Pseudois nayaur) plays a key ecological role in sensitive high‐altitude ecosystems of Central Asia and is among the main prey species for the globally vulnerable snow leopard (Panthera uncia). As the blue sheep has been increasingly exposed to human pressures, it is vital to estimate its population dynamics, protect the key populations, identify important habitats, and secure a balance between conservation and local livelihoods. We conducted a study in Manang, Annapurna Conservation Area , to survey blue sheep on 60 transects in spring (127.9 km) and 61 transects in autumn (134.7 km) of 2019, estimate their minimum densities from total counts, compare these densities with previous estimates, and assess blue sheep habitat selection by the application of generalized additive models (GAMs). Total counts yielded minimum density estimates of 6.0–7.7 and 6.9–7.8 individuals/km2 in spring and autumn, respectively, which are relatively high compared to other areas. Elevation and, to a lesser extent, land cover indicated by the normalized difference vegetation index (NDVI) strongly affected habitat selection by blue sheep, whereas the effects of anthropogenic variables were insignificant. Animals were found mainly in habitats associated with grasslands and shrublands at elevations between 4,200 and 4,700 m. We show that the blue sheep population size in Manang has been largely maintained over the past three decades, indicating the success of the integrated conservation and development efforts in this area. Considering a strong dependence of snow leopards on blue sheep, these findings give hope for the long‐term conservation of this big cat in Manang. We suggest that long‐term population monitoring and a better understanding of blue sheep–livestock interactions are crucial to maintain healthy populations of blue sheep and, as a consequence, of snow leopards.There is a growing agreement that conservation needs to be proactive and pay increased attention to common species and to the threats they face. The blue sheep ( Blue sheep play a key ecological role in high‐altitude ecosystems and represent the staple prey for the globally threatened snow leopard. We show that the Manang area, Annapurna Conservation Area, Nepal, still maintains a relatively high density of these ungulates. Elevation and land cover are the main local determinants of blue sheep habitats. Located in the rain shadow of the Annapurna Range, it is one of the driest areas in the Nepalese Himalaya, with the mean annual precipitation of ~400 mm and most precipitation falling as snow during winter , East Himalayan fir (Abies spectabilis), Himalayan birch (Betula utilis), and black juniper  and Himalayan tahr (Hemitragus jemlahicus) add to the ungulate community at lower elevations , golden jackal (Canis aureus), and red fox , small mustelids (Mustela spp. and Martes spp.), and several species of voles (Alticola spp.) and pikas , goats (Capra hircus), yaks (Bos grunniens), cattle (B. taurus), dzo (B. grunniens × B. taurus), mules , and horses  and in late summer/autumn . To identify potential study units, we placed a grid of 4×4 km cells over the study area. This cell size compromised daily movements and home ranges of blue sheep  and afternoon hours (2:00–6:00 p.m.) when blue sheep activity is high , livestock presence, distance to cliff, distance to stream, distance to settlement, and distance to trail. Elevation was obtained from a digital elevation model of 30‐m resolution , ground‐truthing, and interpretation of satellite images. Locations of settlements were provided by governmental authorities , and we amended this layer by adding long‐term herder camps and by removing abandoned settlements and individual huts. Trails commonly used by tourists and/or local people were derived from OpenStreetMap (https://download.geofabrik.de). These trail locations were checked and modified by ground‐truthing and based on expert judgment.In addition, we assessed livestock presence (1)/absence (0) including large  and small species (sheep and goat). We monitored and mapped livestock from transects using the same methodology as for blue sheep. We created 500‐m buffers around livestock locations to derive areas with livestock presence and absence during the survey. As blue sheep and livestock can graze together , we considered this quite short buffer distance as reasonable to affect blue sheep–livestock interactions  in the 1,500‐m viewshed around the transects. We then sampled from these points with the probabilities obtained from a density function of an exponential distribution parameterized with the observed distances of blue sheep to the transects . As recommended by Barbet‐Massin et al. . In contrast, these parameters were positively correlated in autumn . Therefore, we fitted a linear model where we explained the group size as a function of the intercept and the distance to the transect. The coefficients were 8.498 ± 1.893 (p < .001) for the intercept and 0.009 ± 0.003 (p = .012) for the distance to the transect.We randomly attributed observed group sizes to pseudo‐absence points in spring as we did not find a significant correlation between the group size and the distance to the transect in this season . We decided to retain the slope due to its better comparability across the studies . We retained the distance to settlement since most livestock is gathered around settlements in late winter/early spring and the locations of settlements were complete, yet some livestock could go undetected. Blue sheep observations in forested areas were omitted from GAMs as we assumed a significantly lower detection probability in this land‐cover type . GAMs have been increasingly used in habitat selection analyses  and 61 transects of a total length of 134.7 km in autumn (mean: 2.2 ± 0.2 km/transect). Altogether, we spotted 1,905 blue sheep (143 observations) during the fieldwork in spring and 2,058 blue sheep (146 observations) during the fieldwork in autumn. Thereof, 1,408 individuals (94 observations) were spotted along the transects in spring and 1,648 individuals (108 observations) in autumn. Blue sheep group size ranged from single animals to 86 individuals in spring and to 113 individuals in autumn. Mean group size was 14.4 ± 1.3 individuals in spring and 14.4 ± 1.5 individuals in autumn.2 and 6.9–7.8 individuals/km2 over the surveyed area (180–234 km2) in spring and autumn, respectively  than in Neshyang Valley . In both seasons, blue sheep used a gradient of slope declivity ranging from flat terrain to steep cliffs (>50°), but most animals were spotted in moderately rugged terrain to rather strong slopes . The majority of individuals was observed at 4,200–4,600 m a.s.l. in spring  and 4,300–4,700 m a.s.l. in autumn .We spotted blue sheep in various land‐cover types including grasslands, shrublands, agricultural lands, barren lands, snowfields, and open forests. In spring, the majority (77.5%) of animals used habitats having NDVI values between 0.25 and 0.5 and associated mainly with grasslands, shrublands, and agricultural lands  and autumn , respectively. Elevation and land cover were the only significant variables (p < .05) for habitat selection by blue sheep in both seasons  is similar to previously reported densities in the Manang area both before and after the official establishment of the Annapurna Conservation Area .The main purpose of this study was to estimate the minimum blue sheep density in Manang which would be comparable with minimum densities of this species in other areas and time periods. We applied total counts which, apart from producing such minimum density estimates, are also cost‐effective and reliable for the analysis of population trends in mountain ungulates  and woolly hare (Lepus oiostolus), in Manang adds to an increased dependence of snow leopards on blue sheep abundance  and buckwheat . Theoretically, this result could be affected by non‐detection of livestock if they grazed in secluded places or were released from their night sheds late. But we think that such events were rare and did not influence our main conclusions. Moreover, we assume that livestock densities had a stronger effect on blue sheep than livestock presence/absence. The consideration of livestock densities might have increased model performance, but we could not estimate this parameter from our current data. However, blue sheep–livestock interactions were negative in autumn at larger scales more than 1 km apart Table , thus in5This study in the Nepalese Himalaya demonstrated that quite high densities of blue sheep, a key prey species for the threatened snow leopard, have been maintained in an area in which conservation and development agendas have been combined. Moreover, we describe how elevation and land cover shape habitat selection by blue sheep in the absence of strong hunting pressure by humans, which is relevant for blue sheep management, habitat protection, and potential translocation programs. In light of the importance of blue sheep in high‐altitude ecosystems, we suggest to conduct more research on blue sheep–livestock interactions and to establish a standardized blue sheep monitoring program based on total counts and/or double‐observer counts for the benefit of blue sheep and snow leopards.The authors declare that there is no conflict of interest.MF: Conceptualization; formal analysis; funding acquisition; investigation; project administration; visualization; writing—original draft. RPL: Conceptualization; funding acquisition; investigation; project administration; writing—review and editing. TRG: Investigation; writing—review and editing. JS: Formal analysis; visualization; writing—review and editing. TF: Formal analysis; visualization; writing—review and editing. RRA: Formal analysis; writing—review and editing. MH: Conceptualization; funding acquisition; supervision; writing—review and editing. MW: Conceptualization; funding acquisition; supervision; writing—review and editing. NB: Conceptualization; supervision; writing—review and editing. IK: Conceptualization; formal analysis; funding acquisition; supervision; writing—review and editing.Figure S1. Normalized difference vegetation index (NDVI) of various land‐cover types in Manang based on 203 predefined validation points. The land‐cover types in these locations were distributed throughout the study area and verified by ground‐truthing or analysis of satellite imagery .Click here for additional data file.Figure S2. Sensitivity analysis with the inclusion of forested areas. Relative variable importance (%) in generalized additive models (GAMs) describing habitat selection by blue sheep in Manang based on direct observations in spring and autumn.Click here for additional data file.Figure S3. Sensitivity analysis with different sets of random points. Plots of generalized additive models (GAMs) describing habitat selection by blue sheep in Manang based on direct observations in spring. Shown are the smooth terms based on the model presented in the paper (black) in comparison with the models using two different sets of random points .Click here for additional data file.Figure S4. Sensitivity analysis with different sets of random points. Plots of generalized additive models (GAMs) describing habitat selection by blue sheep in Manang based on direct observations in autumn. Shown are the smooth terms based on the model presented in the paper (black) in comparison with the models using two different sets of random points .Click here for additional data file.Figure S5. Sensitivity analysis with the exclusion of locations near settlements (< 500 m). Plots of generalized additive models (GAMs) describing habitat selection by blue sheep in Manang based on direct observations in spring (a) and autumn (b). Shown are the smooth terms for the normalized difference vegetation index (NDVI).Click here for additional data file.Figure S6. Habitat use by blue sheep in Manang. Shown are the frequencies of distances of adult blue sheep to cliff (a), stream (b), settlement (c) and trail (d) based on direct observations in spring and autumn.Click here for additional data file.TableS1‐S5Click here for additional data file.


