Most tumours of the head and neck are attributable to smoking and alcohol use, but an increasing proportion of head and neck tumours are caused by human papillomaviruses (HPVs). The aim of this study was to use in house molecular assays to detect and genotype HPV in biopsies from patients with histologically confirmed head and neck squamous cell carcinomas. In addition, the results were compared with p16 immunohistochemistry staining, which has been described as a potential marker for HPV infection.Biopsies of squamous cell carcinomas of the oropharynx, nasopharynx, larynx and hypopharynx from 112 South African patients were screened using three PCR assays targeting the L1 and E6 regions of HPV and p16 immunohistochemical staining.HPV was identified in 7 (6.3%) tumours, while 22 (19.6%) had positive p16 immunohistochemical staining. There was concordance between the results obtained using the three PCR assays. There was substantial agreement between the results of molecular tests and p16 immunohistochemistry for hypopharyngeal carcinomas, but only fair agreement for laryngeal and oropharyngeal carcinomas. The study was approved by the Health Sciences Research Ethics Committee, University of the Free State (137/2013B). Written informed consent was obtained from each patient.DNA was extracted from freshly frozen tissue biopsies using QIAamp DNA Mini Kit from QIAGEN according to manufacturer's instructions . Tissue pieces were approximately three to four millimetres in diameter and DNA was eluted in a final volume of 200 µl AE buffer supplied in the kit and stored at −80 °C.2.2Three molecular methods, which included two nested PCR assays targeting the L1 region and an in-house multiplex hemi-nested PCR using type-specific primers that target the E6 gene, were used to screen tissue biopsies for HPV DNA.2.2.1A nested PCR was performed using MY09/11 as outer primers and GP5+/6+ as inner primers The beta-globin gene was amplified concurrently as a control for DNA integrity using PCO4 and GH20 primer pair 2.2.2HPV DNA was amplified using modified PGMY09/11 and GP5+/GP6+ 2.2.3A multiplex PCR reaction was developed using primers that target the E6 gene of low risk HPV types 6 and 11 and high risk HPV types 16, 18, 31, 33, 45 and 58. Primers were designed based on alignment of DNA sequences retrieved from GenBank for HPV types 6, 11, 16, 18, 31, 33, 45 and 58. Each primer sequence and the HPV type targeted is listed in The low and high-risk PCR assays were performed separately, to prevent primer binding competition, with an annealing temperature of 59 °C. One microliter of the first round PCR reaction was used as the template for the hemi-nested PCR. PCR reactions were separated and visualised on 2.5% agarose gels stained with ethidium bromide.2.2.42, Amplitaq Gold DNA polymerase , uracil-N-glycosilase, deoxynucleotides, biotinylated primers and 50 µl of DNA template. An internal control was incorporated in the assay with the inclusion of primers to amplify the beta-globin gene. The reaction was cycled as follows: 50 °C for 2 min, 95 °C for 9 min and 40 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and finally, at 72 °C for 5 min before holding it indefinitely at 72 °C.The Roche Linear Array HPV Genotyping Test  was performed according to manufacturer's instructions on the first 74 samples submitted. The remaining samples were not tested due to financial constraints. Briefly, the PCR was performed using primers provided in the kit. The 100 µl reaction comprised of 50 µl master mix containing MgClThe amplicons were denatured and hybridised to a strip containing specific probes for 37 HPV genotypes and the beta-globin reference. Colorimetric determination was performed using the linear array detection kit. Positive reactions appeared as blue lines on the strip. The strips were interpreted using the HPV reference guide provided.2.3PCR products were excised from the 1% or 2.5% agarose gels and were purified using Wizard®SV Gel and PCR Clean-Up Systems according to manufacturer's instructions . Determination of nucleotide sequence of the amplicon was performed using the Big Dye Terminator sequencing ready reaction kit according to manufacturer's instructions . Purified PCR amplicons were sequenced bidirectionally. Nucleotide sequences obtained for PCR positive amplicons were edited using Chromas Pro version 1.6 and aligned using Clustal Omega 1.2.1 2.4HPV11 DNA extracted in an unrelated study from a patient with confirmed recurrent respiratory papillomatosis was used as a positive control for low risk HPV types in PCR reactions targeting the L1 gene. HPV16 DNA was used as a positive control for high risk HPV types. The PCR targeting the L1 gene is well established and known to amplify multiple HPV types. The specificity of the in house PCR targeting the E6 gene was confirmed using HPV16, HPV18, HPV31 and HPV45 DNA extracted from patients. In the absence of clinical controls for HPV33 and HPV58, DNA controls were prepared by using gene fragments synthesized by GenScript  and supplied in pUC 57 plasmid. DNA template for use as a positive control was prepared by amplifying DNA from the plasmid using a type specific forward primer and M13 primer targeting the plasmid. Amplicons were gel purified and diluted for use as DNA template.2.5Tissue sections of 4 µm were cut from wax blocks of formalin fixed paraffin embedded specimens and stained using the CINtec®-p16 kit  according to the manufacturer's instructions. All stains were performed on a Benchmark XT automated slide preparation system  using diaminobenzidene as a chromogen. The p16 expression was reported as positive if at least 70% of the tumour cells showed moderate to strong nuclear and cytoplasmic staining.2.6t-test, while associations were analysed with Fisher's exact test. Agreement between the results of molecular tests and p16 immunohistochemistry was performed using Cohen's kappa and classified according to the criteria of Landis and Koch Statistical analysis was performed with IBM SPSS version 25. Differences between ages were analysed using the independent samples 3There were 102 (91.1%) males and 10 (8.9%) females. The average age of the patients was 61.5 years (range 