Due to the increasing rates of oropharyngeal cancer, oral HPV infection is a significant concern. Methods for detecting oral HPVs is not standardized as there are different techniques available. We propose that use of oral rinse samples to detect for HPVs is a suitable technique within a clinic setting. Thus, our main objective is to study HPV detection in oral rinse samples.In our study, we used oral rinse sample collection coupled with real-time PCR to detect for HPVs types 16 and 18, and preferentially amplified FAP PCR samples to detect for a broad range of HPVs, in oropharyngeal squamous cell carcinoma (OPSCC), non-OPSCC, and healthy patients.Thirty three percent of 100 cancer patients were positive for any type of HPV; of those 23 were positive for HPV16. Only 1 of 110 healthy controls was positive (this subject was positive for HPV18).Our results indicate that HPV detection in oral rinse samples may be useful as a screening tool to detect HPV-associated oral cancers. Tissues infected by human papillomavirus (HPV) have the ability to evolve into an HPV-associated cancer. This is likely due to a field effect where HPV can modify regular cell functions resulting in malignancy. Though we know how to test for HPV-related cancers, there is currently no standard for detecting HPV infection , and scrAnalyzing p16 expression has been used as a biomarker for HPV-associated OSCC/OPSCC, but studies have reported that p16 overexpression is not always present in cases involving oncogenic HPVs . A recenOPSCC incidence in developed countries has increased significantly and HPV infection is proposed to be the main factor . Risk faWith the rise of OPSCCs it is imperative to have a gold-standard technique in place for oral HPV detection. Collecting samples from the oral cavity and oropharynx for the detection of oral HPVs should be quick, non-invasive, inexpensive, and sufficient in HPV DNA collection. In our study, we investigated oral rinse samples coupled with real-time PCR Taqman assays to detect for HPV types 16 and 18, which is sensitive and specific. To detect for a broad range of HPVs, we preferentially amplified the oral rinse samples from cancer cases, and used fluorescent arbitrarily primed (FAP) PCR, a general PCR method using degenerate HPV primers.Between 2011 and 2013, we recruited 76 OPSCC and 24 non-OPSCC patients from the Seattle Cancer Care Alliance , and 110 healthy subjects from University of Washington Dental Clinic . Non-OPSCC cases included patients with OSCC, laryngeal, sinus, and supraglottis cancers. We screened the schedules of five oncologists in order to identify eligible cancer patients, and discussed our study at their appointment. One patient declined due to mouth sores and sensitivity. 21/100 cancer patients had already begun treatment and of these 21 patients: 18 patients had treatment less than 21 days before sampling, two had over 30 days of treatment, and one patient had treatment for 7 months. Healthy subjects were randomly selected within the student dental clinic, one patient declined to take part in the study. Inclusion requirements for the healthy population included being: cancer-free, not pregnant, HIV-free, and over the age of 16. Each patient signed written consent forms to participate in the study , and answered a simple health questionnaire. Gender, age, race, smoking, alcohol, and marijuana history were recorded for all subjects. Charts for a subset of our patients were reviewed for p16 immunohistochemistry (IHC) and HPV testing.For sample collection, all patients rinsed and gargled for 30 s with Original Mint Scope® mouthwash (Procter & Gamble). Four normal healthy individuals requested to use Crest® Alcohol-free mouthwash due to a history of alcoholism. Oral rinse samples were centrifuged for 15 min. at 4 °C to form a pellet, the supernatant was discarded, and the pellet was placed in -80 °C until further processing. The Puregene® DNA Purification Kit was used to isolate genomic DNA from the buccal cell pellet within the mouthwash samples .All human subjects IRB protocols and regulations were followed under the Fred Hutchinson Cancer Research Center guidelines .HPV16 E7 PrimersForward: CGGACAGAGCCCATTACAATATTReverse: CGCACAACCGAAGCGTAGAHPV16 E7 Probe: TAACCTT(T/C)TGTTGCAAGTGTHPV18 E7 Primers:Forward: CCGACGAGCCGAACCAReverse: TGGCTTCACACTTACAACACATACAHPV18 E7 Probe: AACGTCACACAATGTTTaqman real-time PCR assays were used for detection on the ABI Prism 7900 Sequence Detection System with 40 cycles in a reaction . Absolute quantification was used to determine HPV16 and 18 viral load, and total human genomic DNA in the sample was determined on Alu sequences. Serial dilutions of human genomic DNA and full length HPV16 and 18 plasmids, of known concentrations, were used as standard curves.In order to increase the efficiency of HPV detection, we used the multiply-primed rolling-circular amplification technique (MP-RCA) to preferentially amplify unknown, circular HPV DNA. MP-RCA has been demonstrated to amplify circular DNA templates up to 107-fold . The TemThe FAP PCR published protocol was followed to detect for a broad range of HPV types where primers were developed from conserved L1 regions . We onlyWe categorized smoking history as follows: non-smoker (0 packs); light smoker (<1pack/week); moderate smoker (≥1pack/week ≤1 pack/day); heavy smoker (≥1pack/day). For those who smoked cigars or chewed tobacco we calculated to the equivalent of packages of cigarettes smoked. Alcohol history was categorized as follows: none (never drinks); rarely/occasionally (1 drink every 1–2 months); light ; moderate ; heavy .All data analysis was done using Stata MP 13.1 .p <0.001). Cancer cases were also older than controls, as >80 % of cases, regardless of cancer type, were 50 years of age or older compared to only 63 % of controls (p <0.001). Subjects with OPSCC and non-OPSCC were similar to subjects without cancer with respect to race (p = 0.30) and ethnicity (p = 0.62) and were predominately non-Hispanic Caucasians. Those who had cancer  were more likely to be heavy smokers compared to those with no cancer (32 %). Heavy alcohol history was prevalent in 33 % of OPSCC and in 33 % of OSCC patients (p = 0.015) compared to only 12 % of non-cancer subjects. Marijuana use was mainly observed in cancer cases  and rarely in controls (0.91 %). Cases of both cancer types  were more likely to have detectable HPV than healthy controls (0.9 %), and specifically type 16  detected in their oral rinse samples compared to controls. HPV18 detection did not vary by study group and was detected in a single sample from a control subject (p = 0.63), and none in the cancer cases. Our method for HPV detection had a sensitivity of 33 % and specificity of 99 % (p <0.001).Oral cancer subjects with OPSCC and non-OPSCC were more likely to be male than control subjects without cancer, as at least 83 % of subjects in each cancer type were male, compared to only 46 % of controls (Table p = 0.085) underwent treatment  prior to sampling, which could affect the presence of prior HPV infection. HPV was marginally associated with p16 detection (p = 0.076); of patients who were HPV positive, 96 % had a positive p16 test; only one sample that was positive for HPV tested negative for p16. Amongst HPV negative patients, 79 % were p16 positive. Only two case patients had HPV screening completed before sampling and both were negative for HPV in our tests and the screening.Twenty-two HPV positive samples were tested for p16 prior to sampling, and only one tested negative for p16. However, 26 samples that were negative for HPV were positive for p16 , and HPV testing is rarely done. This is something that needs to be changed during diagnosis due to the increase in HPV-associated OPSCCs.From our results we demonstrated that preferentially amplified oral rinse samples with HPV detection from real-time PCR Taqman assays and FAP PCR, is a usable method overall, which could be used within a clinic setting. More specific studies will need to be done to determine whether mouth rinses do actually reflect, in cases, the type of HPV associated with the cancerous lesion.