Hospital certification is an external assessment mechanism to assure quality and safety systems. Auditors representing the certification body play a key role in certification processes, as they perform the assessment activities and interact with the involved healthcare organizations. There is limited knowledge about the approaches and methods that auditors use, such as role repertoire, conduct, and assessment practice. The purpose of this study was to explore auditors’ practice in hospital certification processes, guided by the following research questions: What styles do auditors apply in hospital certification processes, and how do auditors perceive their role in hospital certification processes?The study was performed in two stages. In the first stage, non-participant observations (59 h) were conducted, to explore the professional practice of three lead auditors in certification processes of Norwegian hospitals. In the second stage, semi-structured interviews were conducted with these three observed lead auditors. The role repertoires and conducts identified were analyzed by using a deductive approach according to a surveyor  styles typology framework.Two distinct auditor styles (“explorer” and “discusser”) were identified among the three studied auditors. Both styles were characterized by their preference for an opportunistic and less structured type of interview practice during certification audits. All three auditors embedded a guiding approach  to their perception and practice of certification audits, interacting with the auditees. The use of group interviews instead of individual interviews during certification audits, was the rule of their professional practice.The auditors’ perceptions and styles demonstrated a multifaceted certification reality, in contrast to what is often presumed as consistent, stringent and independent practices. These findings may have implications for reliability judgements when developing hospital certification programs, and for the refinement of the current framework used here to study the different auditing practices. ISO  9001 Certification is used for external assessment of quality and safety systems in health care. It is often associated with accreditation and other external assessment programs for health care organizations . In thesConcerns have been raised related to arguments of scarce evidence of the benefits from external assessment programs, and the wide use and amount of resources that healthcare services allocate to these programs –14. TherDespite growing evidence of the effects from certification and accreditation in healthcare, little is known about practices and mechanisms involved in the interactional processes between assessment bodies and healthcare organizations in external assessments. In external assessments systems, such as ISO certification, the auditors’ experience, selection of auditors, training, support and motivation may influence the performance, style, and reliability of the auditing (assessment) practices , 24–26. The purpose of this study was to explore the audit practice as perceived and performed by auditors in one certification body involved in hospital certification processes in Norway. The following two research questions guided this study: (i) What styles do auditors apply in hospital certification processes, and (ii) how do auditors perceive their role in hospital certification processes?By exploring the ways in which auditors perceive and perform their role in three hospital certification processes this study contributes with valuable insights into the practices and approaches of a for-profit certification body that often can be challenging to get access to. The exploration reveals different role repertoires and professional conduct among the auditors from the certification body. We discuss how this influences further research and development of future certification processes, and highlight the implications for policy makers.Quality Management Standard – Requirements  [T] hey follow the book since they fulfill the minimum requirements, but should they, when they have those [the requirements] in place, consider whether they can get more out of that arena. (Auditor 1).The auditor cited dialogue in her auditor-auditee encounter as an important means of collecting information and for stimulating and guiding improvement. As part of that audit dialogue, she asked deeper questions to collect additional information:During the on-site assessment in the clinic for internal services, the auditor performed all her interviews with groups of staff from the different departments. The groups never had fewer than four people. The respondents were usually a manager or middle manager, the hospital’s contact person/quality manager, and one or two others “relevant personnel” (the term used in the agenda) from the departments under assessment. All interviews were held in the same room. The auditor used a template to highlight the main requirements in the ISO 9001 standard against which she assessed conformity, and to structure the progress during the interview. The auditor recorded what was said, either in her notebook or on the template sheet. A projector was used in most of the interviews to review and discuss documents and recordings, like policies, objectives, plans and procedures. The auditor usually started with an open-ended question, such as “Can you tell me about the management system?” She invited a conversation about the topic, or had the respondents explain documents on the screen. If the respondents mentioned something that caught her interest, she either asked for verification or a probing question. An example was observed when the auditor asked the auditee about the control of errors and non-conformities. A vague answer from the respondents led her to ask for more explicit documentation, and to see examples of how they correct errors and non-conformities. She directed most of her questions to all the respondents in the room, and was usually answered by the manager (often a middle manager) who was responsible for that topic. The others  followed up if the manager seemed to have left something out of the answer.When we are out, they want us to create added value for the customer, and help the customer to become better at managing risks in relation to their core areas. […] Moreover, in my decision-making process I will then review and ask, in a dialogue with the customer: “What do you see as challenging?”; “Where do you think the problems lie?”; That’s just as important …, that dialogue is very important. (Auditor 1).According to Auditor 1’s perception her main task was to bring added value to the organization. This was not just related to the general understanding of the expected added value from third-party certification , but also to organizational development as part of the certification process. Again, the auditor opened a dialogue.You largely control, but at the same time you’re also a guide. I do not think it is possible to see them isolated. So, you control in the sense that you can kind of check out if things are in place - yes or no. However, since improvement is as central as it is, then together with the customer, in that dialogue, it is to identify things where one in advantage might take small steps to raise oneself further ahead. That becomes the guidance role. I think they go like hand in glove. At the same time, it is important in that guidance role, that you do not give very clear recipes on how things should be done. (Auditor 1).The perception of improvement was emphasized when auditor 1 described her approach within the inspection-guidance axis. She perceived concern over the balance of not adopting a consultancy approach.The most important tool is to ask question, and then to be shown what you have requested. Maybe they do have it, maybe they don’t. And then use what comes to the table in the best possible manner. We may not always follow the preplanned agenda, because one sees that there are strengths in some areas, or weaknesses in other areas where you need to go in-depth. Because, that’s what gives results, getting to the core, and perhaps especially the way one asks questions, so that they themselves see it. It should be themselves who sees it- those are the best audits. When they get a wake-up-call that make them see that this is useful, and they see where they can get better. (Auditor 2).The next auditor stressed the importance of asking questions to elicit information that was needed for the audit. She also perceived her manner of asking questions as an explorative encounter, where the intention was to foster self-reflection.Moreover, it is often appropriate that they are not alone, but that there are two, three of them, so they can ask each other. […] If there are several findings of nonconformity they can feel unfortunate, and think that if someone else had answered maybe it would have been different. They try to represent the organization and they want to be good. That’s what we all want to. (Auditor 2).In her interview strategy, auditor 2 perceived it as important that the respondents were informed, knew the purpose of the session, and felt safe during the audit interview:During her three-day document review of the hospital’s quality management system, the auditor always performed the interview with at least two people from each department, often a manager and a staff member who was involved in quality development in the department. Observations showed that she used a template to guide the interview, but unlike the first auditor she sporadically recorded what she heard and observed. She used a projector during the interviews to review and discuss documents and recordings, like policies, objectives, plans and procedures. Like the first auditor she also often started with an open question when shifting to a new topic. A significant difference from the first auditor was that the second auditor often raised questions about solutions and self-reflection, especially when she observed potential improvements or non-conformities, like “What is a challenge here?” “How and where would a nurse look for this procedure?” If respondents asked for feedback or suggestions about their management systems, the response was often “What is useful for you?” These answers encouraged reflections instead of giving direct suggestions or advice, and also indicated the importance of quality systems as useful and not for the systems in itself. When interviewing the top management, the auditor seemed to base her arguments upon the logic of the quality management standard, as when she was interviewing the top management, represented by the managing director, the vice managing director and the management representative. The observations showed that vice managing director entered in a hurry and asked if it was necessary for him to participate. He agreed to “take part in the beginning” after getting a very short explanation of the objectives for the meeting. The auditor went through the organization’s quality policy, quality objectives, management review and the like in a dialogue-based approach with the respondents, often referring to, and explaining requirements in the ISO standard. The vice managing director turned out to be the most engaged in the whole meeting, and stated that the ISO 9001 management system approach visualized challenges that clearly had improvement potential.Then it’s about getting a review