37.3–85.2 years). The mean age for males was 61.5 years  and for females was 62.1 years , with no significant difference in age between males and females (p = 0.846).Amplification of the beta-globin gene from all samples confirmed the viability of extracted DNA. The MY09/11 and GP5+/6+ primers amplified HPV DNA from 6/112 (5.4%) samples. HPV types 11, 16, 18, and 31 were detected . The PGMThe Roche Linear Array HPV Genotyping Test was performed on the first 74 samples, five of which were positive using the PCR assays. Of the five positive samples, three tested positive for HPV types 16, 18 and 45. These were the same HPV types detected with the PCR assays. The two positive samples that were not detected using the linear array were an HPV11 and HPV31. The linear array did not detect HPV in any of the samples that tested negative for HPV using the PCR assays.There was no significant difference in the age of patients with HPV positive tumours  and HPV negative tumours  (p = 0.620). There was no association between gender and HPV positivity (p = 0.119).Twenty-two (19.6%) samples had positive p16 stains . For the4HPV was identified in 7 of 112 (6.3%) SCCs, including tumours of the oropharynx, nasopharynx, hypopharynx and larynx. Six of these were high risk types.Three molecular assays were compared, namely two nested PCR assays targeting the L1 gene and a hemi-nested PCR targeting the E6 gene. Nested PCR using MY09/11 and GP5+/6+ detected 6/112 (5.4%) HPV types; nested PCR using PGMY 09/11 and GP5+/6+ detected 7/112 (6.3%) HPV types and the hemi-nested PCR targeting the E6 gene detected 7/112 (6.3%). Three of the five samples that tested positive for HPV DNA using the three in-house molecular assays were positive when tested using the commercial linear array while the remaining two samples were negative. These results were unexpected as genotyping confirmed that the samples were HPV11 and HPV31, which are types included in the linear array. As the assay is designed to detect HPV11 and HPV31 it could possibly be surmised that the negative results may be due to a low viral load. Previous reports on viral loads in head and neck tissues have suggested that the viral load can vary and this is an aspect that requires further investigation The p16 stain is often used as a surrogate marker for HPV detection as inactivation of the pRB by HPV E7 induces p16 upregulation There was moderate agreement overall between the results of p16 immunohistochemistry and detection of HPV DNA. Bussu et al. found a poor agreement between p16 immunohistochemistry and the detection of HPV RNA in head and neck SCCs, except for tumours of the oropharynx, where there was a fair agreement HPV-negative p16-positive laryngeal tumours may be associated with a worse prognosis The proportion of HPV positive head and neck tumours varies worldwide Most studies from sub-Saharan Africa have found a low prevalence of HPV in head and neck SCCs Four of the 79 (5.1%) laryngeal carcinomas were HPV positive, 3 for high risk types  and one for a low risk type (HPV11). All three tumours that were positive for high risk types were p16 positive. Hernandez et al. found HPV DNA in 21% of laryngeal carcinomas with a limited correlation with p16 status One of the three nasopharyngeal carcinomas was positive both for HPV18 and p16 and EBER (Epstein-Barr encoding region) immunohistochemistry negative. It has been suggested that HPV may play a role in non-EBV-related nasopharyngeal carcinoma 5Overall there was an HPV prevalence rate of 6.3% in 112 head and neck squamous cell carcinomas in a cohort of South African patients. There was only a moderate agreement between HPV DNA positivity and a positive p16 immunohistochemical stain.


Each year significant numbers of individuals leave military service. The majority successfully transition into civilian life, but a significant minority leave the military either with a diagnosis of PTSD, or go on to report symptoms in later life. Prevalence rates of PTSD in military populations have been extensively studied since the deployments to Iraq and Afghanistan, with data suggesting high rates (in some cases up to 20%) in those who served in these conflicts . There iAt the same time, there is a body of research showing that only a minority of veterans with PTSD are able to engage in help-seeking behaviours . In addiA range of different factors have been identified that could contribute to mental health difficulties in veterans. These include pre-service factors such as growing up in areas of social deprivation or reporting difficult childhood experiences ,11. FactA further consideration is the evidence suggesting that veterans do not respond as well to treatment for PTSD as non-veteran groups do ,15,16. THealthcare features original papers exploring a range of divergent issues surrounding PTSD in veterans and discusses novel ways to support this vulnerable population. For example, Poulsen et al. published a paper exploring the application of nature-based therapy (NBT) for veterans with PTSD [This special edition of ith PTSD . They prAshwick et al. published a paper looking at the relationship between mental health and risk-taking behaviours within a sample of veterans engaged in clinical mental health services . They reThe same research team as Turgoose et al. also published a paper within the special edition that examined the associations between anger, aggression and PTSD in veterans . Whilst Another paper within this special edition looked at potential bias by jurors faced with possible malingering for PTSD in veterans involved with the judicial system . The resAnother paper published titled ‘The Link between PTSD and Functionality among United States Military Service Members Psychiatrically Hospitalized Following a Suicide Crisis’ reported the additive effect of psychiatric comorbidities alongside PTSD on functional impairment .Also within this edition, Steven Koven published a meta-analysis of PTSD interventions . This paIt was our intention that readers of this special edition will be provided with new insights and will hopefully be inspired to contribute to the growing literature to help find better ways to support veterans with PTSD.