Cervical cancer poses a huge health burden, both to developed and developing nations, making prevention and control strategies necessary. However, the challenges of designing and implementing prevention strategies differ for low- and middle-income countries (LMICs) as compared to countries with fully developed health care systems. Moreover, for many LMICs, much of the data needed for decision analytic modelling, such as prevalence, will most likely only be partly available or measured with much larger uncertainty. Lastly, imperfect implementation of human papillomavirus (HPV) vaccination may influence the effectiveness of cervical cancer prevention in unpredictable ways. This systematic review aims to assess how decision analytic modelling studies of HPV cost-effectiveness in LMICs accounted for the particular challenges faced in such countries. Specifically, the study will assess the following: (1) whether the existing literature on cost-effectiveness modelling of HPV vaccines acknowledges the distinct challenges of LMICs, (2) how these challenges were accommodated in the models, (3) whether certain parameters systemically exhibited large degrees of uncertainty due to lack of data and how influential were these parameters on model-based recommendations, and (4) whether the choice of modelling herd immunity influences model-based recommendations, especially when coverage of a HPV vaccination program is not optimal.We will conduct a systematic review to identify suitable studies from MEDLINE (via PubMed), EMBASE, NHS Economic Evaluation Database (NHS EED), EconLit, Web of Science, and CEA Registry. Searches will be conducted for studies of interest published since 2006. The searches will be supplemented by hand searching of the most relevant papers found in the search. Studies will be critically appraised using Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement checklist. We will undertake a descriptive, narrative, and interpretative synthesis of data to address the study objectives.The proposed systematic review will assess how the cost-effectiveness studies of HPV vaccines accounted for the distinct challenges of LMICs. The gaps identified will expose areas for additional research as well as challenges that need to be accounted for in future modelling studies.CRD42015017870.PROSPERO  Cervical cancer poses one of the greatest challenges to women’s health. It is estimated that over a million women worldwide currently have cervical cancer, most of whom have not been diagnosed nor have access to treatment that could cure them or extend their survival . The burThe human papillomavirus (HPV) is the primary cause of cervical cancer and cervical intraepithelial neoplasia (CIN) . HPV is Cervical cancer can be prevented both by primary prevention through HPV vaccination and by secondary prevention through cervical cancer screening. Conventional cytology-based screening, combined with colposcopy and treatment of precursor lesions, has been the gold standard for the secondary prevention of cervical cancer. It has greatly reduced cervical cancer incidence and mortality from cervical cancer in many developed countries. Cytology-based screening is not as widespread in developing countries . For insAlternatives to cytology-based screening include visual inspection with acetic acid (VIA) and newer molecular testing for infection of the cervix with high-risk types of HPV DNA. VIA and HPV DNA testing have proven to be effective screening methods , 13. HPVThere currently are two HPV vaccines in widespread use. Both Cervarix® and Gardasil® offer protection against HPV 16 and 18, respectively, the two most oncogenic types . GardasiThe World Health Organization’s (WHO) recommendation for comprehensive cervical cancer prevention and control strategy includes primary, secondary, and tertiary prevention activities . PrimaryLMICs potentially face a number of challenges in implementing such a cervical cancer prevention and control program. Existing health services may not have the capacity to accommodate additional interventions, and thus, extra cost will be incurred to set up such a program. Resource constraints may necessitate implementation of a program in phases rather than immediate implementation. Resource constraints could also require the introduction of preventive and treatment services only for certain regions rather than country-wide. Sections of the population may systematically avoid participation in such a program. Given these potential challenges, it is therefore important to examine if and how they are accounted for by decision analytic models of HPV vaccines; for example, do analyses adopt a different modelling structure or just change parameters for the constraints mentioned above?The data needed for modelling might be less readily available in LMICs or measured with much larger uncertainty. HPV-related outcome data in many LMICs may have poor resolution and are also often highly aggregated . For exaThere are three types of models that have been employed in cost-effectiveness analyses of HPV vaccination: static models, transmission dynamic models, and hybrid models combining features of both static and dynamic models . A statiAppraising the introduction of HPV vaccines requires estimation of the avertable disease burden, cost-effectiveness of the vaccine compared with alternative uses of the resources, affordability of the vaccine, feasibility of achieving high coverage, likelihood of public acceptability, and political support for vaccination . The lacDoes the existing literature on cost-effectiveness modelling of HPV vaccine acknowledge the particular challenges facing LMICs?How were particular challenges accommodated in the models, e.g., through a different model structure or just by varying parameters?Is the uncertainty among the less readily available essential data/parameters regarding LMICs so large that the model-based recommendation is affected?Does the choice of modelling herd immunity influence model-based recommendations, and in particular, does imperfect HPV vaccination coverage influence model-based recommendations?Two systematic reviews of cost-effectiveness analyses of HPV vaccination in LMICs have been published previously , 19. The. The protocol is registered in the International Prospective Register of Systematic Reviews (PROSPERO) CRD42015017870.This protocol adheres to the Preferred Reporting Items in Systematic Reviews and Meta-analyses (PRISMA) statement . The proStudies based on decision analytical models of HPV vaccination;Studies that considered the cost-effectiveness of HPV vaccination and reported the additional cost and additional health effects in terms of life years gained (LYGs), quality-adjusted life years (QALYs), or disability-adjusted life year (DALYs);LMICs as identified by the World Bank classification of income groups ;Both single- and multi-country studies;The review will include both original research papers and reviews ;Studies to be included in the review could be published in any language;Studies published since 2006.Inclusion criteria are as follows:We will search MEDLINE (via PubMed), EMBASE, NHS Economic Evaluation Database (NHS EED), EconLit, Web of Science, and Tufts CEA Registry. For existing systematic reviews, we will search Cochrane Reviews, Cochrane Database of Abstracts of Reviews of Effects (DARE), and Cochrane Health Technology Assessment Databases. Reviews will be included to reduce the possibility of missing an individual study.A search strategy will be developed for each of the databases. The “Appendix” section provides details of our planned bibliographic database search strategies for MEDLINE (via PubMed), EMBASE, CINAHL, Cochrane Reviews/Cochrane DARE/NHS EED, EconLit, Web of Science, and CEA Registry. The reference lists of all included and relevant articles identified during the search will be reviewed to identify further studies that were missed. Furthermore, we will use the PubMed “related articles” feature. Hand searching of a selection of relevant journals will be conducted as advised by experts in economic evaluation.Titles and abstracts will be screened for inclusion independently by two of the reviewers using the eligibility criteria. Opinion of a third reviewer will be sought to arrive at a consensus in case of disagreement on a study for inclusion.Data will be extracted independently by two of the reviewers from included studies using a predefined data extraction spreadsheet Table . Data toOne of the investigators will assess the validity of included studies using the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement . DetailsThis protocol describes a systematic review for studies on cost-effectiveness of prophylactic HPV vaccination in LMICs. The aim is to assess how cost-effectiveness studies of HPV vaccine accounted for the individual challenges of low- and middle-income countries. The gaps identified in this systematic review will expose areas for additional research as well as challenges that need to be accounted for in future modelling studies. The review will also present current data on cost-effectiveness of HPV vaccines in LMICs as newer studies have been carried out since the publication of the last reviews.


Visual inspection of the cervix after application of 5% acetic acid (VIA) is a screening technique for cervical cancer used widely in low and middle-income countries (LMIC). To improve VIA screening performance, digital images after acid acetic application (D-VIA) are taken. The aim of this study was to evaluate the use of a smartphone for on- and off-site D-VIA diagnosis.Women aged 30–65 years, living in the city of Ambanja, Madagascar, were recruited through a cervical cancer screening campaign. Each performed a human papillomavirus (HPV) self-sample as a primary screen. Women testing positive for HPV were referred for VIA followed by D-VIA, cervical biopsy and endocervical curettage according to routine protocol. In addition, the same day, the D-VIA was emailed to a tertiary care center for immediate assessment. Results were scored as either D-VIA normal or D-VIA abnormal, requiring immediate therapy or referral to a tertiary center. Each of the three off-site physicians were blinded to the result reported by the one on-site physician and each gave their individual assessment followed by a consensus diagnosis. Statistical analyses were conducted using STATA software.Of the 332 women recruited, 137 (41.2%) were HPV-positive and recalled for VIA triage; compliance with this invitation was 69.3% (n = 95). Cervical intraepithelial neoplasia was detected in 17.7% and 21.7% of digital images by on-site and off-site physicians, respectively. The on-site physician had a sensitivity of 66.7% (95%CI: 30.0–90.3) and a specificity of 85.7% (95%CI: 76.7–91.6); the off-site physician consensus sensitivity was 66.7% (95%CI: 30.0–90.3) with a specificity of 82.3% (95%CI: 72.4–89.1).This pilot study supports the use of telemedicine for off-site diagnosis of cervical intraepithelial neoplasia, with diagnostic performance similar to those achieved on-site. Further studies need to determine if smartphones can improve cervical cancer screening efficiency in LMIC. In Madagascar there are 3,194 new cervical cancer cases diagnosed every year and, according to country statistics, 1,804 women died of cervical cancer (estimations for 2012), making cervical cancer a leading cause of cancer death in females [Although VIA is well suited to LMIC, it has important limitations–namely subjectivity and lack of quality control , 4. The Recently, digital imaging after acid acetic application (D-VIA) has become an increasingly important tool for quality control . DigitalAlthough digital images may offer support for physicians, their downside is the need for digital cameras, which are expensive and require substantial field training to operate. New smartphones are an obvious choice for the development of "next-generation" tools for digital image acquisition. Smartphones are “easyuse” devices, allow immediate image capture and can send the images to a remote expert in real time.In view of the growing applicability of this technology, the aim of this study was to evaluate smartphone use for on- and off-site D-VIA diagnosis and measure its diagnostic accuracy against histopathology.This study was conducted by the University of Geneva in collaboration