was poor, precluding this evaluation. There was no significant association between age and tumor grade.The amplification of the beta-globin gene showed that good-quality DNA could be obtained for 420 (89%) of the 474 paraffin-embedded tissue samples. The 54 samples (11%) negative for the beta-globin test were thus excluded from the remaining analyses. Of the 420 samples, 407 (97%) were positive for at least one hrHPV type . Either In considering relative efficacy of the various available vaccines, for this limited analysis, we assumed 100% protection for the hrHPV types included in the available vaccines and no cross-protection between different HPV types. Of the HPV-positive samples (n = 407), 53% had only HPV 16 and/or HPV 18 genotypes detected, suggesting that these cancers would be preventable with vaccination specifically for HPV 16 and 18 only. However, 47% of samples in this cohort harbored other high-risk HPV types in addition to or instead of HPV 16 and 18 , suggest41 In Malawi, cervical cancer is the most common malignancy in women, representing 42% to 45% of all female cancers; survival from cervical cancer is dismal, with a median survival of 9 months and 1-year, 2-year, 3-year, and ≥ 4-year survival of 31.0%, 10.3%, 5.2%, and 2.9%, respectively.37 If given the opportunity, there was unanimous agreement among Malawian women on choosing to vaccinate their children against HPV, suggesting that HPV vaccination would be well received in this population.42There is an abundance of literature on HPV genotyping, which has revealed striking geographic differences in HPV type-specific prevalence among continents, as well as among countries within the same continent. Although infection with only one hrHPV genotype is common in the United States, in some SSA countries, nearly 70% of cervical cancers contain multiple hrHPV genotypes.43 Data on the rates of HPV 35 positivity in cervical cancers and precursors are limited; however, in most reported countries, the rate is less than 6%.48 Some have proposed that HPV 35 may have a lesser oncogenic potential compared with HPV 16 and 18.49 Interestingly, our results align closely with a recent study evaluating HPV types present in HIV-positive Malawian women (not limited to cancer). Although the number of cancers in that study was small , the researchers found that 33% of clinically abnormal cervices harbored HPV 35, but this HPV genotype was detected in only 6% of visually normal cervices,50 suggesting that HPV 35 is indeed carcinogenic but may be more important in the setting of HIV positivity. Indeed, 40% of HIV-positive women from Burkina Faso and Botswana tested positive for HPV 35.24 In HPV genotypes in cervical cancer have not been previously evaluated in Malawi, and with the possible implementation of HPV vaccination, we thought it would be important to document the baseline HPV genotype prevalence in cervical cancers in this population, particularly with respect to the predicted benefit that a vaccine covering just the high-risk HPV 16 and HPV 18 might confer compared with the newer and broader 9-valent human papillomavirus nonavalent vaccine that covers seven hrHPVs, including HPV 16 and HPV 18. Our study has shown that although HPV 16 and HPV 18 are the most common types in Malawi cervical SCC, multiple coinfections are common, and only 53% of the population could be protected by the administration of recombinant human papillomavirus or bivalent human papillomavirus . Many researchers who have undertaken similar studies or meta-analyses evaluating the potential efficacy of the available vaccines did not take into account HPV 35.39 It would have been interesting to know whether those cancers in HIV-positive women were more likely to be associated with multiple hrHPV types, as has been demonstrated in studies in Zambia, Botswana, and South Africa, which showed a median of two to four hrHPVs present in HIV-positive women.51 Similarly, we were unable to assess whether specific HPV genotypes, particularly HPV 35, were associated with HIV infection. Additionally, this study was unable to assess the prevalence of HPV genotypes in women with normal cervical examinations or only precursor lesions; therefore, we were unable to determine a weighted attribution of individual HPV types to the development of invasive cancer.In this study, we were limited by our lack of knowledge of HIV status in the majority of patients; however, on the basis of the literature, at least 50% of Malawian women with cervical cancer may be estimated to be HIV positive.52; however, this effect may wane with time, because the strongest cross-protective effect was demonstrated in trials with the shortest follow-up time,53 and it was relatively less efficacious in those studies with longer follow-up periods.54 Although there is limited (but conflicting) evidence for some cross-protection against HPV 33, 45, 52, and 58 from the HPV 16 and 18 vaccines, notably, there has been no reported efficacy against HPV 35,55 although serum antibodies to HPV 35 have been documented after HPV vaccination.56 The precise impact of HPV 35 is unclear because it is present as a