of the system. What we are talking about …; I can see that awareness, learning, and systematic improvement is helpful for them, because they use their own material to understand these aspects within a context. This is what I may experience is of greatest value, because the report [they receive from us] is short. The process that it entails is perhaps just as important for those involved, so that they over time constantly strive to understand more of the system, systematics, improvement opportunities, in their own organization. (Auditor 2).The auditor perceived verification to be her most important task, which entailed to assess if there was consistency between the systems organizations have and the practices they perform, and to look for improvement and what works well. Her focus on improvement and the certification process was also perceived as valuable from her perspective:Observations and interviews showed that improvement was a central theme of her audit practice. It was embedded in a focus upon motivational factors to bring about organizational improvement. She expressed respect for the limited possibility of her getting a total picture of the organization during a couple of days, and kept in mind that she was not there to tell the organization how things should be done or give advices, but to a great extent provide support for good practice and identify opportunities by giving examples from best practices elsewhere. She perceived that the control dimension was about 30% of her auditing practice. Auditor 2 stated that there might be some differences in the way she performed audits in hospitals than in other sectors, and suggested that underdeveloped management systems might have been a reason for her motivational approach rather than verification.[T] he way I’ve started now, because of what I have experienced in the health sector, where they have placed very much on the quality managers. So, when I put management processes on the [audit] agenda, […] I then ask if all the persons who participates in the management review [from the organization] can participate. Then, it is not just the head of department, or director of administration, or... So, when we assess the minutes from meetings and things like that, all participants should have a possibility to participate – and for me to be able to ask them about their expectations of the system and to get to know them, and so on. So, there’s a direct auditor role. But they have to think themselves to speak. They have to make their, their contributions then -- more active. (Auditor 3).When auditor 3 conducted her interviews, she wanted the participants to be engaged and to contribute to the discussions. Both revision and sharing of experiences were embedded in her dialogue strategy. She strategically used her interview to communicate requirements and meet key personnel in daily practice. As she expressed:Auditor 3 started her three-day recertification of an emergency department by claiming in the opening meeting that she preferred an audit that was based on open dialogue. She often repeated this at the start of every interview. She emphasized that she wanted no surprises in the closing meeting, and that everything should be clarified during the assessment. The audit was arranged so that all the interviews were held in locations where the personnel did their daily work. She said that she wanted to talk to people while they were closest to where they worked. In one audit interview, observations showed that the auditor spoke with only one respondent at some times, but in all others, there were at least two or more other people present. She usually began the interview with a short introduction about the intention for the assessment. She then asked for a description of how things were done in relation to the matter to be discussed. She encouraged the staff to talk about their system, and welcomed dialogue or conversation. One respondent thought she was supposed to be asked questions, and said she had not prepared for an open or “formal” presentation about how “we do things around here.” Auditor 3 often asked direct questions about an issue that had arisen during the conversation, and was then clearly steering the interview. She alternated direct questioning with dialogue. Her point of departure was based on documents or procedures that she had read beforehand in relation to the standard, but never used the standard openly during the interview. Sometimes she drew analogies to her professional background in the process industry, and discussed good practices that she had observed elsewhere. She did not record her interviews, but occasionally took notes when there seemed to be findings related to conformities or nonconformities.[…] because I’ve always kept that one should be objective and not give advice and so forth, but it’s a balancing act when you see that the customers are not completely familiar with the extent of the requirements in the standard. (Auditor 3).Auditor 3 perceived that the certification body expected her to add value when she performed her assessment practice. The practice of adding value seemed to be related to a guidance role. At first, auditor 3 seemed a bit reluctant to focus upon a guiding role or to give advice:When responding to the inspection-guidance axis she expressed an organizational reality that asked for reflexivity towards the maturity of the organization’s competence on what the ISO 9001 standard actually mean for their own management system. She stated that it is not enough “to sit in school and learn the standard” (Auditor 3), the organizations have to translate the content to their own management system as well. Auditor 3 cited examples of how the audits and certification processes drove development and change in hospitals, even though her formal role during certification was assessing the organization. For example, there were often many other control mechanisms that hospitals needed to conform to, and therefore the regular certification activities could become an arena for organizations to seek advice. The auditor perceived that sharing experiences from elsewhere was the best way to avoid giving advices and to balance the audit practice between inspection and guidance.Overall, our findings showed that all three auditors adopted an opportunistic questioning approach, but auditor 3 was more likely to turn to a more direct and closed type of questioning than the others. The first two auditors used a template to guide their interview related to the normative standard, but none used structured pre-planned questions. There were significant differences in how the auditors recorded the interview results. The first two auditors took detailed notes during the interview - the first more frequently than the second. This showed that they were oriented towards the written side of the recording dimension. The third auditor only jotted down a few words occasionally. She was more oriented to the opposite side of the recording dimension: implicit note taking. The two first auditors adopted the conduct of the explorer, while the third adopted the conduct of the discusser. All the auditors perceived both assessment of conformity to the ISO 9001 standard and guidance for improvement as embedded parts of certification audits. They were all concerned about not giving the certified organizations specific advises on how to improve but were familiar with transfer of experiences from elsewhere and giving general guidances. Auditor 2 and 3 were also more concerned with encouraging the certified organizations own reflections of their quality management system, in order to nurture improvement. In the next section, we will discuss this in further detail.In this paper, we explored auditors’ role in hospital certification. By ways of non-participant observation of audit interviews and qualitative interviews with the lead auditors, we have explored different auditor styles and how the auditors perceive their role in hospital certification processes in Norway. In the following we will discuss the findings according to the auditor typology framework .According to the normative standards, certification relies upon objectivity and consistency among auditors , 46. ConIn a study of stakeholders’ views and experiences on accreditation survey reliability  the authThe auditors in this study expressed concern about giving advice, since organizational follow-up on such advices may imply that auditors will audit their own solutions (advices) at a later stage. The normative standards describe consultancy work, like advice and education, as threats to confidence in the process. The three auditors seem familiar (numbers 1 and 2 even more so than 3) with giving suggestions (not “advices” in their opinion) for improvement and transfer experiences from other hospitals, even though it may blur the strict consistency tied to the certification norms. Our results are similar to what is found in studies of regulatory models that seem to be deterrence-oriented in the first place, but more compliance-oriented at the sharp end  , 49. ThiOur findings show that the auditors approached and perceived their role both as assessors of compliance, while educating and stimulating improvement. The preplanned audit agenda was guided by topics from the ISO 9001 standard and directions during interviews was embedded in the requirements among all the auditors. These findings show that the structured certification program contributes to consistency, despite the differences in surveying style, and are in line with findings in other studies , 25.A central proposition in the surveyor style typology is the assumption that auditors perform the same role in different ways. Gaining knowledge about the auditors’ perception of their audit role is therefore important to understand why auditors perform their role differently, especially if we are to follow the suggestions of Greenfield, Braithwaite, and Pawsey  about usSystems audits as performed in ISO 9001 certification are similar to the methods used by inspectors in the Norwegian Board of Health Supervision, who performs system audits founded in the ISO standard for audit practice . The mai. Concerns about interaction are important, because when health professionals participate in assessment contexts that are collaborative and supportive, it can self-reinforce a collaborative quality and safety culture [The auditor practice showed a considerable use of group interviews. Two auditors explained it as a strategic choice, because of respondents’ uncertainty during the assessment, an opportunity to target people who are involved in daily practice, and possibilities for reflections and awareness through discussions. Even though it seems a well-known practice, we have not been able to find suggestions about group interviews in the audit standards , 34, 36  culture . Greenfi culture  also shoThe strengths of the study are the combination of observations and interviews behind the curtains of a certification body. The ability of follow auditors during certification processes and follow up with interviews provide a rich data set on their performance as observed and their perceived experiences. This is an approach lacking in the literature. This is an exploratory study, and the limitations of the study are first, a small sample size with three lead auditors in one certification body. However, choosing one certification body, allows for more details and adds to the knowledge about the practice of auditors representing one organization that may increase the information power  in the rAuditors from a certification body demonstrated a polymorphic certification reality in which guidance and stimulation for improvement were incorporated in the auditors’ styles and perceptions of ISO 9001 certification processes. This contrasts to what is often presumed as consistent, stringent and independent audit practices. A difficulty with the analytical auditor typology framework  is its l