with Madagascar’s Health and Family Planning Ministry and the Saint-Damien Healthcare Centre, in Ambanja, Madagascar. It is part of a long-term research project on cervical cancer prevention in Madagascar that aims to develop a cervical cancer screening and treatment approach for the country. Between January and August 2014, women were invited to perform a human papillomavirus (HPV) self-sampling test (self-HPV) as primary screening. Participants were recruited by advertising flyers, which were widely distributed. Upon arrival to the Saint-Damien Healthcare Centre, the women were checked for eligibility (aged between 30 and 69 years and able to sign an informed consent form), then asked to complete a self-HPV test, after which they completed a questionnaire to provide sociodemographic data and clinical information. Exclusion criteria were: having a positive history of cervical cancer or a total hysterectomy, presenting with any condition that could interfere with visualization of the cervix and being over 20 weeks pregnant.For self-HPV, women were instructed to wash their hands before the procedure. Each participant received a package containing a specimen collection kit. The swab used for self-collection was a simple dry cotton swab. Recommendations were to hold the swab by the end of the handle, to insert the swab into the vagina until they met resistance, by avoiding contact with the external genitalia. Then, they should gently turn the swab three times, remove it, and insert it in its protective sleeve.Genotyping for HPV was performed using the Anyplex II HPV28 detection test , which simultaneously detects high and low-risk HPVs.Women who were HPV-positive were invited to the Saint-Damien Healthcare Centre for further investigation.This study was approved by the Cantonal Human Research Ethics Commission of Geneva  and the Malgasch National Commission for the Ethics of Science and Technology. A written consent form was signed by all participants.Each HPV-positive woman underwent an initial speculum examination and visual cervical inspection without acetic acid by a trained on-site expert. Then, a conventional VIA/visual inspection with Lugol’s Iodine (VILI) was performed. In addition, the cervix was photographed by smartphone. Photographs were taken both before and after VIA/VILI (both termed in the text as “D-VIA”) by a medical student. The same day, D-VIA was emailed blindly to the University Hospitals of Geneva for immediate expert assessment. All women signed the informed consent form, giving permission for cervical image acquisition, transfer and later use of the images for educational purposes.Biopsies of the cervix  and endocervical curettage were performed as routine. Biological samples were sent to a Swiss laboratory for routine processing and histopathological diagnosis. Women deemed to be VIA positive were treated, if eligible .Photos were taken at a distance of about 15 cm from the cervix, with 2× optical zoom. Image capture was conducted by using a smartphone , which has a 16 megapixels camera, with an aperture size of F2.2, focal length of 31 mm and a pixel size of 1.12 μm. The flash mode (LED) was permanently activated. The picture was always taken at the same approximate distance from the cervix, by using a universal digital camera support and bracket, where the smartphone was easily adjusted. Care was taken to avoid the intrusion of hair or the vaginal wall that would mask visualization of the cervix. Smartphone camera face-recognition system focus automatically on the cervix and not on other details.All photographs were taken by a medical student, with no previous experience for VIA/VILI, who was trained for cervical image capture and assisted the physician during the examination. The training was performed in the previous weeks before departure to Madagascar and consisted in five sessions in the operating room with a gynecologist, where pictures of the cervix were taken using the same device and technique. During this training, images were obtained from patients who were going to undertake a conization procedure and who signed an informed consent form for image acquisition. These same images were used for educational purposes.Basic knowledge of how to use a smartphone was required for cervical image capture.Costs associated with the cervical image capture were about $580 US Dollar Three off-site physicians, who each have more than 5 years’ experience in colposcopy, working at the University Hospitals of Geneva analyzed and scored the D-VIA images. Results were scored as either normal D-VIA or abnormal D-VIA requiring immediate therapy or referral to a tertiary center. Each off-site physician was blinded to the result of the on-site physician and each gave an individual assessment, followed by a consensus diagnosis. Only the common assessment was emailed to the on-site physician.An endocervical brush was used for endocervical sample collection and cervical forceps were used for biopsies. Both sample types were fixed in liquid formalin according to standard procedures. Histological results, the gold standard for diagnosis, were classified as negative, cervical intraepithelial neoplasia grade 1, 2 or 3 , or invasive carcinoma.Sensitivities and specificities, using cervical histology as the definitive result, were calculated for each specialist who had graded the photographs, as well as for the on-site expert. Positive and negative predictive values were also determined. Percentage of agreement was calculated for each pair of on-site results and for distant D-VIA results using McNemar’s test. Agreement of results was also assessed using Cohen’s kappa coefficient. Quantitative variables are expressed as means and standard deviations, and qualitative variables are expressed as percentages, unless otherwise stated. Data were analyzed with a statistical analysis software package .A total of 332 women were recruited and performed self-HPV; of these, 137 (41.2%) were HPV-positive. The high-risk HPV types seen in participants of this study were 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73 or 82. Women testing positive for HPV were recalled to the Saint-Damien Healthcare Centre for further examination, although only 95 (69.3%) of the women responded. Of the 95 women HPV positive women being evaluated by D-VIA, 39 (41.9%) had never heard of cervical cancer, 93 (97.9%) had never done a pap test, and 92 (96.8%) had never done an HPV test. Seven (7.7%) participants had someone in their family who had been diagnosed with cervical cancer. Cervical cancer awareness was mostly achieved through health centers, radio broadcasts or by social interaction .A total of eight cervical neoplasia cases were detected by histology: two CIN1, two CIN2, two CIN3 and two invasive cancers . The on-Overall, 17 cases were classified as VIA pathological by the on-site physician (17.7%). The off-site experts, using the digital images, identified 20 pathological VIA cases (21.7%). The inter-observer agreement in the assessment of VIA photography was fair (kappa 0.43) among the experts in Geneva. The agreement between the on-site physician and the consensus diagnosis reached by the Geneva experts was 76.1% (kappa 0.28), which was poor.Consensus diagnosis (after case discussion between the three off-site physicians) reached a sensitivity of 66.7% (95%CI: 30.0–90.3) and a specificity of 82.3% (95%CI: 72.4–89.1). The sensitivity of the physician who performed analysis on-site was 66.7% (95%CI: 30.0–90.3) and the specificity was 85.7% (95%CI: 76.7–91.6). Telemedicine diagnosis consisted of the conjugation of on-site and off-site performances, and compared with the on-site performance, reached a sensitivity of 88.3%  and a specificity of 73.8% . Overall, three out of 95 (3.2%) photographs were considered inadequate for diagnosis.The observed sociodemographic profile of the population included in the current study is typical of African populations and other areas in the world underserved by healthcare services, particularly in the observed low awareness of cervical cancer among the participants and the absence of screening –11. ThisThere is enormous variability in the VIA method and recent data suggest this approach has intrinsic limitations, and training and quality control are mandatory for its success . Using VTo improve the efficiency and objectivity of the VIA approach, a primary self-HPV test was performed and only HPV-positive women were referred for VIA triage. For this group, the expected rate of CIN2+ increases, generally to between 8 and 12% , 15.Quality assurance for VIA is a crucial issue and D-VIA could be a valuable adjunctive procedure . PreviouIn this pilot project, primary self-HPV testing was followed by VIA triage and D-VIA as quality control. The on-site physician had a sensitivity of 66.7% (95%CI: 30.0–90.3), which was identical to the off-site physician consensus 66.7% (95%CI: 30–90.3). Specificity was also similar for the detection of CIN2+, 85.7% (95%CI: 76.7–91.6) and 82.3% (95%CI: 72.4–89.1) for on-site and off-site physicians, respectively. These data show the similarity between results from on-site and off-site healthcare professionals, and suggest this model may be a promising approach for experts to support colleagues in remote areas.A poor agreement between the on-site physician and the consensus diagnosis reached among the Geneva physicians (kappa 0.28) was observed. These data confirm that VIA interpretation can greatly vary between different observers. Subjectivity has also been reported in a study of telecolposcopy  for womeLimitations of the current study include the small sample size and the significant drop-out rate (30.7%) among HPV-positive women. This may have been reduced if a “screen and treat” approach had been implemented. The availability of rapid point-of-care HPV testing might overcome this problem. By undertaking histopathological testing on all HPV-positive women, the sensitivity and specificity of on-site D-VIA and remote expert D-VIA could be directly compared with the pathological findings. A further positive aspect of this study is that only three (3.2%) of the photographs taken were of insufficient quality for diagnosis, which supports the reliability of image acquisition using a smartphone.Generally, VIA method is intended to be performed by non-physician primary health care workers. In this study, a trained physician conducted the examination. Sensitivities between Geneva experts and the on-site physician were similar. The fact that the on-site physician was skilled in VIA may have masked the real potentialities of telemedicine. However, the goal of this study was to verify whether distant evaluation of D-VIA would be as accurate as on-site performance. We do believe these findings could be applied to other contexts where VIA is performed by non-physicians, since the essential is to take good quality images of the cervix by the VIA provider.For taking adequate photographs of the cervix with the Samsung Galaxy S5 smartphone, several aspects need to be considered, as described before in the manuscript. In consequence, procedure standardization should be developed. This would involve standardization of shot angles, the distance from the cervix and the number of pictures taken. As previous studies have demonstrated, poor image quality or slight image manipulation may have an impact on the diagnostic accuracy , 20.In the present study, the second reading of D-VIA was performed by physicians located in Switzerland. Remote healthcare services and technology are quickly becoming routine within healthcare institutions worldwide. In our case, once the program has been well implemented and is shown to be effective, it could be extended to other areas of Madagascar and even to other African countries. In the future, the possibility of the second D-VIA reading to be done by some expert located in an urban area of Madagascar or other African countries should be contemplated in order to combine screening with telemedicine. In our institution, a Telemedicine network (RAFT)  is alreaIn conclusion, our data support the feasibility of telemedicine for detecting cervical intraepithelial neoplasia and cancer, with diagnostic accuracy similar to on-site results. Further studies are needed to determine if smartphones have the potential to improve cervical cancer screening efficiency in LMIC.