Pipe integrity is a central concern regarding technical safety, availability, and environmental compliance of industrial plants and pipelines. A condition monitoring system that detects and localizes threats in pipes prior to occurrence of actual structural failure, e.g., leakages, especially needs to target transient events such as impacts on the pipe wall or pressure waves travelling through the medium. In the present work, it is shown that fiber-optic distributed acoustic sensing (DAS) in conjunction with a suitable application geometry of the optical fiber sensor allows to track propagating acoustic waves in the pipeline wall on a fast time-scale. Therefore, short impacts on the pipe may be localized with high fidelity. Moreover, different acoustic modes are identified, and their respective group velocities are in good agreement with theoretical predications. In another set of experiments modeling realistic damage scenarios, we demonstrate that pressure waves following explosions of different gas mixtures in pipes can be observed. Velocities are verified by local piezoelectric pressure transducers. Due to the fully distributed nature of the fiber-optic sensing system, it is possible to record accelerated motions in detail. Therefore, in addition to detection and localization of threatening events for infrastructure monitoring, DAS may provide a powerful tool to study the development of gas explosions in pipes, e.g., investigation of deflagration-to-detonation-transitions (DDT). In recent years, the integrity of pipelines and piping systems in industrial plants has moved into the focus of public concern as the global piping infrastructure grows and ages. Moreover, to meet increasingly higher standards in technical safety, environmental compliance and failsafe performance the demand for a holistic pipe monitoring system are expected to rise even further in the future. Such a system should ideally be able to detect, classify, and localize a wide range of different threatening pipe conditions prior to the occurrence of leakages over the whole length of the pipeline and in real time. In the long run it should replace time-consuming and costly routine inspections, which are currently taking place at predetermined time-points .Since piping systems in industrial plants typically are highly branched and stretch over long distances the use of conventional point sensors is usually not practicably feasible to realize a monitoring system without gap. Therefore, the principle of distributed fiber optic sensing (DFOS) ,2 has coPresent DFOS techniques established in pipeline monitoring are widely based on distributed temperature sensing (DTS) and focus on leak detection ,4,5. DTSBesides the established methods to measure temperature and strain, distributed acoustic or vibration sensing (DAS/DVS) has lately received considerable attention as a means to detect and localize third party threats to pipelines, i.e., approach of vehicles, digging, and mechanical manipulation on the pipe ,6,7,8. TThe principle of distributed acoustic/vibration sensing relies on the detection of Rayleigh backscatter caused by naturally occurring inhomogeneities along an optical fiber by employing the measuring scheme of coherent optical time domain reflectometry (C-OTDR) ,22. BasiAlthough, strictly speaking, the term distributed acoustic sensing would imply detection of the full acoustic field including the phase, in many application fields it has become a convention to collect all C-OTDR schemes under the term DAS. For brevity, we will therefore employ this designation in the following.Undamaged pipe: incidents causing (dynamic) circumferential changes of the pipe or initiating propagation of acoustic waves in the pipe wall .Damaged pipe: changes inside the pipeline causing altered flow generated noise .Developing damage of the pipe: crack formation and propagation in the wall with accompanying acoustic emissions.In the sense of a holistic monitoring system described above, the so far not utilized potential of DAS as a means for continuous condition monitoring of pipes by detecting and localizing acoustic signals that point to certain damage scenarios, has been investigated in the interdisciplinary research project AGIFAMOR (Ageing Infrastructures—Fibre Optic Monitoring of Pipes) at BAM from 2015 to 2018 . The feaWhile the results for scenarios 2  and 3 2 are descSince the better part of the events falling into the first category is highly transient, e.g., impacts on the pipe wall or pressure waves travelling through the pipeline, it is necessary to develop a scheme for data analysis that makes do without any temporal averaging. Especially for steel pipes, which are commonly used, localization is additionally aggravated since each event causes acoustic waves propagating inside the pipe wall with the very high speed of sound around 5000 m/s and very little dampening. This means that dynamic strain changes are transmitted to the sensor fiber along many meters along the pipe within milliseconds, which could significantly reduce the spatial resolution of the measuring system. Therefore, tracking of propagating waves on a very fast time-scale is necessary to reveal the signal onset and the location of the initial source of disturbance.In any implementation of a (distributed) fiber-optic sensing system the application of the optical sensor fiber to the structure to be monitored is crucial. This is especially true for cases, where (dynamic) strain has to be transmitted to the fiber. Nevertheless, in most of the established applications of DFOS for pipeline monitoring, standard optical telecommunication cables are employed which are usually installed in the ground with some spatial gap to the pipeline. This very cost-efficient sensor installation is suitable for detection of temperature changes caused by leakages (DTS) and the above-mentioned detection of third-party intrusion by recognizing vibration patterns transmitted via the ground (DAS). However, to enable a true condition monitoring system that is sensitive to small changes in the piping system itself and amongst others needs to pick up circumferential changes of the pipe, installation of the optical fiber sensor directly on the pipe is a prerequisite.The optimization of the application method was one of the main aims of the project AGIFAMOR and in a first investigation comparing different ways of linear fiber application along a pipe the importance of a high quality fiber installation was underlined . AdditioAnother way to provide close contact is helical wrapping of the fiber sensor around the pipe. This type of application has already been employed when carrying out DSS measurements on pipes ,30 and hOn the down side, helical application of the optical fiber sensor to the pipe reduces the overall accessible length of pipe and is therefore expected to be of interest for short to medium pipe lengths. Additionally, it is noted that the bandwidth of the system is reduced in comparison to a linear fiber application on the same pipe length as the maximum pulse repetition rate depends inversely on the length of the fiber under test.However, in the sense of a feasibility study, we focus on optimal conditions with respect to spatial resolution and sensitivity. Therefore, in the work presented here, a bare fiber is used wrapped helically around the pipe