The acute-on-chronic liver failure associated with hepatitis B virus (HBV-ACLF) was a type of clinical syndrome with rapid deterioration of liver function. It was characterized by short-term elevated bilirubin, ascites, prolonged clotting time, hepatic encephalopathy, organ failures, and high short-term mortality. It was important to predict and evaluate the disease early. This study intended to comprehensively analyze the prognostic factors of patients with ACLF associated with HBV DNA infection through clinical manifestations and laboratory tests, and to establish a corresponding prediction and evaluation model for further clinical guidance.A total of 220 patients were first diagnosed with HBV-ACLF and admitted to and treated at the Department of Infectious Diseases of the First Affiliated Changhai Hospital of the Second Military Medical University from 2009 to 2018. These patients’ records were collected and divided into two groups: (1) 120 patients who were improved and discharged were classified as good prognosis group and (2) 100 patients who died or underwent liver transplantation were classified as poor prognosis group. By analyzing baseline characteristics and clinical indicators of the two groups, the main potential factors affecting prognosis were identified and the corresponding prognostic evaluation model was established. This model’s advantages and disadvantages were compared with classic prognostic scoring systems.p) = 1/(1 + eZ−) was established, where z = 10.0127 + 0.3687 × NEUT (%) − 0.0082 × PLT + 1.8157 × hepatic encephalopathy. The area under receiver operating characteristic (ROC) curve was 0.89, specificity was 80.83%, and sensitivity was 81%. The newly established prognostic model was compared with other three scoring systems including model for end-stage liver disease (MELD), MELD-Na, and ALBI scores. The results showed that the specificity, sensitivity, and area under the ROC curve of the newly established model were significantly higher than the other three scoring systems.The proportion of ascites and the proportion of hepatic encephalopathy of poor prognosis group were significantly higher than those of good prognosis group. The total bilirubin, creatinine, white blood cell count, and NEU (%) levels of poor prognosis group were significantly higher than those of good prognosis group, and the international normalized ratio, albumin (ALB), alanine aminotransferase, Na, Cl, RBC, and PLT levels of poor prognosis group were significantly lower than those of good prognosis group. A new prediction model LR(p) had better prediction accuracy than the other three scoring models of MELD, MELD-Na, and ALBI and could more accurately assess the prognosis of HBV-ACLF, but in the later stage, it was still necessary to expand the sample size for verification.Hepatic encephalopathy, NEU (%), and PLT levels were independent risk factors for predicting the prognosis of HBV-ACLF. The new prediction model LR( It is cThe morbidity of hepatitis B in the Asia-Pacific region is especially high; therefore, HBV infection becomes the most important cause of ACLF. ACLF progresses rapidly, and the mortality is as high as 50–90% . If earlDue to the complicated occurrence and progression mechanism of AFLC, the disease evolved rapidly, and the lesions could involve multiple organs and tissues. The assessment of a single indicator could not fully and accurately reflect the severity and prognosis of the disease. Therefore, it was necessary to further establish a corresponding multifactor prognosis model through the statistical method to improve the specificity and the sensitivity of the prediction. Currently, many classical models were used to evaluate ACLF, and some models were modified and improved based on classical models, but there were still some shortcomings and deficiencies ,9.The earliest Child-pugh (CTP) score had been used clinically for more than 50 years. The evaluation indicators included serum total bilirubin, albumin level, hepatic encephalopathy, ascites, and prothrombin time (PT). So far, it was still one of the classic models used by the internal medicine department and the surgery department to quantify and assess the liver reserve function of patients with cirrhosis. However, there were certain defects. For example, ascites could be affected by diuretics, and albumin level could be changed by intravenous infusion. In addition, the renal function was not reflected in the model, which caused the CTP score not to fully reflect the severity and prognosis of the disease. Model for end-stage liver disease (MELD) and its derived MELD score were originally used to predict the mortality within 3 months after surgery in patients undergoing transjugular intrahepatic portosystemic shunt, which were subsequently found to be used to determine the prognosis and prioritization of liver transplantation. Compared with the CTP score, MELD was more refined and more accurate in predicting the prognosis of end-stage liver disease. Hence, Biggins et al.  improvedThis study retrospectively analyzed the acute-on-chronic liver failure associated with hepatitis B virus (HBV-ACLF) patients admitted to and treated at the Department of Infectious Diseases of the First Affiliated Changhai Hospital of The Second Military Medical University from 2009 to 2018. Through data mining the baseline information, laboratory indicators, and related factors affecting prognosis of HBV-ACLF patients with different prognosis, we established a predictive model, compared it with other models, and investigated its application value.22.1The medical records of inpatients with first diagnosis in line with the HBV-ACLF criteria at the Department of Infectious Diseases from 2009 to 2018 were retrieved in the clinical data record system of the First Affiliated Changhai Hospital of The Second Military Medical University. The main laboratory test data, changes in the condition and clinical medication during hospitalization, and conditions at discharge were also collected. This study was approved by the Ethics Committee of Changhai Hospital, and all patients signed the informed consent form.2.2Guidelines for the Prevention and Treatment of Chronic Hepatitis B ) + 3.8 × loge (TBil [mg/dL]) + 11.2 × loge (INR) + 6.4 × cause . This model was recommended by the US Organ Distribution Network in 2002 as a standard for liver function assessment and prognosis judgment in patients undergoing liver transplantation surgery, thus enjoying wide application. On this basis, Biggins et al. [R = MELD + 1.59 × (135 − Na [mmol/L]).The MELD score was first proposed by Malinchoc. The scoring model could be used to evaluate the need for transjugular portal-body bypass in patients with cirrhosis. In 2001, Kamath et al. revised the formula: Albumin–bilirubin (ALBI) score was a recently developed new model that combines serum albumin and bilirubin to assess the severity of liver dysfunction. The previous study  showed t2.7The prognostic scoring system included MELD score, MELD-Na score, and ALBI score. MELD score formula : 3.78 × 2.8Guidelines for the Diagnosis and Treatment of Liver Failure [The prognostic criteria of patients in this study were based on  Failure  issued b2.9The data from 220 patients were included in the statistical analysis, including 120 cases in good prognosis group and 100 cases in poor prognosis group.t-test or Mann–Whitney U-test. Categorical variables were summarized using frequency and percentage and compared using Chi-square test or Fisher’s exact test.Continuous variables were tested for normality with the Kolmogorov–Smirnov test. Data are presented as mean ± standard deviation or median (percentile 25 to percentile 75). Categorical variables are expressed as number. When appropriate, continuous variables were compared using Student’s P value of <0.1 (defined “a priori”) were considered relevant and included into the multivariate logistic regression analysis. This second analysis was used to define the factors that were independently associated with poor prognosis. All the analyses were performed using R software. A P value of <0.05 was considered to be statistically significant.For all possible determinants of poor prognosis, univariate logistic regression analysis was performed. All variables with a Receiver operating characteristic (ROC) curves were constructed for variables that significantly predict poor prognosis. Areas under the curve with the 95% confidence interval (CI) were calculated.Logistic regression analysis was performed with R 3.5.1 for macOS . We have complete patient records in our study without any missing values.33.1A total of 302 HBV-ACLF patients hospitalized at the Department of Infectious Diseases of Changhai Hospital from January 1, 2009, to October 2018 were included in the study. According to the inclusion and exclusion criteria, 42 cases were excluded due to complication of other hepatitis, tumor, or other underlying diseases, and a total of 260 cases were included in the study. Of the 260 patients, 40 cases were not included in the statistics due to clinical data loss. The list of related data was deleted. Of the remaining 220 patients, there were 190 males and 30 females. According to the prognosis of patients, they were divided into good prognosis group and poor prognosis group. There were 120 cases in the good prognosis group, accounting for 54.55%, and 100 cases in the poor prognosis group, accounting for 45.45% see .3.2p = 0.1). The age of poor prognosis group was significantly higher than that of recovered group . The incidence of hepatic encephalopathy was also significantly higher than that in recovered group (P < 0.001). There was no statistically significant difference in sex ratio between the two groups. Among the two groups of patients with different prognoses, the TBil, Cr, WBC, NEU, and INR levels of poor prognosis group were significantly higher than those of good prognosis group (P < 0.05). The ALB, ALT, Na, Cl, RBC, and PLT levels of good prognosis group were significantly higher than those of poor prognosis group (P < 0.05). There were no statistically significant differences in AST, AKP, TBA, γ-GT, PA, Glu, or other indicators between the two groups (see The proportion of ascites in poor prognosis group was significantly higher than that in recovered group (oups see .3.4p) = 1/(1 + eZ−), where z = 10.0127 + 0.3687 × NEUT (%) − 0.0082 × PLT + 1.8157 × hepatic encephalopathy. The area under the ROC curve was 0.89, 95% CI: 0.85–0.93, specificity was 80.83%, and sensitivity was 81%.In this study, demographic information, baseline clinical indicators, as well as other potential parameters that might affect prognosis in the two groups were subjected to analysis. Many potential parameters for poor prognosis in HBV-ACLF patients were defined, including advanced age, ascites, hepatic encephalopathy, high TBil, Cr, WBC, NEU, and INR levels and low