L. crispatus and L. iners, whereas for SIL, it was Sneathia spp. and for CC, Fusobacterium spp. We found higher median cervical levels of IL-4 and TGF-β1 mRNA in the CST dominated by Fusobacterium spp. These results suggest that the cervical microbiota may be implicated in cervical cancer pathology. Further cohort studies are needed to validate these findings.Cervical cancer (CC) is caused by high-risk human papillomavirus persistence due to the immunosuppressive tumor microenvironment mediated by cytokines. Vaginal microbiota determines the presence of certain cytokines locally. We assessed the association between cervical microbiota diversity and the histopathological diagnosis of each stage of CC, and we evaluated mRNA cervical expression levels of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ across the histopathological diagnosis and specific bacterial clusters. We determined the cervical microbiota by high throughput sequencing of 16S rDNA amplicons and classified it in community state types (CST). Mean difference analyses between alpha-diversity and histopathological diagnosis were carried out, as well as a β-diversity analysis within the histological diagnosis. Cervical cytokine mRNA expression was analyzed across the CSTs and the histopathological diagnoses. We found a significant difference in microbiota's diversity in NCL-HPV negative women vs those with squamous intraepithelial lesions (SIL) and CC.When β-diversity was evaluated, the CC samples showed the highest variation within groups (p<0.0006) and the largest distance compared to NCL-HPV negative ones (p<0.00001). The predominant bacteria in women with normal cytology were  Cervical cancer (CC) is the fourth most common cancer in women and the seventh overall worldwide, with an estimated 485 000 new cases and 236 000 deaths in 2013. CC caused 6.9 million disability-adjusted life-years (DALYs) in 2013 .The vast majority of HR-HPV infected women never develop CC because an adequate immune response is capable of controlling the infection and preventing its progression to precancerous lesion . This suChlamydia trachomatis as a co-factor for CC development [It has been recently proposed that abnormal vaginal microbiota plays an important role in the development of cervical neoplasm . An epidelopment . Similarelopment , 18.There are still several gaps in knowledge regarding the association between vaginal and cervical microbiomes and CC development . BacteriCC is a long-standing disease, and there are stages previous to it during which the conditions of the cervical and vaginal environment are modified, including vaginal acidity and cytokine pattern that lead to a local immunosuppression state. The presence of Lactobacilli, a low vaginal pH (< 4.5) and antimicrobial peptides are part of the defense mechanisms present in the vaginal microenvironment . When anConsidering that it has been proved that specific species of cervicovaginal microbiota modulate the inflammatory immune response in the female genital tract of healthy black South African women , it is pCentro de Atención para la Salud de la Mujer del Estado de Morelos) in Mexico between June 2008 and November 2011, and from cervical squamous cell carcinoma cases (n = 171 HPV-positive samples), recruited from the Gynaecology Service at the National Cancer Institute (INCan) in Mexico City, between September 2010 and December 2011. The Bioethics and Research Committees at INCan (reference number. INCan/CC/326/10CB/609) and at the National Institute of Public Health  approved the baseline study during which the biological bank was built. In addition, all participants gave their informed consent to use their biological samples for further research studies. Every subject was interviewed for lifestyle, socio-demographic and reproductive factors known to be associated with increased risk of CC [A cross-sectional study was carried out, using samples from a biological bank built between 2008 and 2011 from newly diagnosed cases of squamous intraepithelial lesions (SIL) (n = 268 HPV-positive samples); women with a negative Papanicolaou and a normal colposcopy as controls , recruited from the Women’s Health care Center in the State of Morelos , whereas among CC patients the most prevalent genotype was HPV 16.For methodological convenience, we selected 32 cases for this study, non-cervical lesions , SILs (n = 4 HPV-positive) and CC (n = 8 HPV-positive). Inclusion criteria for the subsample selection related to the medical history were: patient´s recruitment on the same day of menstrual period , the non-use of douches and no sexual activity in previous days of the sampling. Also, not having records of antibiotic or antifungal use in the last 30 days previous to sampling. Other inclusion criteria included: molecular HPV + diagnosis; DNA and RNA purity and integrity suitable for sequencing and quantifying mRNA by qRT-PCR, and being Mexican with Mexican parents and grandparents to ensure similar ancestry. The only exclusion criterion was not having sufficient reads. “We considered 1 000 as the threshold, after performing the bioinformatics sequence analysis. The studied population’s socio-demographic and reproductive-sexual characteristics are presented in Samples were taken from women’s cervix seven days after menses withdrawal. The biological bank used for this study is composed of DNA samples and cDNA extracted from cervical epithelial scraping swabs from women diagnosed with NCL and from fresh cell biopsies from women diagnosed with SIL and CC. Genomic DNA was extracted from cervical epithelial scrapings and biopsies previously digested with proteinase K, using the Genomic DNA Purification Kit . All possible measures were taken to avoid cross-contamination of samples during DNA extraction. DNA concentration and purity were evaluated by Thermo Scientific NanoDropTM 1000 Spectrophotometer (260/280) and DNA integrity was determined by electrophoresis in agarose gels at 1%. Total RNA was isolated from cervical samples using the Trizol reagent from Invitrogen. cDNA synthesis was carried out in the presence of 200 U of M-MLV reverse transcriptase and 2.5 ug of total RNA using standard conditions. The PCR reactions were performed in a reaction volume of 25 μL containing 1 μL of cDNA, 0.2 mM of dNTPs, 15 pmol of each primer, 2.5 μL of reaction buffer and1U of Taq DNA polymerase recombinant. Primers for the human housekeeping glyceraldehyde-3-phosphate dehydrogenase GAPDH (450 pb) were used to verify cDNA integrity.2O served as a negative control. All products were analyzed by electrophoresis in acrylamide gels at 6%. Positive samples were visualized in an agarose gel at 1.5%. The DNA band obtained was extracted and purified with the MinElute Gel Extraction Kit  and sequenced using the Sanger method. The HPV sequences were analyzed by BLAST. HPV was categorized according to its phylogenetic patterns into low- and high-risk types: HPV16 and HPV18. HPV status was confirmed with the AnyplexTM II HPV HR Detection assay from Seegene®, based on multiplex real-time PCR, TOCE and DPO primer pairs technology, according to the supplier’s instructions. [Cervical specimens were previously tested for HPV. Viral DNA fragments from the samples were amplified by PCR using consensus primers MY09/MY11 , LIC1/LIuctions. This study was conducted according to the principles expressed in the Declaration of Helsinki. It was approved by the Research, Ethics and Biosafety Committees at INSP (CI:1143). Written informed consent was obtained from all participants.A qRT-PCR amplification was performed in duplicate for the gene expression analysis with the following TaqMan probes: GAPDH (ID-Hs99999905_mL), IL-4 (ID-Hs00174122_mL), IL-6 (ID-Hs00174131_mL), IL-10 (ID-Hs00961622_mL), TGF-β1 (ID-Hs00961622_mL), TNF-α (ID-Hs00174128_mL) and IFN-γ(ID-Hs00174143_mL). The amplification mix was prepared by adding 100 ng of each cDNA sample to a final reaction mixture of 10 μl containing 5μl of TaqMan PCR Master Mix for expression, 0.5 μl of probe and 3.5 μl of DNase-free molecular grade water. The amplification cycles were as follows: 94°C for 10 minutes, 40 cycles at 94°C for one minute, 54°C for one minute, 72°C for one minute and 30 seconds, followed by 72°C for 15 minutes. GAPDH was used to normalize the amount of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA present in each sample . Periphe4, 1mM dNTP, 0.2 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and 5 pmol/μL of the primers. The amplification program was the following: 94°C for 3 minutes, then 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 30 seconds, followed by 68°C for 5 minutes. Amplicons were visualized by electrophoresis in agarose gel at 1%. The DNA bands obtained were purified by MinElute Gel Extraction Kit .Amplicons of ~456 bp containing V3-V4 variable regions from 16S rRNA genes were obtained for DNA libraries preparation. Primers 347F Forward 5'-GGAGGCAGCAGTRRGGAAT-3' and 803R Reverse 5'-CTACCRGGGTATCTAATCC-3', described by Nossa, were used . A first4, 1mM dNTP, 0.2 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and 5 pmol/μL of primers 347F and 803R. Amplification cycles were the following: 94°C for 3 minutes, then 15 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 30 seconds, followed by 68°C for 5 minutes. Gel electrophoresis was performed in agarose gel at 1.5% to visualize the integrity of the amplified products. DNA fragments were purified by MinElute Gel Extraction Kit and afterwards quantified and assessed. Amplicons were pooled in eight libraries and mixed in an equimolar manner. Subsequently, the libraries were purified with Ampurebeads XP . Emulsion PCR titration and yield calculation were then performed to determine the volume of beads to load into sequencing plates. Subsequently, massive sequencing was performed on the Genome Sequencer Titanium Roche-454 (AppliedScience) platform.Amplicons were re-amplified and processed with the same primers linked to adapter A of the 454 sequencing protocol followed by a 6-mer multiplex identifier and by the primer 347F. The reverse primer was the same for all the reactions linked to adapter B of the 454 sequencing protocol. The reaction conditions were as follows: 0.0625 ng of each DNA sample to a final reaction mixture of 30 μl containing 1X reaction buffer, 2.5 mM MgSOThe raw sequences of the V3-V4 region from the 16SrRNA gene generated for each amplicon were processed with the Roche-454 complementary software, and *.sff files were generated. High quality reads were selected with the following criteria: reads with more than five consecutive bases with scores under 20 on the Qphred scale on a 30 bases moving window were discarded. Quality control (QC) was supervised with Thomas Girke’s R script that uses a fastq Quality.R library to plot the distribution of the rating assigned to each base according to its position on the Qphred logarithmic scale . Once quThe sequences were grouped into operational taxonomic units (OTUs) using the PyNAST algorithm for sequence alignment. All sequences which had a similarity of 99% or more were considered a single OTU . A reprede novo assembly and to be considered in the diversity calculus. To determine if microbiota composition was able to cluster samples by diagnosis, an unsupervised hierarchical clustering based on the Bray Curtis dissimilarity between OTU abundance of each sample (S1 Table) was performed with R packages and plotted as a heatmap. Alpha diversity was described using a phylogenetic diversity (PD) whole tree and a Shannon diversity index (H´) calculation and their rarefaction. A distance matrix and principal components (PC) were calculated with UniFrac to define beta diversity [Sequences that did not align to the reference were extracted to another file for iversity . Moreoveiversity .We classified microbiota composition according to community state type (CST) based on the predominant taxa found in the samples. A cervical CST is a cluster of community states  that are similar in terms of the kinds and relative abundances of the observed phylotypes . The cluχ2 and Kruskal-Wallis test for categorical and continuous variables, respectively. T-student tests were carried out to determine the mean difference between the Shannon diversity index or the phylogenetic diversity whole tree and the histopathological diagnosis. Logistic regression models were used to determine the association between