under test without any additional glue to exclude influences arising from the adhesive. Two helical configurations are investigated in the following. The first corresponds to a fully distributed sensor system, where the fiber is continuously wrapped around the pipe with a defined pitch, while the second constitutes a quasi-distributed system, where the fiber is applied in spaced segments of densely wound fiber. The latter resembles the deployment of point sensors—as was for example investigated by Cui et al.  for acouFor a fundamental investigation of the capability of DAS in conjunction with a suitable sensor application geometry to detect and localize threatening pipe conditions in an undamaged pipe, as summarized under scenario 1, the present study was carried out in two sets of experiments.In the first set of experiments presented in this paper, we compare the fully distributed and quasi-distributed sensor configuration with respect to their capability to localize transient acoustic events occurring on a pipe. As a showcase application mechanical impacts on the outside of the pipe wall are analyzed. Different procedures for data evaluation and automatic impact detection and localization are introduced. Consequently, localization accuracy and precision are analyzed for different sensor configurations, data evaluation procedures and experimental settings of the DAS system.In the second part an example for monitoring critical conditions occurring inside the pipe, i.e., propagation of pressure waves, is presented. Data evaluation procedures introduced in the first part are employed to detect and localized such events. Additionally, we focus on separation of different acoustic signatures occurring at the same time and capturing of non-linear propagation.In all experiments a sensing system was used consisting of a standard single mode optical fiber  without jacketing and a commercial direct detection C-OTDR-interrogator . This system is specified for a maximum range of 40 km with optical pulse lengths between 10 ns and 1 µs and has a maximum pulse repetition rate of 100 kHz. The settings chosen in this study are specified for each experiment in the following paragraphs. For optimal signal transduction, the optical fiber was pre-strained and directly applied onto the surface of the pipe under investigation by helical wrapping using an inhouse designed wrapping device. Uniform and defined pre-strain was achieved by employing a loosely hanging fiber spool with an attached weight onto the sensor fiber during the wrapping process.Tp used to interrogate the fiber were optimized for each experiment. Longer Tp entail higher sensitivity but lower spatial resolution c is the speed of light in vacuum and neff is the effective refractive index of the optical fiber mode. Pulse lengths were chosen between 30 ns and 100 ns, corresponding to an expected spatial resolution of approximately 3 m to 10 m along a straight fiber stretch. With respect to localization along the pipe, spatial resolution is additionally increased by a factor given by the pitch of the helically wrapping of the fiber sensor and the outer diameter of the pipe. To capture transient processes on a short time scale with high precision the pulse repetition frequency fp was set to 80 kHz—lose to the maximum possible setting of the interrogator—for experiments targeting acoustic waves in the material and to 40 kHz for experiments targeting pressure waves in the medium. According to Nyquist’s theorem, this allows for registering of signals up to 40 kHz or 20 kHz, respectively. It is noted that the maximum monitoring distance Lm along the optical fiber inversely depends on the repetition frequency given by fp may be adjusted to lower values depending on the specific monitoring task and required bandwidth.Optical pulse lengths Position along the fiber is usually given in bins, where one bin corresponds to the portion of fiber from which the interference pattern is analyzed within the interrogator. With the system used one bin represents either 0.57 m or 0.68 m of fiber depending on the digital sampling settings (180 or 150 mega samples per second) for the two sets of experiments, respectively.Raw data recorded by the C-OTDR system was further processed using a home-written Python program. Transient events propagating along the pipe appear as edges within the three-dimensional plot of signal intensity versus time and position along the fiber. For optimal detection of these edges, we employ edge filtering by numerical differentiation of raw data along the time axis. Step width for calculating estimated derivatives was optimized for each experiment.After preparing data in this way, edges were automatically detected by thresholding the signal intensity within a certain bin or summed up intensity within a range of bins with respect to the noise level. The threshold level was set as a multiple of the standard deviation of the noise. From the signal onsets detected in this way the propagation velocity and/or localization of events was deduced by either slope fitting in the case of a fully distributed setup, or by calculation of the runtime difference for the quasi-distributed setup. The whole process of data analysis is summarized in For sensor ranges that can be approximated as localized sensors, data processing was carried out based on the procedure of random quadrature demodulation (RQD) proposed in  for thisNumerical simulation of the propagation of acoustic waves in cylindrical waveguides was carried out using the MATLAB toolbox PC disp (Pochhammer–Chree dispersion) . Pipe geFor laboratory-scale tests a stainless-steel pipe of 6.25 m length, inner diameter of 40.7 mm and outer diameter of 48.3 mm was simultaneously equipped with two separate sensor configurations. The pipe was open on both ends. Each configuration could be connected to the C-OTDR system independently. They are schematically depicted in Configuration 1 represents a fully distributed system, where the fiber is helically wrapped around the pipe on the full length. Pitch of the fiber was 2 cm, so that 7.9 m of fiber correspond to 1 m on the pipe. Altogether 39.5 m of fiber were applied to the pipe in this manner.Configuration 2 corresponds to a quasi-distributed setup with two localized sensors regions at the beginning and at the end of the pipe. Each of the two localized sensors sections consists of approximately 30 m of fiber densely wrapped around the pipe covering a region of 3.7 cm to 3.8 cm. They are separated by two loose coils of fiber (15 m each), which are decoupled from the pipe, to ensure separation of signals originating from the two sensor regions. The distance between the centers of the two sensor regions amounts to 5.98 m.In each setup, a coil of 200 m of fiber was introduced between the measuring system and the applied fiber stretch under test.Short mechanical impacts were produced by manually hitting the pipe with a metallic device either on the front face (A) or on the side (B) as indicated in Propagation of pressure waves within the medium inside a pipe was caused by gas explosions in a laboratory-scale gas-filled pipe. The setup for these experiments was placed in an underground bunker dedicated to pipe tests involving explosive gas mixtures. The pipe equipped with sensors and the ignition device was set up in a separate room whereas