ALB, ALT, Na, Cl, RBC, and PLT levels. Univariate and multivariate logistic regression analyses were used to determine independent risk factors for poor prognosis see . The ROC4As we all know, HBV-ACLF, a clinically serious syndrome, has numerous occurrence and development factors. The results of this study showed and confirmed some factors affecting its prognosis.The age factor was significantly associated with HBV-ACLF. This study found that the age of poor prognosis group was significantly higher than that of good prognosis group. Therefore, age might be a potential parameter influencing the prognosis of HBV-ACLF. In this study, white blood cell level and neutrophil ratio were positively correlated with the patients’ mortality. The white blood cell and neutrophil levels of poor prognosis group were significantly higher than those of good prognosis group. Elevation of white blood cell and neutrophil levels suggested an aggravation of infection or inflammation. The previous study  suggesteIn terms of liver function, patients with liver failure may develop ascites due to the decreased ability of the liver synthesizing protein, low colloid osmotic pressure, portal hypertension, aldosteronism, increase in antidiuretic hormone, insufficient effective circulating blood volume, and many other factors. In addition, hepatic encephalopathy is a serious complication of end-stage liver disease, which plays a promoting role on the prognosis of ACLF. In this study, the incidence of ascites and hepatic encephalopathy in the poor prognosis group was significantly higher than that of good prognosis; therefore, the occurrence of ascites and hepatic encephalopathy suggested a poor prognosis of ACLF patients.Refractory hyponatremia and persistent damage of the renal function are common clinical manifestations in ACLF patients. Especially in the late stage of ACLF, hyponatremia is irreformable, and the continuous increase in urea nitrogen and creatinine levels is still easy to cause hepatorenal syndrome. In this study, the creatinine level of poor prognosis group was significantly higher than that of good prognosis group, while the blood sodium level was significantly lower than that of good prognosis group. Biggins et al. included serum sodium in the MELD score and improved it, confirming the importance of changes in the blood sodium level in predicting the prognosis of ACLF patients. Therefore, monitoring of blood sodium and serum creatinine levels should be strengthened during clinical diagnosis and treatment.ACLF patients suffer from severe liver damage, massive destruction of liver cells, reduction in liver transaminase synthesis, decrease in blood coagulation factors, and decline in bilirubin metabolism due to various factors, which leads to decrease in the serum transaminase level, elevation of the bilirubin level, and coagulation disorders. The liver function and coagulation function in both groups were significantly abnormal, but the total bilirubin level and INR level of poor prognosis group were significantly higher than those of good prognosis group, while the ALT level was significantly lower than that of good prognosis group.The HBV-ACLF scoring system can help clinicians to predict the early prognosis of the disease, assess the severity of the disease, and provide guidance for the selection of active and effective treatments. However, due to different factors such as genetic polymorphism, race, and disease occurrence and development, each scoring system has a certain degree of defects. This study assessed HBV-ACLF patients with clinically common scoring systems such as MELD, MELD-Na, and ALBI. For example, ascites could be affected by diuretics, and albumin level could be changed by intravenous infusion. In addition, the renal function was not reflected in the model, which led to the CTP score not fully reflecting the severity and prognosis of the disease. Therefore, the CTP score was not included in the study. The results showed that MELD, MELD-Na, and ALBI scores had also good predictive value for ACLF patients.p) = 1/(1 + ez−), where z = 10.0127 + 0.3687 × NEUT (%) − 0.0082 × PLT + 1.8157 × hepatic encephalopathy. The AUC was 0.89 (95% CI: 0.85–0.93) for logistic regression, 0.78 (95% CI: 0.72–0.84) for MELD, 0.79 (95% CI: 0.73–0.86) for MELD Na, and 0.69 for ALBI scores (95% CI: 0.62–0.76). Delong’s test was used to compare the ROC curves. The AUC of our logistic regression was significantly higher than those of MELD (p < 0.01), MELD Na (p < 0.01), and ALBI (p < 0.01).This study determined the related potential factors affecting the prognosis of ACLF and established a new prediction model through univariate and multivariate logistic regression analysis: LR, and PLT level are independent risk factors for predicting the prognosis of HBV-ACLF. Hepatic encephalopathy is also one of the serious complications of ACLF. In addition, as for individual indicators, the neutrophil ratio and platelet level are independent predictive factors for the prognosis of HBV-ACLF and both have a high predictive value. The neutrophil ratio indicates the severity of the infection or inflammatory response, while platelets reflect the coagulation function of the patients. The aforementioned aspects are of great significance in the occurrence, development, and clinical diagnosis and treatment of ACLF. The number of samples included in this study was derived from a single center, which may have an impact on the accuracy of the scoring system, and multicenter large sample size would be required for further validation of the prediction system in the future. In addition, this study was a retrospective study. All the enrolled patients were Asians of yellow race, so the race was relatively limited.The mechanism of occurrence and development of ACLF is complicated, and it is easily interfered by various factors during the progression of the disease, and the disease can involve in multiple organs, thereby affecting its prognosis. In addition to traditional assessment indicators, many new indicators and new prediction models are emerging, providing clinicians with a variety of lessons. Individual indicators or individual models are still unable to fully assess the prognosis of the disease. In future, to more accurately judge the prognosis of ACLF, on one hand, is necessary to explore new indicators with higher sensitivity and specificity; on the other hand, it is necessary to carry out statistics on large-scale clinical data and to formulate more reasonable prediction models.


Mental health and its correlates were examined in U.S. Vietnam War veterans approximately fifty years after the War. The 2016-2017 VE-HEROeS  was a mail survey of the health of U.S. Vietnam War veterans who served between February 28, 1961 and May 7, 1975 and matched US non-veteran controls. ‘Veteran status’ represented wartime experience for three cohorts: ‘theater’ veterans with service in Vietnam, Cambodia, or Laos, non-theater veterans with service elsewhere, and non-veterans with no military service. Veterans and non-veterans, aged 58-99 years, were randomly selected from a veteran sampling frame (n=9.87 million) derived from the Department of Veterans Affairs’ USVETS dataset and a commercial address database, respectively. Questionnaires were mailed to 42,393 veterans and 6,885 non-veterans; the response rate for veterans was 45%  and 67%  for non-veterans. Weighted bivariate and multivariable analyses were conducted to examine poor overall mental health, via the SF-8TM Mental Health Component Summary score (MCS), and other mental health measures by veteran status and socioeconomic, health, and other military characteristics. Nearly 50% of all theater veterans reported poor overall mental health (MCS<50). Prevalence of mental health measures was greatest for theater veterans and successively decreased for non-theater veterans and non-veterans. Key correlates significantly (P< 0.02) associated with poor MCS included veteran status, race/ethnicity, income, physical health, health perception, trauma, distress, depression, posttraumatic stress disorder (Primary Care DSM-5 PTSD screen), and drug use. Results indicate a high burden of poor mental health among those who served in-theater.


Background: The literature reports conflicting results regarding the effect of human papillomavirus (HPV) genotype 16 (HPV-16)/18 (HPV-18) positivity on cervical cancer (CC) prognosis.Aim: To conduct a meta-analysis to examine the effect of HPV-16/18 positivity on the prognosis of patients with CC.Methods: PubMed, Embase, and the Cochrane Library were searched for available papers published up to March 2020. The main outcome was the hazard ratio (HR) of overall survival (OS) or disease-free survival (DFS) comparing HPV-16 or HPV-18 positivity and negativity. The random-effects model was used for synthesizing survival outcomes.Results: Nine studies and 2,028 patients were included. Four studies reported OS in HPV-16 positivity, and no association was found between HPV-16 positivity and OS to CC . Three studies reported DFS in HPV-16 positivity, and no association was found between HPV-16 positivity and DFS to CC . Two studies reported DFS in HPV-18 positivity, and no association was found between HPV-18 positivity and DFS to CC . One study reported progression-free survival (PFS) in HPV-18 positivity, and an association was observed between HPV-18 positivity and PFS to CC . The sensitivity analyses showed that one study biased the analysis of the association between HPV-16 and OS, and another study biased the association between HPV-16 and DFS.Conclusion: The presence of HPV-16 and HPV-18 positivity appears to have no significant association with prognosis in CC in either OS or PFS. The presence of HPV-16 or HPV-18 positivity has no significant association with prognosis in CC in either OS or PFS. Cervical cancer (CC) is a malignancy originating in the transformation zone of the cervix, most commonly in squamous cells . It is tInfection with high-risk human papillomavirus (HPV) is a major risk factor for the development of CC , 4–8. ItNevertheless, despite the sound pathogenic effect of HPV-16 and HPV-18 for CC, the prognosis of HPV-16 and HPV-18 positivity in patients with CC has not been established. A recent meta-analysis revealed that HPV DNA positivity is associated with good overall survival (OS) and disease-free survival (DFS) in patients with CC . SimilarGaining a more comprehensive insight into how the HPV type affects survival is important. We herein hypothesized that HPV-16/18 positivity is associated with poorer prognosis in patients with CC. To test our hypothesis, we conducted this meta-analysis and systematically reviewed the existing literature.This meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The seaThe study characteristics , treatment parameters , and outcome (OS and DFS) were extracted by two authors independently. Any discrepancy was solved by discussion.The main outcome was the hazard ratio (HR) of OS or DFS comparing HPV-16 or HPV-18 positivity and negativity on DFS or OS.The quality level of evidence of all articles was assessed independently by two authors according to the Newcastle–Ottawa scale (NOS) for cohort study . DiscrepThe risk estimates of each study were reported as HR or relative risk (RR). We treated RRs as HRs. When possible, multi-adjusted HRs were used in the meta-analysis.Q-test and the I2 index. An I2 > 50% and