the histopathological diagnosis and the Shannon diversity index and the histopathological diagnosis (NCL independently of HPV status and SIL/CC) and PD whole tree, adjusting by age, parity, contraceptive method and HPV-genotype.Relevant variables were compared between NCL vs SIL and NCL vs CC, using We estimated alpha diversity risk as an odds ratio (OR) with 95% confidence interval (CI). The estimated mean difference of bits units (Shannon diversity index) in cervix between SIL or CC and NCL independently of HPV status was evaluated by a linear regression analysis adjusting by age, contraceptive method and HPV-genotype. We evaluated the variation of weighted UniFrac distances using a Kruskal–Wallis test to check the beta diversity within each histopathological diagnosis group. A Wilcoxon Mann-Whitney test was carried out to evaluate the statistical significance of weighted UniFrac distances between SIL/CC vs NCL-HPV negative.Cervical expression of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA was analyzed by histopathological diagnosis (NCL vs SIL and NCL vs CC) using the Wilcoxon Mann-Whitney test. Cervical expression of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA was analyzed across CST clusters by means of the Kruskall Wallis test. Finally, we performed direct correlation analyses between the Shannon diversity index and the cervical expression of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA. We performed all the statistical analyses using Stata statistical software, version 13.0 .Fig A in S1 File) show that after calculating H´in more than 400 reads, subsample diversity did not increase; therefore, 1000 sequences had sufficient sequencing depth for this study. On the unsupervised heatmap , samples from the NCL cluster together and independently of the HPV status, showed that HPV-negative women had a higher proportion of Lactobacillus crispatus (46%) and a smaller one of Lactobacillus iners (14.9%), whereas HPV-positive women had proportions of 13.3% and 2.1%, respectively. Interestingly, these proportions seemed to switch with the presence of HPV. Furthermore, L. crispatus was found in smaller proportions in SIL (14.4%) and CC (1.3%), whereas L. iners dropped to 2.1% in SIL and was not detected in CC.After QC, there were 311863 high-quality reads unevenly spread through the samples. To address this issue, 1000 random subsamples were obtained from the raw data of each sample, and they were used for further bioinformatics analysis. Alpha diversity rarefaction curves , and it decreased gradually across the HPV-positive (6.9%), SIL (8.1%) and CC (3.3%) groups. S. agalactiae represented more than 90% of the microbiota composition of two of the samples. Pseudomonas oleovorans was observed in a relative abundance of 19% only among HPV-positive women with NCL. Bacteria from the Fusobacteriales order were found only in the SIL and CC groups. In the SIL group, Fusobacterium spp. displayed a relative abundance of 6.3%; Sneathia spp., 26.6%; Shuttleworhia satelles, 8.7%; and Megasphaera elsdenii, 10.4%; whereas the relative abundance of the same microorganisms in the CC group was as follows: 14%, 12.9%, 0%, and 2.2%, respectively. Fusobacterium necrophorum was only observed in the CC group (14.2%). This points to the fact that microbiota diversity and composition are different among the analyzed groups. To confirm this, alpha and beta diversity were analyzed.Other species of Table 2. CST I isdominated by L. crispatus (21%), CST II by L. iners (17%), CST III by Pseudomonas oleovorans (10%), CST IV by Sneathia spp. (17%), CST V by G. vaginalis (7%), CST VI by Streptococcus agalactiae (7%), CST VII by F. necrophorum (7%), and CST VIII by Fusobacterium spp. (14%). Except for CST VI, all the samples were clustered according to the histopathological diagnosis. CST I was composed mainly of HPV-negative women with NCL; CST II and III were predominantly composed of HPV-positive women with NCL; CST IV was predominantly composed of SIL cases; CST V was composed mainly of women with NCL regardless of their HPV status; CST VIII was composed predominantly of CC cases, and CST VII included only cases of CC .The cervical communities were classified into eight CSTs according to the dominant bacteria, as shown in Fig A in S1 File) showed a higher diversity in the CC and SIL groups than among the HPV-negative and -positive women with NCL, we found that diversity per se, which accounts only for richness and relative abundance, is not sufficient to determine the stage of developmentof CC. Nevertheless, when it comes to indices that consider phylogenetic metrics as a PD whole tree, we found a significant difference regarding phylogenetic diversity between HPV-negative NCL and SIL and between HPV-negative NCL and CC  (Table 3). In other words, the composition of the cervical microbiota is different between groups. The box plots in Fig 3show the distribution of the Shannon diversity index and the PD whole tree across histopathological diagnosis groups. The Shannon diversity index shows an increasing trend but it is not significant. The PD whole tree shows a significant difference between HPV-negative NCL and SIL and between HPV-negative NCL and CC. SIL and CC groups display the most diverse cervical microbiota. We did not find an association between the histopathological diagnosis and the Shannon diversity index (NCL regardless of HPV status and SIL/CC), nor between the PD whole tree and the histopathological diagnosis according to the logistic regression analysis (Table 4). In assessing the association between the Shannon diversity index for the cervical microbiome samples and the histopathological diagnosis, the mean estimated difference of bit units between CC and NCL was 1.11  (Table 5). This confirms that microbiota diversity in CC cases is higher than in the NCL group.Even though rarefaction curves for the Shannon index . The three PCs where plotted in 2D for pairwise comparison; PC1 accounted for 33.44% of the variance between samples; PC2 for 26.85% and PC3 for 10.38%. The presence of Fusobacterium spp., Sneathia spp. and Megasphaera spp. was related to PC1. The presence of Bifidobacteria spp. and Pseudomonas spp. was related to PC2. The absence of Pseudomonas spp., Fusobacterium spp. and the presence of bacteria from the Bifidobacteriaceae family were related to PC3, according to the calculated factor loading matrix (Table A in S1 File).The PCoA results consistently showed that the cervical microbiome is notably different in every stage of the natural history of CC  and the largest distance compared to HPV-negative NCL samples .When beta diversity was evaluated within each histological diagnosis group, the CC samples showed the highest variation within the groups .Cervical expression levels of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA from NCL vs SIL and NCL vs CC cases normalized to GAPDH mRNA are shown in Fusobacterium spp., and the difference was statistically significant  .To see if these findings could be explained mainly by the presence of Fusobacterium spp., the microbial composition of samples from CST I, CST IV and CST VIII were compared with their respective IL-4, TGF-β1 and INF-γ expression level . The highest level of IL-4 was found in sample CC2, whereas the highest level of TGF-β1 was found in sample CC8, which is composed of 87.95% Fusobacterium spp.When cervical expression of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA was evaluated across CST clusters, we found higher median cervical levels of IL-4 and TGF-β1 mRNA relative to GAPDH in CST VIII dominated by Finally, when we analyzed the direct correlation between the Shannon diversity index and the PD whole tree and the cervical expression of IL-4, IL-6, IL-10, TGF-β1, TNF-α and IFN-γ mRNA, we did not find any correlation.Fusobacterium spp.. As expected, we found high abundance of Lactobacillus spp. in the cervix of HPV-negative women without lesions, and L. crispatus was the most abundant species. In the cervix of HPV-infected women without lesions, the most abundant species was L. iners. Two of the CST detected in this study , which were described only in HPV-negative and -positive women with NCL, were similar to those reported by Ravel et al. and Smith et al. [Gardnerella vaginalis, was found in a healthy Hispanic population in Smith’s study [Several factors are implicated in the development of CC, including bacterial co-infection .The mainh et al. , 32. Mor’s study .Table 6). Previous reports on vaginal microbiota state that bacterial diversity among Chinese and Korean HPV-positive women is more complex than among healthy women [L. crispatus is more beneficial than L. iners. [L. iners has been found more often in women with vaginal dysbiosis by HIV, HPV and HSV-2 than L. crispatus [L. iners, L. crispatus produces hydrogen peroxide. This could explain why L iners is found in a higher proportion than L. crispatus among HPV-infected women, since hydrogen peroxide has been recognized as critical in maintaining a healthy vaginal microbiota [Our findings agree with the latest studies of HPV-infected women’s vaginal microbiome  and CC in HPV-positive subjects has been documented [Sneathia spp. was the most abundant species in the cervix of women with SIL and it was less abundant in patients with CC. Taken together, these results indicate that the presence of Sneathia spp. is a characteristic trait of patients with HPV-positive SIL.n cohort . Bacterind women . A correcumented . In our Megasphaera elsdenii and Shuttleworthia satelles have not been previously reported in women with SIL. This was somewhat unexpected as Megasphaera type 1 and Sneathia amnionii are considered good predictors of BV [Shuttleworthia satelles. The only finding is that it has been isolated from the human oral cavity and from one patient with endocarditis and prosthetic valve infection [rs of BV , which irs of BV . Little nfection .Fusobacterium spp. is significantly more abundant than in the early stages (HPV-negative or -positive NCL),and F. necrophorum was only observed in the CC group. In agreement with our results, Fusobacteria and Atopobium vaginae were also predominant in the vaginal microbiota of Korean women with high risk of cervical intraepithelial neoplasia (CIN) [Remarkably, our findings on the late stages of CC showed that ia (CIN) .Fusobacterium species are anaerobic Gram-negative bacteria that are part of the normal flora in the human mouth and the gut mucosa [Fusobacterium spp. have been recognized as opportunistic pathogens in inflammatory diseases in both mouth (periodontitis) and gut (bowel disease) [Fusobacterium spp., have been linked to colorectal cancer [t mucosa . On the disease) . Furtherl cancer , 64. Moll cancer –67.Fusobacterium spp. induces its pathogenic effects. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating the E-cadherin/β-catenin signaling via its FadA adhesin, thus modifying the tumor-immune microenvironment [Fusobacterium spp. appear in high proportion in CC, it is possible that the FadA gene levels could be overexpressed in cervical cancer patients. Likewise, previous reports demonstrate an abnormal distribution of tumor-suppressor E-cadherin, which functions through beta-catenin, in different histological types of CC [Some studies have shown the molecular mechanisms by which ironment , 69. Theironment . Moreoveironment . Given tes of CC –72.Fusobacterium enhances the systemic level of IL-4 [Fusobacterium infection could play a key role in the development of an immunosuppressive microenvironment characterized by anti-inflammatory cytokines (Th2 cytokine profile), such as IL-4 and TGF-β1, in HPV-transformed cells from the uterine cervix.In terms of the aim of our study, finding out whether there is a relationship between cervical microbiota and a specific cytokine profile in women with different stages of CC, we found significant differences between CST VIII (conformed by SIL and CC cases) and high levels of cervical IL-4 and TGF-β1 mRNA. Several reports, including some from our laboratory, have shown high mRNA levels of TGF-β1, IL-4, and IL-10 in cervical biopsies of patients with SIL and CC , 34. Sim of IL-4 . Thus, aFusobacterium spp., may induce Th2 immunity through the RORγτ+ Treg and Th17 cells, present in the cervical epithelium [Likewise, it was demonstrated in a recent study that the microbiota regulates Th2 immunity through the induction of type 3 RORγτ+ Tregs and Th17, acting as a key element to balance the immune responses of the epithelial cells . T-lymphithelium . The traithelium .Another Th2 cytokine implicated in cervical immunosuppression in CC patients is IL-10 . In thisOn the other hand, cervical high levels mRNA of IFN-γ had been found in CC cases , 82; in The evidence reported in literature indicates that the expression levels of IFN-γ differ at local and systemic level in women with CC, 25. RecFig 8. After a HR-HPV infection in a normal epithelium, the microbiome switches its composition from being dominated by L. crispatus to L. iners. With the progression to a SIL, the diversity of the microbiota increases with the appearance of Sneathia and other Fusobacterium spp. When CC appears, Fusobacterium necrophorum is present. This change in composition and diversity could be explained by the immunosuppressive microenvironment triggered by the viral infection, and it contributes to maintain a positive feed-back loop between the cytokine profile and the cervical microbiota.After everything that has been discussed, we arrive to the following possible mechanisms leading to cervical cancer progression, as illustrated in In conclusion, we suggest that some members of the cervical microbiota are possible modifiers of the cytokine profile of the cervical microenvironment during the development of SIL and CC. Currently, a Mexican cohort study is being carried out to support these findings. Further studies are needed to delve into the mechanism used by bacteria to promote cervix immunosuppression in CC. Evidence is accumulating and it points toward a major role of the microbiota in the immune system modulation of the female genital tract .Fusobacterium spp. in cervical carcinogenesis. Fusobacterium spp. either contributes by shifting Th1 immunity to Th2 or by a direct effect on the E-cadherin/β-catenin signaling pathway on cervical HPV-transformed cells. The findings reported here may allow, after validation, to build up new diagnostic strategies with microbiological markers for patient identification during the early stages of cervical cancer.We consider that this opens new trends for understanding the role of S1 FileFig A, Alpha diversity rarefaction curves Table A, Factor loading matrix for cervical microbiota composition.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Genetic variants of human papillomavirus types 16 and 18 (HPV16/18) could differ in their cancer risk. We studied the prevalence and association with high-grade cervical lesions of different HPV16/18 variant lineages in a case-control study including 217 cases  and 116 controls (no CIN2 or CIN3+ in two-year follow-up). HPV lineages were determined by sequencing the long control region (LCR) and the E6 gene. Phylogenetic analysis of HPV16 confirmed that isolates clustered into previously described lineages: A , B , C , and D . Lineage D/lineage A strains were, respectively, detected in 4/82 control patients, 19/126 CIN3+ cases , 6/1 glandular high-grade lesions , and 4/5 invasive lesions . HPV18 clustered in lineages A  and B . Lineage B/lineage A strains were respectively detected in 1/23 control patients and 2/5 CIN3+ cases . In conclusion, lineages A of HPV16/18 were predominant in Spain. Lineage D of HPV16 was associated with increased risk for CIN3+, glandular high-grade lesions, and invasive lesions compared with lineage A. Lineage B of HPV18 may be associated with increased risk for CIN3+ compared with lineage A, but the association was not significant. Large well-designed studies are needed before the application of HPV lineage detection in clinical settings. The causal association of high-risk types of human papillomavirus (HR-HPV) with cervical cancer has been well established HPVs contain a circular double-stranded DNA genome of approximately 7.9 kb that consists of eight protein-coding genes divided in an early region  and a late region  and two noncoding regions (the noncoding region [NCR] and the long control region [LCR]). Since complete HPV genomes were available and phylogenetic analysis was applied to the study of HPV, variant lineages would be defined by an approximately 1.0% difference between full genomes of the same HPV type and variant sublineages by a 0.5–1.0% difference between lineages. HPV lineage distribution is related to geographic or race distribution For HPV16, phylogenetic analysis confirmed the description of four lineages In case of HPV18, three lineages have been described Some non-lineage-specific single nucleotide polymorphisms (SNPs) appear independently of lineage, like 350G polymorphism in the E6 region that has been reported to be frequent in the E sublineages  and in AA  sublineages of HPV16.Nucleotide changes in HPV16 and HPV18 lineages may interfere with the viral oncogenic potential, and it is unknown whether any modification in the amino acids of the viral capsid may affect the efficacy of vaccination.Several investigations have suggested the influence of viral genetic variation between HPV16 and HPV18 lineages in viral persistence and the development of cervical cancer The aim of the present study was to know the prevalence and association with high-grade cervical lesions of different HPV16/18 variant lineages in Galicia, Spain.This study received approval from the Ethics Committee of Clinical Investigation of Galicia . All study participants were informed about the purpose of the survey and were asked to sign a consent form before taking part in the study. Confidentiality was ensured during data collection and subsequent publication of the results.Women attending the Department of Obstetrics and Gynecology of two university hospitals in Galicia, Spain (Vigo and Ourense), for cervical cancer screening (2006–2012) positive for HPV16 (n = 333) or HPV18 (n = 63) were included in a case-control study .Between 2009 and 2010, samples from 4058 women were processed for HPV detection in the Microbiology Departments of these hospitals. The presence of HR-HPV was detected in 1644 women. HPV16/18-positive women were offered to be enroled in a prospective study. Two hundred and eighty-seven out of 658 HPV16/18-positive women were included. Patients were followed for two years with cytology and virological detection every six months to assess viral persistence and progression of the cervical lesions. Colposcopy was performed in case of abnormal cytology, and biopsy was taken in case of abnormal colposcopy. Follow-up was discontinued during pregnancy and continued four months after delivery. Patients with intraepithelial neoplasia grade 2 (CIN2) or CIN grade 3 diagnosis or worse (CIN3+) at any time during the study received treatment  with the corresponding cytological and virological monitoring after treatment. Main exclusion conditions were conization in the previous 12 months, missing follow-up, or treatment for CIN grade 1.They were designated as cases  or controls .Besides, 109 HPV16-positive cases diagnosed from 2006 to 2008 and 2011 to 2012 in the same department of the University Hospital of Vigo were included.Data for age at enrolment and age at the first worst histological diagnosis of CIN2 or CIN3+ were collected.Patients answered a voluntary self-questionnaire composed of questions regarding sociodemographic variables such as race, smoking, and hormonal contraceptives use and questions regarding sexual behavior like age at first sexual contact in order to calculate years since first sexual contact at enrolment.All women were suggested to be tested to know their HIV serological status. This data was collected when available.The study was approved by the Ethics Committee of the Clinical Investigation of Galicia .Cytological examinations of Pap smear were reported using the 2001 Bethesda Reporting System Cervical biopsy specimens were sampled under a colposcopic guide. Additional biopsies were studied in case of conization or hysterectomy. The worst histological diagnosis throughout the clinical course of the patient was considered in the classification of the lesion.insitu (CIN3-CIS), invasive squamous cell carcinoma (SCC), adenocarcinoma insitu (AIS), and adenocarcinoma (ADCA) were referred here as CIN3 or worse (CIN3+). Squamous high-grade lesions included CIN3-CIS and SCC. Glandular high-grade lesions included AIS and ADCA. Invasive lesions included SCC and ADCA.Diagnoses of moderate dysplasia-cervical intraepithelial neoplasia grade 3-carcinoma Two different storage media and two different tests were used for HPV detection and genotyping. Samples were maintained at 2–8°C and processed within 24–72 hours after collection. QIAamp MinElute Media Kit  was used for DNA extraction. The extracted nucleic acids were stored at −20°C. An aliquot of the original sample was also stored at −20°C.In the case of 138 patients, endocervical samples were collected in ThinPrep PreservCyt Solution . After HPV detection with AMPLICOR HPV detection kit , genotyping was carried out using the Linear Array HPV Genotyping Test  according to the manufacturer’s instructions.In the case of 100 patients , endocervical samples were collected in TE buffer pH 8.0 Molecular Biology grade  and genotyping was carried out using the Linear Array HPV Genotyping Test according to the manufacturer’s instructions.http://blast.ncbi.nlm.nih.gov/).In the case of 158 patients , endocervical samples were collected in TE buffer pH 8.0 Molecular Biology grade. Nested PCR was used to amplify the L1 conserved region as described previously HPV lineages were characterized in samples at enrolment. For their identification, 751 bp from the LCR-E6 region of HPV16 and 956 bp from the same region of HPV18 were amplified with the primers described previously PCR products were purified with YM-100 PCR filter units  or PCR cleanup filter plates . DNA concentration was quantified on a NanoDrop ND-1000 spectrophotometer  and adjusted to 20–40 ng/µl. Amplicons were automatically sequenced in a 5 µl reaction volume using the Sequencing BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit  and then purified with the Montage 96-SEQ . All the primers used in amplification and sequencing reactions were listed in L1 region was characterized in samples at enrolment. A total of 423 bp from the L1 region of HPV16 were amplified by nested PCR with primers described previously  [23] incPCR products were purified and sequenced with internal primers as described above (see the [Sub] lineages Characterization Based on LCR/E6 section). The prevalence of nucleotide changes in the studied region was described. Representative sequences from each group of highly related sequences (<0.4 differences) obtained in this study were deposited in GenBank (KJ549646-52).Odds ratios (OR) and corresponding 95% confidence intervals (95% CI) were estimated to assess the strength of association between HPV lineage and cervical lesion risk. Available epidemiological characteristics were described for different groups of HPV lineages. For all calculations of means, standard deviation was calculated. Means were compared using T-Student test. Qualitative variables were compared with the Fisher or the chi-square tests .The increased risk for CIN3+, squamous high-grade lesions, glandular high-grade lesions and invasive lesions associated with certain HPV lineages was calculated using Stata 12 . p-value was <0.05.Statistical tests performed in the present study were considered significant whenever two-sided Lineage was established in 333 patients. A total of 217 cases and 116 controls were eligible for final analysis. Results of HPV persistence and progression at the end of the two-year follow-up of patients without histological lesion at enrolment are shown in Some epidemiological characteristics that may be related to cervical lesions risk are briefly described in The four lineages of HPV16 were found in the studied population  and 3. Hp = 0.04, control group as reference), 6/1 glandular high-grade lesions , and 4/5 invasive lesions   .vs. 35.5±9.8, p = 0.003). HIV coinfection was detected in 6/198 (3%) HPV16- infected women (two patients diagnosed of CIN2 and four diagnosed of CIN3+).Average age at the histological diagnosis of HPV-16 positive CIN3+ cases was 35.0±9.0 (n = 137) for CIN3-CIS, 46.0±18.0 (n = 7) for SCC, 44.8±12.0 (n = 5) for AIS, and 52.0±7.1 (n = 2) for ADCA. Patients with glandular high-grade lesions (n = 7) were older than those with squamous high-grade