control of gas filling and ignition as well as data acquisition was carried out remotely. The pipe used for explosion tests was a stainless-steel pipe of 5.54 m length, inner diameter of 30 mm and outer diameter of 38 mm, which was sealed on both ends and contained two sensor penetrations.After evacuation of the pipe different gas mixtures were introduced into the pipe to the desired pressure level (0 to 10 bar overpressure). Consequently, ignition was carried out at one end of the pipe employing a melting wire. Gas mixtures used were 4.2% vol. propane in air and 28.5% vol. hydrogen in air.Overall guide values of pipe temperature were monitored using a conventional thermocouple placed close to the sensor penetrations on the surface of the pipe. Maximum temperatures throughout the course of the test series amounted to around 50 °C.As a first test of the ability of the system to localize short mechanical impacts on the pipe, the pipe was hit on the front face  m/s in agreement with the above value within the scope of the measurement uncertainties. The observed greater uncertainty when hitting the side of the pipe may be a result of less effective excitation of the present acoustic modes than for excitation on the front face.For shorter pulse lengths  and impacts in position B the signal-to-noise ratio is not sufficient for automatic edge detection and derivation of velocities. For these cases, acoustic propagation velocities are estimated manually from visual recognition of the edges. The derived values are approximately 5100 m/s for the main mode and 3300 m/s for the second mode using 50 ns pulses and 5100 m/s for the main mode using 30 ns pulses. All of these are in good agreement with the ones derived for position A.Localization of the source is readily obtained in all cases, as indicated in Localization of short impacts on a pipe equipped with a quasi-distributed fiber-optic sensor configuration as described in In each sensor region, the signal is detected simultaneously over all bins and is recurring periodically as the acoustic wave is reflected on both ends of the pipe. Therefore, the two sensor regions indeed act as point sensors with respect to the propagating acoustic waves, and signal processing using summation over the range of each localized sensor  is a valid approach. The potential of such an approach is illustrated in Additionally, the recurring nature of the signal may be used for data evaluation by correlation of the two time traces presented in If excitation takes place on the side of the pipe  and are discarded for further processing of the data. A linear fit of the remaining points yields a velocity of the pressure wave of (380 ± 10) m/s. The average velocity determined by the two conventional pressure sensors is 406 m/s and is compatible with valued derived by DAS measurements.For ambient pressure, i.e., 0 bar initial overpressure, similar signal features are observed as in the experiments using propane in air as seen when comparing At 2 bar initial overpressure a more complex signal shape is observed b. The auFirst, a higher acoustic intensity is noticed leading to much more pronounced signal features of the structure-borne waves (marked in yellow) in the filtered data so that automatic signal evaluation is possible—albeit with a rather high uncertainty. The deduced velocity amounts to (4850 ± 1360) m/s in agreement with former results.Moreover, propagation of the pressure wave along the observed length of pipe is non-linear and may be roughly divided into two linear regions with different slopes. In the first region as seen from the place of ignition  and shows an overall agreement with the gradient of the onset points.For comparison of the propagation velocities of structure-borne acoustic waves inside the pipe material determined by distributed acoustic sensing to theoretical predictions, a numerical simulation of the propagation of acoustic waves was carried out for the chosen pipe geometry. The calculated dispersion curves for the different acoustic modes expected in a cylindrical waveguide are presented in Interestingly, the (frequency independent) group speed of the torsional mode T  is 3130 m/s indicating this as the second mode observed in some of the experiments with velocities in a range of 3000 m/s to 3300 m/s.2 leakage from transportation pipes. Propagation velocities of acoustic waves traveling in the pipe material were determined in a laboratory scale experiment on a steel pipe of comparable dimensions as the ones used here, i.e., 6 m length, 50 mm outer diameter, and 46 mm inner diameter, employing two acoustic emission sensors and Nielsen-Hsu pencil lead break tests as excitation source. Two main modes with velocities of 5070 m/s und 3268 m/s were derived in agreement with the findings of the present study.Recently, a study  was presIn the first set of experiments, which were carried out with a fully distributed sensor configuration, the determined position of the disturbance and the localization precision can be directly read from a spatial-temporal representation of raw or adequately filtered data. Localization accuracy is basically determined by the accuracy of the mapping of bins to actual positions along the pipe. This can be done in a calibration procedure after installation of the fiber where the fiber is tapped in crucial places and the observed signals are assigned to the position. This procedure may be carried out with the shortest pulse length available yielding an expected accuracy determined by the spatial resolution of the system. Here, this amounts to When employing a quasi-distributed sensor configuration knowledge of the propagation velocity in the material is a prerequisite to map the observed events to a position on the pipe based on the time difference of signal onset. In practice, this value could be either taken from a theoretical consideration or from a calibration procedure with localized signals in known positions.For the sake of analyzing localization accuracy here, the relative deviation of the experimentally determined speed values from the theoretical value confirmed by numerical simulation is used as a measure for the deviation from the actual point of impact. The localization accuracy is depicted in A summary of the deduced localization uncertainty for different sensor configurations and evaluation procedures is given in However, the positions of impact determined for the quasi distributed configuration by evaluation of signal onsets hints at an additional systematic deviation to one side of the points of impact corresponding to systematically underestimated time differences between the two localized sensors. This is also evident in the trend to somewhat higher speed values  deduced Overall, the quasi distributed sensor configuration tends to produce smaller localization uncertainties, especially at longer pulse lengths than the fully distributed one for the setup with a short pipe segment used here. This is attributed to the possibility of signal averaging over a certain bin range and resulting higher signal-to-noise levels. However, for longer pipes it is expected that signal intensities and localization precision will deteriorate if the distance between disturbance and the localized sensors increases. This effect was also observed by Cui et al.  for locaFor selection of an optimal application