single infection in only a fraction of patients. Importantly, in the context of HIV positivity, studies have shown that immunogenicity and cross-protective serologic responses have been documented against HPV 31, 33, and 45 in HIV-positive patients,60 suggesting that HPV vaccination should be effective in this subpopulation.Some data exist regarding potential cross-protection in HPV 16 and 18 vaccines with non-16 and 18 HPV types, with the strongest evidence for cross-protection for HPV 31 from both bivalent human papillomavirus vaccine and recombinant human papillomavirus61 Recently, groups have analyzed two-dose versus three-dose schedules for administration of bivalent human papillomavirus vaccine and recombinant human papillomavirus, and found that a two-dose schedule is more cost effective, unless the cost of the third dose is significantly decreased, and have even raised the possibility of exploring the efficacy of one-dose schedules. However, this analysis has not yet been performed on the new 9-valent human papillomavirus nonavalent vaccine.63 Another potential method in cost-effective prevention of cervical cancer, particularly in resource-poor countries, includes the recently reintroduced concept of prophylactic ablation of the cervical squamocolumnar junction.66 The squamocolumnar junctions are thought to be the cells initially infected by HPV and thus give rise to clinically significant precursor lesions and carcinoma; by ablating this region, the potential to develop lesions is drastically reduced, regardless of the HPV type present.69 However, the feasibility, safety, and efficacy of this approach have yet to be proven.In considering a more widespread use of HPV vaccination, particularly in resource-poor countries, cost effectiveness is important.In conclusion, this study is the first large study characterizing HPV genotypes present in invasive SCC of the cervix in Malawian women. Our study has shown that although any HPV vaccination will likely have a significant effect on the future incidence of invasive cervical cancer, if given the option, broader vaccine coverage might be more beneficial in this population. Furthermore, the high prevalence of HPV 35 suggests that a significant subset of cancers may not be prevented with any currently available HPV vaccine. This study highlights the importance of studying cross-protection against HPV 35 with the clinically available HPV vaccines. If additional future multivalent vaccines are to be designed, a careful evaluation of HPV genotypes present in invasive cancer in those countries harboring the highest burden of disease, and thus having the most to gain from a broader HPV vaccine, should be considered.


Gammapapillomavirus 19.A novel human papillomavirus (HPV TG550) isolated from the oral rinse of a Chinese male resident was fully characterized. The L1 open reading frame of HPV TG550 shares 82.5% nucleotide sequence similarity with its closest relative, HPV166, and clusters within the species group  Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, and Nupapillomavirus, within the family Papillomaviridae. The Alpha-PV genus contains all of the genital oncogenic HPV types associated with cancers (Human papillomaviruses (HPVs) are a heterogeneous group of circular double-stranded DNA viruses with a size around 8 kb and infect the epithelial surfaces of genital and cutaneous sites of humans. A distinct HPV type is defined when the complete L1 open reading frame (ORF) is less than 90% similar to those of the characterized types . Current cancers , 4.de novo assembled; a total of 11 valid contigs  covered nearly 80% of the complete genome. Type-specific primers were designed based on NGS contigs to amplify the complete genome in three overlapping fragments. The complete genome sequence was assembled and validated using a proofreading polymerase, followed by Sanger sequencing using a primer-walking strategy.In this study, we characterized the complete genome of a novel HPV type isolated from the oral rinse of a 22-year-old Chinese male resident without visible oral lesions. Viral DNA was initially detected using a multiplexed next-generation sequencing (NGS) assay targeting a 125-bp fragment within the L1 ORF , HPV162 (JX413108), and HPV166. The nucleotide sequence of a small region of the HPV TG550 L1 ORF shares 97 to 99% similarities with three previously reported partial sequences, SIBX58 , SE9 , and SE71 .This novel HPV genome (HPV TG550) is 7,216 bp in size, with a GC content of 37.4%. It contains five early genes  and two late genes (L1 and L2). The complete L1 ORF shows 82.5% nucleotide sequence similarity with its closest relative, HPV 166 , meeting the criteria to be considered a distinct HPV type. Phylogenetic trees using either the L1 ORF or the complete genome sequence unambiguously cluster HPV TG550 into the species group The identification of novel HPVs reveals high genomic diversity of papillomaviruses. Further studies on the epidemiology, evolution, and pathogenesis of this novel virus are warranted.MF176072.The complete genome sequence of HPV TG550 is available in GenBank under the accession number