lesions (n = 144),  patients. Coinfection by two different lineages of the same genotype was not detected in this study.Specific regions of the HPV genome differ in their power to discriminate between all different lineages. In the present study, part of the LCR and E6 region of the HPV16 and HPV18 genome were used for (sub)lineage assignment as these fragments were found to be able to distinguish between all HPV16 and 18 sublineages In patients with glandular high-grade lesions, the lineage D of HPV16 was the most commonly found, as previously reported by other authors Two lineages of HPV18 were found in both cases and control patients. These data differ from those found in another Spanish study The distribution of HPV16 lineages around the world has been reported to be highly geographically/ethnically specific and their relative risk for cervical cancer has been suggested to be population dependent For HPV16 data analysis, we combined the CIN3+ cases from a prospective study and the CIN3+ supplemental cases drawn from the same population at a different period of time for increased analytic power. Differences in mean age, proportion of CIN3, or glandular high-grade lesions were not significant. Besides, lineage D was similarly distributed between prospective study cases and supplemental cases, justifying combining the two groups.The common 350G polymorphism is a no lineage-specific SNP localized in the E6 oncogene. It was studied within the European sublineages of HPV16, but no differences were observed in the prevalence of this SNP in relation with the presence of cervical lesions or the age of the patients. Previously reported differences With regard to HPV18, there is a lack of evidence of the lineages’ role in cancer pathogenesis. Although it is the second most common HPV in cervical cancer after HPV16, it is much less prevalent, and the precancerous lesions are infrequently detected, so progression studies are not easy to perform. Variation at nucleotide 104 was associated to a higher activity of the E6/E7 promoter HIV prevalence was 3% in HPV16-infected women. Given the relationship between HIV and HPV in cervical cancer, it might be interesting to take into consideration the HIV coinfection in future studies of cervical cancer risk of HPV lineages.The study of the L1 region individually provides limited information on viral lineage but it could be helpful if specific changes in this region were related with severe disease. A part of HPV16 L1 region, commonly used for the detection of HR-HPV in clinical specimens, was sequenced in a limited number of patients previously characterized by LCR/E6 sequencing. In this study, eight amino acid substitutions were identified. The two more common non-synonymous SNPs found in the L1 region were 6695C and 6803T. The 6695C SNP leads to the replacement of Thr at position 353 by Pro. The 6803T polymorphism that results in a substitution of Thr by Ser at position 389 was present in samples that clustered in lineage D in this study. The detection of the 6803T polymorphism was associated to an increased risk for high-grade glandular and invasive lesion compared with the detection of 6803A. It may be interesting to conduct further studies to investigate the usefulness of this specific SNP detection using quicker and cheaper methods than nucleic acid sequencing analysis. The incorporation of a lineage-specific SNP detection to the current L1-based genotyping methods could help to get a deeper knowledge of the CIN3+ risk of HPV16.Understanding the genetic basis of the special carcinogenicity of some HPV16 and HPV18 lineages may help us to discover interactions between the virus and the host that could be important to achieve a better control of HPV infection and cervical cancer. Few epidemiological studies of HPV lineages referring to the Spanish population have been published so far One limitation of the present study is the sample size that restricted the analysis of some histological types of cervical lesions and the analysis of some lineages not commonly found in Europe. Different methods were used for HR-HPV genotyping; this could lead to differences in HPV16/18 detection. Another limitation is the two-year follow-up because some patients included in the control group could have progressed in a longer follow-up. It would be interesting to perform collaborative studies including a larger number of patients to confirm the obtained results.In summary, lineage A of HPV16 was predominant in Spanish women. Lineage D was associated with increased risk for CIN3+ compared with lineage A. Further large well-designed, age-adjusted studies are needed before the application of HPV16 lineage detection as a high-grade lesion biomarker in clinical settings.Table S1Primers for characterization of HPV16/18 LCR/E6 region and HPV16 L1 region.(DOC)Click here for additional data file.Table S2Two-year follow-up of women without cytological lesions at enrolment. The two-year follow-up of women that were attending the Gynecology and Obstetrics Department for cervical cancer screening. Virological and cytological control every six months to assess viral persistence and progression to moderate or high-grade cervical lesions. ASC-US , LSIL , HSIL , ASC-H . Persistence: Virological persistence. Progression: CIN2+ at enrolment or progression to CIN2+ in follow-up.(DOC)Click here for additional data file.Table S3Frequency of SNP 350G of European (E) HPV16. CIN2: Cervical intraepithelial neoplasia grade 2. CIN3-CIS: CIN grade 3-carcinoma in situ. SCC: Invasive squamous cell carcinoma. AIS: Adenocarcinoma insitu. ADCA: Adenocarcinoma. CIN3+ includes CIN3-CIS, SCC, AIS, and ADCA. 350K: mix of 350T and 350G.(DOC)Click here for additional data file.


This study presents numerical simulations and experimental verification of a vibration-based damage detection technique. Health monitoring of a submerged pipe's girth-weld against an advancing notch is attempted. Piezoelectric transducers are bonded on the pipe for sensing or actuation purposes. Vibration of the pipe is excited by two means: (i) an impulsive force; (ii) using one of the piezoelectric transducers as an actuator to propagate chirp waves into the pipe. The methodology adopts the empirical mode decomposition (EMD), which processes vibration data to establish energy-based damage indices. The results obtained from both the numerical and experimental studies confirm the integrity of the approach in identifying the existence, and progression of the advancing notch. The study also discusses and compares the performance of the two vibration excitation means in damage detection. Therefore, periodic visual inspections have to be carried out by skilled divers or remote operating vehicles (ROVs) . Such ini.e., visual inspection), is usually a cumbersome and costly practice. Vibration-based approaches have therefore been developed and been proven to be relatively successful in detecting damage [Health monitoring of a large network of offshore pipelines, even at a preliminary stage  and corrosion along onshore and offshore pipelines –11. In tet al. [Chen et al.  developei.e., impact and chirp excitation methods) in damage detection are compared. Additionally, the study examines the sensitivity of the method's diagnostic capability to the presence of fluid by which the pipe is pressurized. The paper begins with providing a brief introduction on the vibration-based damage detection strategy adopted in this work. This is followed by the details of the developed numerical model and the experimental setup used in the study. Finally, a discussion on the outcome of the damage detection is presented.In the present work, numerical simulations and experimental verification of a vibration-based damage detection methodology (developed in our research group ) for hea2.et al. [Huang et al.  introducet al. . The reaet al.  for a det0 is the signal duration. Subsequently, damage indices can be established as follows:Thereafter, the EMD has been used in data interpretation in various disciplines . For insHealthyE, and DamagedE are the energy terms calculated from vibration signals of individual sensors gathered at the initial state  and the subsequent  state of a given structure, respectively. Sensors producing relatively higher damage indices infer presence and the location of damage. Progression of damage can also be quantified by noting the increasing value of damage indices monitored as a function of time. Several experimental case studies accompanied by numerical simulations have been conducted to verify the integrity and effectiveness of the proposed methodology [In the above equation, hodology –4,15,17.3.3.1.An aluminum pipe was considered in this case study. The material properties and dimensions of the pipe are listed in ES is the compliance matrix, d is the piezoelectric coupling matrix, and εS is the permittivity of the piezoceramic. The density of the piezoceramic was taken as 7500 kg/m3. Individual piezoelectric transducers could serve as a sensor and an actuator. They could turn into an actuator once a surface charge is defined for one of their surfaces.To monitor pipe's vibration, eight piezoelectric sensors were bonded to the pipe, 5 mm away from either sides of the girth-weld in the configuration depicted in 3, and 2.13 GPa, respectively, and 1.2 kg/m3 and 101,000 Pa for air. The “TIE” constraints were also used to form the interfaces of the pipe and external and internal fluids. The constraints correlated the pipe's surface displacement degrees of freedom to the neighboring fluid elements' pressure degrees of freedom [ABAQUS's acoustic element, AC3D8  was used to model the surrounding water and the internal fluid in the computational domain; 1280 and 640 elements constructed the medium and the internal fluid, respectively. Density and bulk modulus values of water were taken as 997 kg/m freedom .The question was raised whether one could model the pipe within an effective water depth (EWD), as opposed modeling a large volume of water, hence reducing the computational cost without sacrificing the accuracy. To establish the appropriate EWD, one can establish a boundary such that the reflected waves from that boundary would not affect the pipe's vibration. In other words, the reflection from the boundary would become negligible once the boundary is located adequately far from a vibrating structure .h/OD, was observed. h is the height of water above the pipe, and OD is the pipe's outer diameter.Two different boundary conditions were considered as the exterior boundaries of water in order to establish the EWD; they were (i) radiation and (ii) rigid-wall see . The radEWD = h/OD = 4 is considered in the analysis, hereafter.The results shown in In addition, a mesh convergence study was conducted to ensure the accurate representation of the submerged pipe model. The mesh density was increased to a level such that the variation in the first-three eigenvalues remained below 1%.3.2.Health monitoring of the submerged pipe was accomplished by conducting a series of transient dynamic analysis solved by the implicit solution algorithm of the ABAQUS. Two excitation approaches were adopted in the damage detection trials. First, an impulse force, similar to the force that was produced by a pneumatic hammer that was used in the experiment, was prescribed to the pipe by defining a force with magnitude of 1000 N; this force was assumed to be acting over a very short period of time (0.0003 s). The other alternative excitation method was achieved by using one of the piezoelectric transducers as an actuator. In this way, a chirp signal was propagated into the pipe by the actuator. The adopted chirp, defined as a surface charge in the piezoelectric actuator, contained a frequency range of 10–5000 Hz, varied linearly over 0.05 s. The schematic representations of the actuation units are elucidated in i.e., 5 kHz) through the chirp. The pipe's vibration was registered via the piezoelectric sensors' output voltage.The transient analysis was performed with an increment of 0.00002 s. In the other words, the pipe's vibration was sampled at 50 kHz (1/0.00002 s), so that it could accommodate the highest excitation frequency , was initially considered. However, a time-sensitivity analysis was performed and revealed the insensitivity of the damage detection's outcome to the selected time modulation of the chirp signal. Therefore, the