geometry in an industrial environment these advantages and disadvantages must be considered alongside issues concerning accessibility of pipes, overall range to be monitored and the required spatial resolution.Moreover, for practical pipes, which often have a larger diameter than the ones inspected here, the localization precision and accuracy would increase when employing similar fiber pitch as in the experiments shown here. However, in this case the monitoring distance along the pipe would be decreased and/or the bandwidth restricted further. Therefore, it is expected to be favorable to adjust the fiber pitch to larger values in order to cover longer stretches of pipeline, as in many practical cases lower spatial resolution than the one reached here should be sufficient.In the set of experiments carried out to detect and localize propagating pressure waves in pipes it was found that the fully distributed fiber-optic sensor configuration is indeed able to track such waves continuously along the pipe. Besides the intended use in condition monitoring this opens new perspectives in the fundamental study of gas explosions.In general, gas explosions can be subdivided into two groups with tremendous differences in their consequences on pipeline structure. While deflagrations are characterized by velocities below the speed of sound in the medium and explosion pressures on the order of 10 bar above initial pressure, detonations propagate with supersonic velocities and pressures an order of magnitude larger usually causing catastrophic damage. Due to the complex interplay between fluid dynamics, heat transfer and turbulences depending on the specific gas mixture and pipeline configuration, determination of occurrence and location of deflagration-to-detonation transitions (DDT) is still a topic of current research ,38.pex being the explosion and p0 the initial overpressure, this is characteristic of a deflagration. A similar situation was found in the experiment with a gas mixture of hydrogen in air ignited at ambient pressure.Here, both measurement techniques, DAS and piezoelectric pressure transducers, confirmed a (constant) velocity in the range of the acoustic speed inside the medium after ignition of a gas mixture of propane in air. Together with the observed pressure values of The advantage of using a fully distributed sensor as represented by the distributed acoustic sensing system, emerges in cases where a non-linear propagation takes place. This is illustrated in the experiment where a gas mixture of hydrogen in air was ignited at 2 bar initial overpressure. The supersonic propagation velocity and pressure values of Therefore, DAS, together with an appropriate sensor configuration, may be used as tool for fundamental studies on the behavior of gas explosions in pipes. As shown in the first part of this paper, the present helical sensor configuration may allow to localize such events with a spatial resolution down to ~20 cm. This would correspond to installing 30 piezoelectric pressure transducers on a pipe of 6 m length to achieve the same resolution.In the present study, the capability of DAS to detect and localize transient events on pipes was investigated. It was found, that in conjunction with a fitting sensor configuration of the optical fiber, mechanical impacts can be identified and localized along the pipe. Depending on the favored application, a fully distributed configuration, represented by continuous helical wrapping of the sensor fiber around the pipe or a quasi-distributed configuration consisting of short local sections of densely wound fiber, may be favorable. The localization accuracy and precision in general depends on the chosen fiber configuration, experimental settings  and the procedure of data evaluation. For the parameters tested here, values down to 20 cm were achieved. In summary, the methods presented in this study may be understood as a toolbox for practical implementation of DAS on pipes.In a second set of experiments the technique was applied for spatially continuous tracking of pressure waves in pipelines following gas explosions. It was demonstrated that the employment of DAS with a fully distributed sensor configuration not only allows for observation of propagation with a constant velocity but also of accelerated motion. In contrast to conventional testing of gas explosions with local pressure sensors, the proposed technique may provide a means to identify the point of deflagration-to-detonation transition without gap and without a priori knowledge of its location.The approach is therefore of interest not only in condition monitoring of pipes but also in investigations of the behavior of gas explosions in closed pipe systems.


The presence of grain scratches in wet blue hides (76.3%) was significantly higher than in pickled sheep (67.2%) and wet blue goat (59.1%) skins. The major slaughter defects included flay cuts or scores, holes, poor pattern and vein marks, with a higher occurrence in wet blue goat skins  than in wet blue hides (22.8%) and pickled sheep skins (11.1%). The most prevalent post-slaughter defects were grain cracks (14.9%), hide beetle damage (8%), damage caused by heat or putrefaction (3.7%) and machine-induced defects (0.5%). Grain cracks (27.04%) and hide beetle damage (13.9%) in wet blue goat skins were significantly more common than in wet blue hides and pickled sheep skins. These defects cause depreciation in the value of the hides and skins. Statistically significant (P < 0.001) higher rejection rates were recorded for wet blue hides (82.9%) than for pickled sheep skins (18.3%) and wet blue goat skins (8.5%). Improved animal health service delivery, effective disease control strategies and strong collaboration between stakeholders are suggested to enhance the quality of skins and hides.Skins and hides are perishable resources that can be damaged by parasitic diseases and human error, which result in downgrading or rejection. This study was conducted to identify defect types and to determine their prevalence in pickled sheep and wet blue goat skins and wet blue hides. Each selected skin or hide was examined for defects in natural light and the defects were graded according to established quality criteria in Ethiopian standard manuals. Major defects were captured by digital photography. The major pre-slaughter defects included scratches (64.2%), cockle ( Ethiopia has 53.4 million cattle, 25.5 million sheep and 22.7 million goats. These numbers illustrate a considerable potential for the leather industry in the country , with export earnings for the country ranging from $405 to $590 million between 1998 and 2004  of the raw stock, and insufficient collection and preparation for further processing and export.ekek), which is due to an allergic skin hypersensitivity reaction to parasitic infestation, grain scratches, pox lesions, warts, tick damage, branding, age (shrinkage) and poor substance . The major slaughter defects are flay cuts (scores), holes (a complete perforation of the skin or hide resulting from a knife or flaying appliance), poor pattern (an asymmetric skin or hide due to bad opening cuts or distortion during drying because of uneven tension), and vein marks (traces of blood vessels in the skin where the blood was not completely drained). Post-slaughter defects include heating or putrefaction , hide beetle damage, machine damage and grain crack , which is located in Wukro in eastern Tigray, a northern regional state of Ethiopia. It is about 830 km from the country's capital city, Addis Ababa, and 47 km from Mekelle, the capital city of the region. The tannery obtains raw sheep and goat skins and hides mainly from the Tigray region, as well as from Addis Ababa, Gondar, Wollo and the Amhara region . Cockle lesions were seen mainly on pickled sheep skins (68.0%) and wet blue hides (5.7%), with only 1.0% of wet blue goat skins being affected. Pickled sheep skins were more than 200 times more likely to be affected by cockle than wet blue goat skins and the difference was statistically significant . Comparable defects due to pox were observed more often on wet blue goat skins (10.8%) and wet blue hides (8.1%) than in pickled sheep skins (1.2%). Wound or scar damage was seen more often in cattle hides (66.1%) than in sheep (11.0%) and goat (6.1%) skins. This defect was statistically significant and wet blue hides were 34.22 times more likely to be affected than wet blue goat skins . Wet blue hides and pickled sheep skins were, respectively, 22% and 90% less likely to be affected by pox or LSD virus than wet blue goat skins . Defects due to branding were more common on wet blue hides (28.8%) than on wet blue goat skins (0.8%) and pickled sheep skins (0.1%). This difference was statistically significant, with wet blue hides almost 50 times more likely to have branding marks than wet blue goat skins . Defects caused by poor substance in pickled sheep skins (9.8%) were significantly more common (P < 0.001) than in wet blue goat skins (0.8%) and wet blue hides (0%). Tick damage was observed only in wet blue hides (22.7%). The presence of warts was limited in both skins and hides.The presence of grain scratch in wet blue hides (76.3%) was significantly higher than in pickled sheep (67.2%) and wet blue goat skins (59.1%). Compared with wet blue goat skins, wet blue hides and pickled sheep skins were respectively found to be 2.8% and 1.4% more likely to be damaged by scratches  in wet blue goat skins (28.7%) than in wet blue hides (22.8%) or pickled sheep skins (11.1%), which were respectively 23% and 51% less likely to be damaged by knife cuts than wet blue goat skins . Holes were the major defect in wet blue hides (17.5%) and more common than in the skins of pickled sheep (6.7%) and goats (5.6%). A higher proportion (P < 0.001) of poor pattern was seen on wet blue goat skins (3.7%) than on pickled sheep skins (0.5%) or wet blue hides (0.0%). Wet blue hides presented with vein marks (11.5%), whereas no vein mark defects were observed on wet blue goat skins and pickled sheep skins.The major slaughter defects were flay cuts or scores (23.8%), holes (7.0%), and poor pattern and vein marks (0.9%). Defects due to flay cuts and scores occurred significantly more often , hide beetle damage (8%), heat or putrefaction (3.7%) and machine damage (0.5%). Grain crack was the major post-slaughter defect in wet blue goat skins (27.04%). Severe damage was caused by hide beetles in wet blue goat skins (13.9%) and in wet blue hides (4.6%). No beetle damage was found in pickled sheep skins. Putrefaction of hides (21.8%) was significantly more common than in wet blue goat and sheep skins . Post-slaughter defects observed in pickled sheep and wet blue goat skins included damage caused by heat or putrefaction (3.5%) and machine processing (0.9%), respectively.Damage due to single or combined defects was responsible for skins and hides being downgraded or rejected, with 82.9% of wet blue hides, 18.3% of pickled sheep skins and 8.5% of wet blue salted and dry goat skins being rejected .P < 0.001). Grain scratch resulted in the downgrading and rejection of 67.2%, 71.2%, 49.4% and 72.3% of pickled sheep skin, wet blue or salted goat skin, wet blue or dry goat skin, and wet blue hides, respectively (data not shown). Pox accounted for 1.5%, 15.5%, 6.8% and 8.3% of pickled sheep skins, wet blue or salted goat skins, wet blue or dry goat skins and wet blue hides, respectively, being rejected. Cockle caused rejection in 68.0%, 1.8%, 0.3% and 5.6% of pickled sheep skin, wet blue or salted goat skin, wet blue or dry goat skin and wet blue hides, respectively. Branding caused rejection in 0.05%, 0.9%, 0.6% and 28.8% of pickled sheep skins, blue or salted goat skins, wet blue or dry goat skins and wet blue hides, respectively. Cracked grain resulted in rejection in 49.1% wet blue or dry goat skin. Hide beetle damage caused downgrading or rejection of 25.2% and 4.4% of wet blue or dry goat skins and wet blue hides, respectively.There was a statistically significant difference in rejection rate across the three product types. Wet blue hides and pickled sheep skins were, respectively, 52.49 and 2.42 times more likely to be rejected than wet blue goat skins , poor substance, brandings, and wounds and scars resulting from pox, LSD or tick infestations. These observations are in agreement with those of a study by Kidanu (The production of good-quality leather depends on the quality of raw stock. Defects in leather are costly from a production point of view and greatly reduce the sale value of the leather. Damage and defects can occur from birth to the completion of the leather processing (Leach & Wilson y Kidanu . Howevery Kidanu  reportedy Kidanu  reportedy Kidanu  and Tefey Kidanu  reportedy Kidanu  indicatePox and LSD lesions were observed on wet blue goat skins, wet blue hides and pickled sheep skins. The animals infected by pox or the LSD virus develop blisters with small red spots, followed by the appearance of papules, vesicles, pustules and pox lesions, or deep, yellow, pea-sized oval ulcers that cause permanent scarring Boden . Accordiet al.The major slaughter defects found in this study were flay cuts or scores, holes, poor pattern and vein markings. Flay cuts and scores are caused by the careless use of a knife during skinning of the carcass because of poor technique ESGPIP . This waThe post-slaughter defects observed in this study included grain crack, hide beetle damage, and putrefaction, heat or machine defects. Similar post-slaughter defects in hides and skins have been reported previously ESGPIP . Grain cet al.et al. (The mentioned defects lead to downgrading or rejection of skins and hides. In this study, a large proportion of wet blue hides were found to be rejected, followed by pickled sheep skins and wet blue goat skins . At the .et al.  documentIt is not realistic to expect animal hides or skins to be perfect and defects are almost always present to some extent. Such defects cause depreciation in the value of the hides and skins and the consequence is that farmers, traders and the tanning industry suffer considerable financial losses. We found pre-slaughter defects to be more common than slaughter and post-slaughter defects combined. To improve the quality of hides and skins, animal health service delivery needs to be strengthened and there is a need for effective strategic planning, monitoring and assessment of disease control programmes. Awareness creation, training, and increased collaboration between the Bureau of Agriculture, international and regional organisations, research institutions and the Ethiopian leather associations can address unnecessary losses in the leather industry.