a Q-test P < 0.10 indicated high heterogeneity, and the random-effects model was used; otherwise, the fixed-effects model was applied. P < 0.05 were considered statistically different. We did not assess potential publication bias by funnel plots and Egger's test because the numbers of studies included in each quantitative analysis were <10, in which case, the funnel plots and Egger's test could yield misleading results . HRs and corresponding 95% confidence intervals (CIs) were used to compare the outcomes. Statistical heterogeneity among studies was calculated using Cochran's  results .n = 34), outcome (n = 9), population (n = 48), exposures (n = 8), full text not accessible (n = 2), meta-analyses (n = 2), and non-English (n = 10).Therefore, nine studies were included , 22–28 . Those sP = 0.675; I2 = 91.6%, Pheterogeneity < 0.001)  , Table 2< 0.001) , 25, 26,P = 0.984; I2 = 0.0%, Pheterogeneity = 0.853; P = 0.002; Two studies reported DFS in HPV-18 positivity , 24, andRegarding the association between HPV-16 and OS, the sensitivity analysis showed that omitting Pilch et al.  affectedThe literature reports conflicting results regarding the effect of HPV-16/18 positivity on CC prognosis. Therefore, this meta-analysis aimed to examine the effect of HPV-16/18 positivity on the prognosis of patients with CC. The results suggest that the presence of HPV-16 and HPV-18 positivity appears to have no significant association with prognosis in CC in either OS or PFS. The presence of HPV-16 or HPV-18 positivity has no significant association with the prognosis of CC (either OS or PFS). This is in contradiction to the aggressive feature of HPV-16/18-positive lesions during the development of CIN to CC.In HPV-associated CC, tumorigenesis is driven by the E6 and E7 oncogenes from the viral DNA integrated into the host cells , but in It is now well-known that the different HPV types differ widely in terms of epidemiology and potential for CIN and CC –36. AmonThe results of the present meta-analysis must be considered in light of its limitations. We failed to conclude the prognostic effect of HPV-16 and HPV-18 in CC because the eligible studies had conflicting results. Of note, a number of studies could not be included because they did not report results specifically for HPV-16 or HPV-18. To our knowledge, there are only a few studies that investigated the prognostic effect of HPV-16 and HPV-18 in patients with CC, and the survival outcomes in each study were reported differently. Nevertheless, the non-eligible studies also had conflicting conclusions. Despite that nine studies were included in the meta-analysis, never more than four studies were analyzed together for a given outcome. One study reported the risk estimates as RR instead of HR. We treated the RR as HR for the analysis purpose, but it could introduce a bias. The nine studies were all observational studies, decreasing the strength of the conclusion, but a randomized control trial is not possible in this context. In addition, false-negatives could not be taken into account because of nonuniform reporting or non-reporting among the included studies. Finally, the risk estimates of survival outcomes were not reported at the same duration after the patients were discharged from the hospital.In conclusion, the presence of HPV-16 and HPV-18 positivity appears to have no significant association with prognosis in CC in either OS or PFS, but the sensitivity analysis indicated that the study by Pilch et al.  has a stAll datasets generated for this study are included in the article/XC substantially contributed to conception or design, contributed to acquisition, analysis, or interpretation of data, drafted the manuscript for important content, critically revised the manuscript for important intellectual content, and gave final approval. PZ substantially contributed to conception or design, contributed to acquisition, analysis, or interpretation of data, drafted the manuscript for important content, and critically revised the manuscript for important intellectual content. SC contributed to acquisition, analysis, or interpretation of data. HZ drafted the manuscript for important content. XDC gave final approval. All authors contributed to the article and approved the submitted version.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.


Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (p > 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (p > 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) . HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME. Human papillomavirus (HPV) infection is often associated with benign diseases and malignant tumors affecting the upper respiratory tract, including respiratory papillomatosis and oropharyngeal cancers ,2. SeverCSOM is a middle ear disease that relies on chronic inflammation. Different pro-inflammatory cytokines, such as TNF-α, IL-1β, IFN-γ and IL-6, have been found to be up-regulated in the middle ear mucosa sampled from CSOM patients . HoweverCOMC is a form of expanding growth consisting of keratinizing squamous cell epithelium . There iThere is emerging evidence that HPV infection can occur in different anatomical sites. Since HPV infects epithelia , all anaTherefore, in this study, HPV sequences, viral DNA load and HPV genotypes were investigated in middle ear specimens from patients affected by COM, including CSOM and COMC, as well as in normal middle ear (NME) specimens.p > 0.05; p > 0.05; HPV sequences were investigated by quantitative PCR (qPCR) in COM specimens (n = 52), including CSOM (n = 38) and COMC (n = 14), as well as in NME specimens (n = 56). Overall, HPV DNA was detected in 23% (12/52) of COM and 30.4% (17/56) of NME specimens  in CSOM (n = 10) and 7.47 copy/cell (range 6.28–8.67 copy/cell) in COMC (n = 2) specimens. The difference in the HPV DNA load between COMC and CSOM as well as between COMC and NME was statistically significant  middle ear mucosa specimens. Twenty-nine qPCR products from CSOM (n = 10), COMC (n = 2) and NME (n = 17) were sequenced. DNA sequencing confirmed the presence of HPV in all specimens analyzed. HPV genotypes detected were HPV16, HPV18 and HPV11. Specifically, HPV16 genotype was present in 100% (2/2) COMC, 50% (5/10) CSOM and 52% (9/17) NME, while HPV18 genotype was detected in 20% (2/10) CSOM and 23.5% (4/17) NME. HPV11 genotype was detected in 20% (2/10) and 5.9% (1/17) CSOM and NME, respectively. The simultaneous presence of HPV16 and HPV18 genotypes was detected in 10% (1/10) and 17.6% (3/17) CSOM and NME, respectively. To evaluate the involvement of HPV co-factors in the etiopathogenesis of COM, a univariate analysis was performed between HPV-positive specimens and age, smoking and gender . ResultsIn this study, HPV DNA sequences, viral DNA loads and genotypes were investigated in COM specimens, CSOM and COMC, as well as in NME specimens.CSOM is a middle ear disease, which depends on chronic inflammation. The etiology of CSOM is largely unknown. High- and low-risk HPV sequences in 30.7% of CSOM have been previously reported . SimilarCOMC is a disorder of the middle ear consisting of keratinizing squamous cell epithelium. Studies of HPV in COMC reveal widely differing prevalence rates, ranging from 3% to 70% ,31,32, wThe role of HPV infection in middle ear disease onset remains to be elucidated. In this context, it should be remembered that some HPV-infected subjects, due to their genetic or immunological characteristics, are more prone/susceptible to viral activity resulting in disease, as reported for HPV-positive uterine cervix tissues . It is pStaphylococcus aureus [Pseudomonas aeruginosa [Notably, it cannot be excluded that the presence of concomitant pathogenic agents, during HPV infection, could potentially increase host susceptibility to developing inflammatory middle ear diseases. Indeed, a number of case–control studies have reported an association between pathogenic infectious agents, such as s aureus ,40 and Pruginosa ,42,43 wiThe difference in HPV DNA prevalence, in available studies, could be related to (i) differences in sampling  versus formalin fixed ,19,20,21In this study, for the first time, HPV sequences were detected in NME. Indeed, HPV sequences were found in 30% of NME, indicating that the middle ear mucosa is an additional epithelial tissue susceptible to HPV infection. High- and low-risk HPV genotypes  were detected in the middle ear mucosa. This result is in agreement with previous studies, which reported HPV infection in several normal tissues ,44. AlthMiddle ear mucosa specimens were collected from patients (n = 52) suffering from COM (mean age ± standard deviation (SD), 47.6 ± 16.2 yrs), including CSOM (n = 38)  and COMC (n = 14) . NME specimens (n = 56)  were collected from patients undergoing ear surgery for cochlear implantation or stapedoplasty. The exclusion criterion was no previous ear surgery. Middle ear mucosa specimens were collected during surgery, or by middle ear swab through a tympanic perforation with a micro-otoscopy. The study was performed in accordance with the Declaration of Helsinki (2008). Institutional Review Board (IRB) approval was obtained from University Hospital of Ferrara Ethical Committee . Informed written consents were obtained from patients.β-globin gene [DNA was isolated according to standard procedures ,46. Briebin gene . DNA sam8 to 10 copies, of specific recombinant plasmid to calculate the viral DNA load [β-globin gene was used to determine the human cell equivalents of each sample under qPCR analysis. HPV DNA load values were reported as viral copies per human cell equivalents (copy/cell). Negative controls were the two samples used during the DNA extraction  and two qPCR controls, including HPV-free human DNA and a non-template control [HPV DNA load was quantified by qPCR assay, using SYBR green, with the CFX96 Touch™ RT-PCR Detection System . DNA samples were analyzed by qPCR for HPV DNA sequences using the universal primer pair GP5+/GP6+, as reported previously . BrieflyDNA load . Human β control . Sampleshttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi) [HPV genotypes were determined by direct sequencing analysis in HPV-positive middle ear samples. qPCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) . Purifieast.cgi) . The prevalence of HPV DNA in CSOM, COMC and NME specimens was evaluated by a two-sided chi-square test. Viral DNA load values were analyzed with the D’Agostino−Pearson test, and means were compared with the non-parametric Kolmogorov−Smirnov test. Univariate analysis was employed to compare the features of CSOM, COMC and NME patients, such as age, gender and smoking, in association with HPV. Statistical analyses were performed using Graph Pad Prism version 5.0 for Windows  ,56. P vaIn conclusion, this study shows that HPV sequences are present in CSOM, COMC and normal middle ear specimens. We also show that the high-risk HPV16 and HPV18 are the main genotypes detected in CSOM, COMC and NME specimens. Lastly, although HPVs have been detected in CSOM, COMC and NME with similar prevalence, high-risk HPV DNA load was higher in COMC compared to CSOM and NME. Taken together, these data indicate that CSOM and COMC epithelia and normal middle ear mucosa are target tissues for HPV infection. It remains to be assessed whether the higher HPV DNA load detected in COMC is significant for a putative pathogenic role of HPV in this middle ear disease.