aforementioned chirp was applied over 0.05 s in order to reduce the computational cost without sacrificing the accuracy.4.3 laboratory tank. The pipe was clamped at both ends as shown in An aluminum pipe with the same material properties and dimensions listed in Eight flexible piezoelectric actuator/sensors  were bonded to the pipe in either sides of the girth-weld as shown in (1)ImpactA waterproofed pneumatic hammer was designed and fabricated in-house to generate the required impulsive forces. (2)Chirp wavesIt effectively manages the consistency of excitation, thus minimizing the discrepancy in repeated trials' measurements; the force generated by an impulse hammer is vulnerable to severe inconsistencies due to several factors, including small deviations in the impact location and the magnitude of generated force.i.e., chirp, burst, and random).It allows exploitation of a wide range of frequencies, and selection of various types of excitation signals  was used to generate the chirp signals. The signals were then amplified via a power amplifier , before being injected to the piezoelectric actuator. The chirp signals contained frequency range of 10–5000 Hz, varied linearly over 0.5 s. The piezoelectric-based excitation method provides the following main advantages over the impulsive excitation, as typically generated by an impact hammer:As stated earlier, two excitation methods were considered for conducting the damage detection trials:The vibration signals gathered through the sensors were digitized by a data acquisition system . The sampling rate was set to 50 kHz.5.An experimental study was conducted in our laboratory tank to verify the integrity of the developed numerical model before beginning the damage detection process. For that, the experimentally measured eigenvalues of a submerged pipe were compared against those obtained from the simulation. The aluminum pipe was hanged in the tank by soft elastic ropes emulating a free-free boundary condition. One of the piezoelectric transducers was used to propagate the chirp signal along the pipe. The pipe's vibration was recorded by one of the piezoelectric sensors, whose output was a voltage-signal. The experiment was simulated by the numerical model described earlier. The eigenvalues of the submerged pipe were determined by applying the FFT to the pipe's forced vibration signals obtained from the experimental study and the model. The first-three eigenvalues of the submerged pipe, obtained via the two approaches, were compared as tabulated in 6.i.e., 19% of the wall thickness) was introduced adjacent to the girth-weld and was then propagated to a depth of 4 mm  in 1 mm increments. In the experiments, the notches were created by a jeweler saw. Two different conditions were considered for the pipe's internal loading conditions: (i) the pipe pressurized with air (P = 1 MPa) and (ii) with water (P = 5 MPa). Health monitoring of the pipe's girth-weld was attempted by utilizing the outlined damage detection algorithm. A notch of 1 mm depth , as well as for each incremental damaged state. The vibration signals at each run were recorded through the sensors. An in-house MATLAB-code was developed to include the adopted damage detection algorithm (EMD_EDI). The code performed the following tasks on the vibration data:(i)Filtered the signals (ii)Extracted the IMFs of vibration signals through EMD see (iii)Calculated the first IMF's energy using (iv)Established the damage indices using The numerical simulations included execution of a transient analysis to capture the dynamic response of the pipe at its initial state , and trace its advancement. Half of the sensors yielded higher damage indices as the notch depth increased. The only exception was in the case shown in The charts shown in It can be inferred from the results that the relative location of the sensor and actuators with respect to the notch is responsible for the variability seen in the results. Those sensors receiving their vibration waves passing through the notch outperformed the rest of the sensors in terms of damage detection accuracy. This statement can be further explained by referring to Based on the analysis of the results, one can postulate that the diagnostic capabilities of half of the sensors were affected by the reflection of the waves from the notch. Conducting a systematic parametric study on pipes with larger diameters could shed more light on the observed behavior and validity of the noted postulation.7.2.i.e., when the pipe was excited by an impact). Comparatively, the advancement of the notch could be traced more accurately when the chirp method is used to excite the pipes.The results of the damage detection tried by the chirp method qualitatively confirm the findings of our previous trial, as noted above  did not produce the largest EMD_EDI. This is because, it was located in-between the source of excitation and the notch, thus a low diagnostic capability was already anticipated based on the observations explained in Section 7.1.The diagnostic capability of the method in damage localization was also investigated under the both excitations approaches. The numerical results illustrated in In comparison to the numerical results, the EDIs obtained by processing of the experimental data produced somewhat controversial results in terms of damage localization. The results presented in Taking the above observations into account, it could be suggested that one requires a minimum of two piezoelectric sensors for accurate health monitoring of such damage conditions . A more effective health monitoring, however, could be achieved by conducting two separate trials, using either of the excitation techniques. In the case of vibration excitation by an impulse force, the two tests would consist of impacting the pipe at both sides of the girth-weld, and recording the associated vibration of each impact. In the case of chirping, the sensors can alternate their tasks in the form of a sensor and actuator, respectively. In this way, upon completion of the two tests, the existence and severity of damage could be more effectively discerned by noting the largest EMD_EDI obtained from the two trials. The conclusion holds also true for the numerical model, so long as the chirp excitation method is used as the means of excitation; otherwise, at least four sensors (two placed on each side of the girth-weld) are required for a successful damage detection, since it was observed that some of the sensors could not sense the existence of the damage see ,b.i.e., compressible (air) or incompressible (water)). It can therefore be concluded that the variable damping introduced by the presence of fluid inside the pipe would not weaken the method's diagnostic capability.In all, both the numerical and experimental studies provided satisfactory results with respect to identification of an advancing notch, regardless of the fluid-type the pipe carried  was selected for replicating the impulse force in the numerical study. Furthermore, due to unavoidable circumstance, the pneumatic hammer produced double-impact at each loading application. As a result, the time resolution of the repeated force could not be retrieved accurately within each test. Therefore, it was idealized as a single impulse force in the numerical simulations.8.In the experimental investigation, the repeatability of the measurements was established by comparing the energy terms resulting from the applied excitation from one trial to the next for total of ten trials. The consistency of the impact force was determined to be 92% and 98% for the pneumatic hammer and piezoelectric actuator, respectively. The deviations in the measurements are believed to be due the inevitable noise associated with the instruments and laboratory noise, as well as due to minor inconsistencies produced during the hammer impacts.i.e., 40 N·m). It was also of interest to examine the simultaneous effects of the noise and disturbance associated to the pipe's constraints  on the repeatability of the measurements. For that purpose, the clamping torque of the bolts securing the collar on one of the pipe's ends tested in air see  was reduMoreover, owing to the damping of the propagated waves, it is anticipated that the observed variations would be smaller for cases where the supports are located farther from the inspection zone.i.e., 0–5 MPa) were assigned to the internal surface of the healthy pipe. At each pressure level then, the pipe was excited by an impulse load and a chirp wave according to the configuration elucidated in i.e., 5 MPa).Operational variability such as abrupt changes in the internal pressure of the pipe could also affect the integrity of a vibration-based damage detection trial. The additional stiffening generated by the internal pressure can variably modify the overall stiffness of the pipe, thereby its vibration response. As a result, erroneous energies, causing false alarms, could be developed. To investigate the criticality of this issue, the sensitivity of the proposed energy index to such operational variability was examined. For that, different levels of pressures  to process the recorded vibration signals and establish the energy-based damage indices (EMD_EDIs). The integrity and efficiency of the technique were evaluated by detection of an advancing notch that was formed in the girth-weld of two mating aluminum pipes. The results of the numerical study were compared against those obtained from the experimental investigations. Reasonable agreement was obtained between the damage detection indices produced by the numerical and experimental case studies. Examination of the EMD_EDIs produced through sensors' data revealed encouraging evidence with respect to detection of the presence and advancement of damage, regardless of the type of fluid used to pressurize the pipe.The investigation also examined the performance of two vibration excitation approaches, namely: the impact method and chirp excitation method. The results demonstrated the effectiveness of the chirp excitation method over the impact method for damage detection of the submerged pipes. It was also revealed that method could sustain its integrity and reliability in the presence of some common environmental and operational variability . Finally, it was concluded that health monitoring of the submerged pipe's girth-weld could be efficiently accomplished with a minimum of two transducers, one acting as a sensor and the other as an actuator, bonded on each side of the girth-weld.


Tretinoin , is an active metabolite of vitamin A [in vitro production embryos systems, TTN acts improving cytoplasmic maturation process in oocytes, developmental competence in early embryos, and quality in blastocysts [γ) for TTN in oocyte, hatched blastocysts, and cumulus cells [in vitro maturation media, and its influence in reactive oxygen species (ROS) production in two-to four-cell stage embryos. In conclusion, tretinoin- loaded lipid-core nanocapsules added in in vitro maturation media highly protects embryos at early stage of development against oxidative stress.The improvement of itamin A , that mestocysts . Studiesus cells . This mous cells . The aimin vitro maturation medium supplemented with 0.25, 0.5 and 1 μM of TTN-LNC or TTN. Control groups of COCs matured without treatment and treated only with blank lipid-core-nanocapsules (LNC) were also examined. The oocytes were in vitro fertilized in order to evaluate the ROS levels in embryos produced by the different treatments. The ROS formation was evaluated in two-to four-cell stage embryos as previously described [The experimental groups were established, and cumulus-oocyte complexes (COCs) were matured in oocyte in vitro. The studies at the molecular level using oocyte competence markers are alternatives to clarify the function of lipid-core nanocapsules in in vitro maturation and in embryonic development.ROS production was lower in embryos derived from oocytes matured in the presence of TTN or TTN-LNC. Both treatments TTN and TTN-LCNC protect the cell more effectively against oxidative damage reducing the ROS production. A significant reduction (p <0.05) in ROS production was detected in the presence of TTN-LCNC compared with controls. There was no difference between the concentrations in TTN and TTN-LNC groups. Tretinoin-loaded lipid-core nanocapsules offers an increased protection against oxidative stress in embryos produced