In context of the ongoing multi-centric HPV vaccine study in India, unvaccinated married women (N = 1484) aged 18–23 years were recruited in 2012–2015 as age-matched controls to the vaccinated women and followed up yearly. We assess type-specific prevalence, natural history and potential determinants of human papillomavirus (HPV) infection in these unvaccinated women. Cervical samples were collected yearly for at least four consecutive years. A Multiplex Type-Specific E7-Based polymerase chain reaction assay was used to detect 21 HPV types.HPV prevalence was 36.4% during 6 years. Most common HPV types were 16 (6.5%) and 31 (6.1%). Highest persistence were observed for HPV 35 (62.5%) and 52 (25%). New HPV acquisition rate was 5.6/1000 person-months of observation (PMO), highest for HPV 16 (1.1/1000 PMO). Type-specific clearance rates ranged between 2.9–5.5/100 PMO. HPV 16 and/or 18 infections were 41% (95% CI 4–63%) lower among women with 2-<3 years between marriage and first cervical sample collection compared to those with <2 years. HPV prevalence and acquisition rates in young Indian women were lower than their Western counterparts. HPV 16 infections being most common shows the importance and potential impact of HPV vaccination in India. Women with 2–3 years exposure had reduced risk possibly due to higher infections clearance. A working group of the International Agency for Research on Cancer (IARC) categorized 12 types of Human Papillomavirus (HPV), phylogenetically all belonging to the alpha genus, as ‘definitely carcinogenic’ to humans . The stuTM, Merck, NJ, USA) to that of two and three doses. A total of 17,729 girls received three doses, two doses or a single dose of the quadrivalent vaccine by April 2010. Follow up of the recipients of different doses of the vaccine and a cohort of age-matched unvaccinated women is ongoing, which is expected to generate valuable evidence on the long term efficacy of a single dose of the vaccine to inform the cervical cancer eliminations strategies being formulated by the World Health Organization (WHO).IARC initiated a multi-centric study in India in 2009 to compare the efficacy of a single dose of quadrivalent HPV vaccine  HPV types and two low-risk types  in young sexually active healthy women from seven different provinces of India. We present the data on the type-specific acquisition of new HPV infection, their persistence and clearance. Longitudinal investigation into the natural history of HPV infection allowed us to additionally identify the key determinants of incident infection.A total of 1540 eligible healthy married unvaccinated women, from the same rural and urban communities that the vaccinated participants belonged to were invited, of which 1484 accepted participating. The present manuscript reports HPV infection in this cohort of 1484 unvaccinated married women recruited at ages 18–23 years. The ongoing trial aims to compare HPV infection rates and incidence of cervical lesions related to HPV infection among recipients of different doses of the vaccine. The vaccinated participants received first dose of the vaccine between September 2009 and April 2010 when they were 10–18 years old and were unmarried. The unvaccinated women were recruited and provided their first cervical cell samples between April 2013 and June 2015, matched for age and study site with the then married vaccinated participants. At recruitment, the trained health workers and nurses of the study interviewed the eligible women for sociodemographic and reproductive information and took their first cervical cell samples. The health workers and nurses then visited every participant in her household every year enquiring about their general health and wellbeing. Like in the vaccinated groups, medically significant events, pregnancy, antenatal and postnatal events, delivery, and migration details were obtained and recorded through the household visits, relatives, the network of social workers, healthcare providers and hospitals records. The yearly follow up is still ongoing. The detailed study protocol and the preliminary findings on vaccine efficacy have been described elsewhere ,8.TM medium  from the women at their recruitment visit and yearly thereafter, until four annual samples were collected. If the fourth cervical sample was positive for any incident HPV type, an additional sample was collected to be tested for persistence. The samples were tested at the Rajiv Gandhi Centre for Biotechnology (RGCB), India, by the HPV type-specific E7 Polymerase Chain Reaction bead-based multiplex genotyping  to detect 12 high-risk , seven possible/probable high-risk  and two low-risk  types of HPV.Cervical cell samples for HPV genotyping were collected in PreservCytAs a quality control exercise, a Global HPV DNA proficiency panel  providedThe ethics review committees of IARC and the participating centers approved the protocol.The analysis of HPV genotyping, based on the cervical cell samples collected from 29 April 2013 to 10 June 2019 in the unvaccinated cohort, is reported in this manuscript. The following outcomes are evaluated:Type-specific HPV period prevalence estimated from HPV types present in the participant’s first cervical cell sample  and new ones detected in samples other than the first (newly acquired infections). The period prevalence reported in this analysis is based on the cumulative HPV infections accrued during 6 years of follow-up.Type-specific HPV persistence defined as having the same HPV type in two consecutive samples taken at least 10 months apart. This analysis included women with at least two sample collections.Type-specific newly acquired HPV infections, which involved women who tested negative for a particular HPV type in their first sample.Type-specific HPV clearance defined as having a negative HPV test result for a particular type after being identified as positive for that same type in an antecendent sample from the same individual.Combinations of high-risk HPV types depending on whether they may be protected, cross-protected or not protected by the vaccines  were assessed for prevalence, new acquisition and clearance, in addition to the individual types.The effect of participants’ baseline socio-demographic characteristics and cervical sample collection patterns on any HPV infection and HPV 16/18 grouped outcomes was assessed. The sample collection patterns included three variables. The first variable was delayed sample collection. A participant was defined as having a delayed collection if she had a gap of 18 months or more between any consecutive sample collection dates. A participant who had less than four consecutive sample collections as per protocol, and whose due sample collection was delayed by more than 18 months by the date of our data analysis (10 June 2019), was also defined as having a delayed collection. The second and third variables were the number of cervical sample collections per participant, and the gap between the dates of marriage and first sample collection.Counting of the HPV type-specific person-months of observation (PMO) for each woman in the prevalence and incidence analysis began at date of first sample collection (baseline/recruitment visit) and ended either at the date of detection of the specific HPV type of interest, or at last negative HPV test/sample collection. Women who had only one sample collection were considered to have been followed up for one day. The rates of newly acquired infections were calculated by dividing the number of new infections by the total follow-up time at risk for an individual HPV type and expressed per 1000 person-months together with their exact Poisson 95% confidence intervals (CIs). The rates were assessed based on the individual HPV type, and rates of grouped HPV infections types were also expressed per 1000 person-months, as the ratio of number of infections to the total combined follow-up time a woman was at risk of acquiring each HPV type in the respective group. The cumulative probability of acquiring a new HPV infection was assessed using Kaplan-Meier estimates of the cumulative hazard function among cohort members who were negative for a particular HPV type tested in their first sample.PMO for clearance of each HPV type for a woman was estimated starting from the date the HPV infection was first detected to the date the woman turned negative, or to the last date the woman was detected with a persistent HPV infection for that type. Again, type-specific HPV clearance rates and associated exact Poisson 95% CIs were calculated and expressed per 100 person-months, and Kaplan-Meier estimates were obtained for the cumulative probability of HPV clearance over the study period.The analysis to evaluate the effect of the socio-demographic factors and cervical sample collection patterns on the grouped HPV infections outcomes was carried out at the infection level rather than the woman level to increase power . This analysis was done using generalized estimating equation (GEE) regression models to account for lack of independence between infections occurring within the same individual. All statistical analyses were carried out in Stata 15.1 .The study was approved by the International Agency for Research on Cancer (IARC) Institutional Review Board Committee on 18 February 2008 (Project No. 07–39) and by the IARC Ethics Committee on 27 March 2013 (Ref. IEC 07–39). Written informed consents were also obtained from all the recruited women.Out of the total 1484 unvaccinated women recruited at eight study sites from seven different provinces, 229 women had one, 202 had two, 312 had three, 735 had four and six had five cervical samples assessed for HPV genotyping till June 2019. The median time of follow-up was 36.9 months . These participants had a mean age at recruitment of 20.3 years (SD: 1.1), 878 (59.2%) had high school or higher formal education and majority of them belonged to low income group. The mean age at marriage was 19 years and three-quarters had their first cervical cell sample two years or more after marriage .In women negative for any of the 21 HPV types tested on their baseline cervical cell sample collection, the acquisition rate of new HPV infections was 5.6 per 1000 PMO. The HPV type-specific acquisition rates of new infections are presented in The cumulative probability by follow-up time of new HPV infections was assessed for HPV 16 and/or 18, HPV 31, 33 and/or 45, high-risk types other than HPV 16/18/31/33/45 and any of the 21 HPV types . Women wThe effect (obtained from the GEE regression models) of the women’s baseline socio-demographic characteristics and cervical cell sample collection patterns on the grouped HPV infection outcomes is shown in The risk of any HPV infection was significantly (2.08-fold) higher in Mizoram compared to Pune, Maharashtra . There wIn the quality control exercise based on the Global HPV DNA proficiency panel 2017, RGCB in-house Luminex assay was proficient for the detection of HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68b.Our study has systematically evaluated the prevalence and natural history of HPV infection over a median period of nearly 4 years in a young sexually active cohort of women. More than one third of the women were infected over time with any of the 21 HPV types; HPV 16 being most frequently detected followed by HPV 31, 58 and 56 in that order. Over 90% of the infected women cleared the infection by 36 months irrespective of the HPV type. The clearance rate was significantly higher in the initial months of infection–a phenomenon well-documented by previous studies . This miThe longitudinal nature of our study helped assess HPV persistence which is the necessary cause of cervical neoplasia. This is the largest cohort so far in India in which HPV genotyping was systematically studies both for prevalence and natural history. Undoubtedly, the most important risk factors of HPV infection are related to sexual practices of the participating women and their partners. Indian society is still conservative and discussion regarding sexual practices, especially premarital or extramarital sex, is a taboo in most places. We did not collect any data on sexual practices to avoid embarrassing young women and their possible non-participation. We used gap between married and first cervical sample collection as representation of exposure time assuming marriage is a proxy for sexual debut.Furthermore our study participants were not selected using any systematic sampling methodology and may not be truly representative of the country or the provinces they belong to. We decided not to screen this very young cohort of women for cervical cancer to avoid harms. It is possible that some of them may have had high grade cervical premalignant lesions at study entry and the data may not be exactly comparable to the outcomes of the studies that excluded such women. However, the proportion of high-grade lesions is likely to be low in these young Indian women.Some of the persistent infections reported in our study could be due to reinfection of the cervix, which theoretically could over-estimate the rate of persistence. However, we have followed the definition of persistent infection used in majority of the natural history studies to ensure comparability of data .Type-specific persistence of high-risk HPV is the best known predictor of a woman developing high-grade cervical precancers in future . The defThe reported HPV prevalence among apparently normal women varies widely with geographical location. A meta-analysis of 194 studies conducted between the years 1995 and 2009 estimated the HPV point prevalence among more than a million women with normal cervical cytology . The ageHPV prevalence reported by Indian studies varied from 2.3% to 36.9%, reflecting the heterogeneity of women included in the studies . High prIndia is generally considered to be a conservative society as far as sexual practices are concerned. The National Family Health Survey  collecting data to estimate the critical indicators of health reported that only 2.5% of unmarried women between 15–19 years of age had sex ever and only 0.4% of sexually active women aged between 20 and 24 years had multiple partners . In spitLike our study, HPV 16 was the most prevalent type among normal women in previous studies irrespective of the study location, with prevalence varying between 2.5% in Asia and 5.8% in North America  and prevThe rate of acquiring a new HPV infection in our study (5.6/1000 PMO) was similar to the rate (5.0/1000 PMO) reported in another study from North India among 16–24 year old women, but much lower than that reported in the Western countries ,32. The Indian women have a high burden of cervical cancer. The primary explanation is that women in India do not have access to population-based organized screening for cervical cancer and only a miniscule proportion of adolescent girls has been covered by HPV vaccination. Our study clearly shows that the Indian women are at high risk of being infected with HPV, especially HPV types 16 and 18. Interestingly, the center reporting highest prevalence (Mizoram) and lowest prevalence (Ahmedabad) in our study also had the highest and lowest incidence of cervical cancer among all the sites . Policy-


Immunosuppression is a risk factor of persistent human papillomavirus (HPV) infections, which might lead to development of (pre)malignant lesions of the cervix and lower anogenital tract. Results of HPV DNA testing using cervicovaginal self-samples are comparable to those that are clinician-obtained and therefore might be used in cervical screening. The aim of this study was to assess the prevalence of high-risk HPV (hrHPV) infections, their risk factors and the genotypes distribution among women undergoing immunosuppressive therapy. Women undergoing immunosuppressive therapy for at least three months due to solid organ transplantation or autoimmune disorders were asked to self-collect samples for HPV testing using cervicovaginal brushes and complete questionnaires regarding cervical cancer risk factors. HPV DNA detection and genotyping were performed using Genotyping kit HPV GP version 2. hrHPV was detected in 26/90 (28.9%) specimens. Genotyping revealed a broad range of hrHPV, with type 16 being the most common genotype (11/26). The components of bivalent/quadrivalent or nonavalent vaccines cover all genotypes present in 4.4% and 17.8% women, respectively, and occur as a co-infection with other types in 12.2% and 23.3% of women, respectively. The only feature significantly associated with being hrHPV-positive was having at least two lifetime sexual partners. The high prevalence of hrHPV infections among immunosuppressed women emphasizes the need for regular cervical cancer screening with HPV DNA testing, which might be performed on self-collected specimen. Infection with high-risk human papillomavirus (hrHPV) is a risk factor of precancerous lesions and cancers of the cervix—cervical intraepithelial neoplasia (CIN) and cervical cancer—as well as intraepithelial neoplasia and cancers of the vulva, vagina and anus. Most human papillomavirus (HPV) infections are transient, resolving spontaneously within 12–24 months .A self-sampling-based study was also conducted by the same study group and described by Hinten et al. who assessed the prevalence of HPV infection in renal transplant recipients both pre- and post-transplantation, revealing an increase in prevalence of hrHPV from 19% before transplantation to 31% after transplantation [Hinten et al. reported a statistically significant increase in hrHPV prevalence after transplantation and therefore implementation of immunosuppressive therapy, which was not related to changes in relationships or sexual behavior. Such change was not observed by the authors in the control group of women who did not undergo renal transplantation, so it is suspected that implementation of immunosuppressive therapy causes reactivation of a latent HPV infection .There is, however, no agreement between authors in terms of the increase in hrHPV prevalence among immunocompromised women. Some report this prevalence to be higher than in the general population ,18,19,20Similar to Meeuwis et al., we observed that having at least two sexual partners has a positive correlation with being hrHPV-positive . Such teSimilar to Hinten et al., we decided to consider genotypes 53, 66, 67, 68, 70 and 73, which carcinogenicity is still being discussed on by the International Agency for Research on Cancer, as hrHPV ,22. ImmuThe most common genotype in our cohort was type 16, similar to Verroux and Meeuwis ,17. In oThe surprisingly low prevalence of HPV 18 (2/90) was also observed by Meeuwis et al. 2/218) and Roensbo et al. who noted no women were positive for this genotype ,21. Howe8 and RoeNot only was the hrHPV prevalence high in the studied cohort, but also a large variety of hrHPV genotypes was detected, both as single type infections as well as multitype. Multitype infections, which are considered a risk factor of HPV persistence, were detected in 34.6% of hrHPV-positive women, while Meeuwis et al. observed 27.1% of women with multitype infections .As many as 23.3% of women from our cohort could benefit from being vaccinated with a nonavalent HPV vaccine. Briefly, 17.8% of women were positive exclusively for the genotypes included in the nonavalent vaccine and this group would possibly benefit from this vaccination most. However, taking into consideration the recently limited access to the nonavalent vaccine and a reported cross-protection of bivalent vaccine from non-bivalent vaccine HPV strands, vaccination with the bivalent vaccine should be advised when otherwise not possible . It is wThe limitation of the study was the wide age range of the enrolled women (20–69 years) and that this was not a case-control study due to funding reasons. What is more, the interpretation of the acquired data in comparison to literature is limited by differences in HPV genotyping tests, age range of patients, as well as type and duration of immunosuppressive therapy.A high prevalence of hrHPV and a significant proportion of multitype infections that are of higher risk of persistence and progression to precancerous lesions and cancers of the cervix and lower anogenital tract were detected in the studied population of iatrogenically immunocompromised women. This emphasizes the need for regular annual gynecological cancer screening visits and HPV DNA testing, which may be performed using self-sampling devices. Healthcare providers treating immunocompromised patients ought to educate them on cervical and lower anogenital tract cancers’ risk factors and consider advising them to limit their number of lifetime sexual partners.As the majority of the detected hrHPV genotypes were of the nonavalent HPV vaccine—preventable types—it can be assumed that this group of women would benefit from such vaccination. Therefore, the nonavalent HPV vaccination should be recommended to immunocompromised women. As the number of women undergoing immunosuppressive therapy is increasing, implementation of a government-refunded HPV-vaccination program should be considered in such vulnerable populations, especially in the pre-transplantation period in an attempt to decrease the cervical cancer burden.