The aim of this paper was to conduct a baseline survey of HPV infection in unvaccinated women in Xinjiang Uyghur Autonomous Region before the mass use of HPV vaccine.Between 2008 and 2018, the HPV genotype detected by a PCR-based hybridization gene chip assay of 37,722 women who were from Gynecology Department and Health Management Center of the First Affiliated Hospital of Xinjiang Medical University were tested HPV genotype by a PCR-based hybridization gene chip assay. All statistical analysis methods were performed with this statistical software including Python version 3.6.1, R Software 3.5.1 and Excel 2011.The total positive rate for HPV was 14.02%, the most prevalent genotypes were HPV 16 (3.79%), HPV 52 (2.47%), HPV 58 (1.76%), HPV 53 (1.35%) and HPV 31 (0.72%). The single infection (11.34%) and high-risk HPV (HR-HPV) infection (9.72%) was the main prevalence of HPV. Age-specific HPV distribution was presented as a bimodal curve, while the youngest age group (≤25 years) presented the highest HPV infection rate (20.78%), which was followed by a second peak for the 36–40 age group. According to the ethnic stratification, the HPV infection prevalence ranging from the high to low was: Mongol (16.36%), Hui (15.15%), Kazak (14.47%), Han (14.43%), Other (14.37%), Uygher (10.96%). From 2009 to 2013, the HPV infection rate fluctuated but did not changed much. It peaked in 2014 and then fell significantly, reached the bottom point in 2017 and rose slightly in 2018. In 2015, the infection rate of HPVl6 and 52 in the population was almost the same (both 3.40%) the infection rate of HPV52 type (3.31%) was higher than that of HPVl6 type (2.18%) and became the dominant type in 2016.We present data regarding the prevalence and type distribution of HPV infection, which could serve as the valuable reference to guide nationwide cervical cancer screening. These baseline data enable the estimates of maximum HPV vaccine impact across time and provide critical reference measurements which are important to the assess of clinical benefits and potential harms in HPV vaccination and the increase in non-vaccine HPV types. Cervical cancer (CC) is the fourth most common cancer in women worldwide. Every year, approximately 570,000 new cases and 310,000 deaths of CC are reported around the world, 85% of which have taken place in developing countries . These nHuman papillomavirus (HPV) infection is the most common sexually transmitted viral infection worldwide, with approximately 75% of sexually active men and women exposed to HPV during their lives . HPV is The discovery that the persistent HR-HPV infection causes cervical cancer and the HPV is a necessary cause of cervical cancer , 11 has Cervical cancer vaccines have been available in more than 130 countries and regions around the world over the past decades and are widely vaccinated among females. Based on good clinical protection and safety data from HPV vaccines, the world health organization (WHO) has also issued recommendations to encourage the use of HPV vaccines in the appropriate population to reduce the incidence of cervical cancer. Data released by the National Bureau of Statistics of China showed that the total population of the mainland was 1.375 billion at the end of 2015, among which the female population was 670,480,000(48.76%), the proportion of women aged 0–45 was about 84.02%, suggesting that a large number of women were at an age appropriate for HPV vaccination. The first approved HPV vaccine was officially launched on July 31, 2017 in China. At present, only a small number of females of the right age have been vaccinated against HPV-related disease in China, especially in Xinjiang. Up to now, there was no large sample study on the genotype prevalence of HPV in Xinjiang. The aim of this paper was to conduct a baseline survey of HPV infection in women in Xinjiang before the mass use of HPV vaccine. It would lay a foundation for the observation of the efficacy of HPV vaccine and HPV genotype change after vaccination in the future.A total of 37,722 females aged 13–89 years were enrolled in this study from November 2008 to July 2018. All participants were from Gynecology Department and Health Management Center of the First Affiliated Hospital of Xinjiang Medical University. The patients visited the hospital for various reasons, including physical examination, infertility, vaginitis, cervicitis, undiagnosed abdominal pain, genital warts and cervical intraepithelial neoplasia. Clinical data were collected from the participants, and a molecular survey of HPVs was conducted. All the women enrolled in this study had sexual life history, and the study excluded those who were pregnant at the time of enrollment, who had been vaccinated against HPV, who had undergone hysterectomy, who suffered from cervical cancer and received radiation and chemotherapy, and who had undergone colposcopy in the 24 months prior to the enrollment. All participants were told to refrain from sexual activity and avoid washing the genitals for 48 h before sample collection. Eligible women were included in the study after signing an informed consent form.Quality control of HPV genotyping and data input procedures were implemented throughout the study, which was conducted in accordance with the Good Epidemiological Practice Guidelines and the Principles of the Declaration of Helsinki and was approved by First Affiliated Hospital of Xinjiang Medical University ethics committee. Quality control of HPV genotyping included sample quality requirements, test quality requirements, test report entry and issuance, and molecular diagnostic item analysis performance standards. The sample container was a disposable HPV collection tube. When receiving the sample, there was a clear identification and the uniqueness of identification was ensured. The sample for secondary testing could be traced back to the original sample, and the label on the sample was consistent with the label on the application form. Samples were rejected under the following circumstances: if the application form was filled with illegible handwriting or there was doubt, the sample storage solution was less than 1 ml, and the volume of the sample transferred to the secondary catheter was less than 1 ml. The original sample, nucleic acid extractive and/or nucleic acid amplification products stored in − 80 °C refrigerator, were used for recheck. Test Quality included External Quality Assessment (EQA) and Internal Quality Control (IQC). HPV test in our hospital participated in the quality evaluation system of the clinical test center of the national health and family planning commission, and all of them were qualified. Qualified laboratory staff operate were in strict accordance with standard operating procedures, and weakly positive and negative quality control products were randomly placed among clinical samples for each test to monitor the accuracy of the experiment. Laboratory staff recorded the experiment process in detail, analyzed the quality control results, if the results were out of control, the reasons for the control would be judged, the corrective measures would be taken, and then the corrective results would be evaluated. The person in charge of the laboratory should regularly supervise the results of internal quality control. When the experimental data were input, double check was used. The original experimental records were retained for each experiment. For the failed experiment and unsatisfactory results, the reasons were analyzed and the recheck was conducted. Molecular diagnostic item analysis performance standards: After equipment fault repair, analysis system comparison: 5 samples, covering the test interval, at least 4 samples measurement results bias<±7.5%. Retention sample retest criteria: according to the stability requirements of the project, the longest duration samples were selected, with 5 samples covering the test interval and at least 4 samples with the test result bias<±7.5%. Test criteria for inter-batch difference of reagent: 5 samples tested with old batch number, covering measurement interval , and at least 4 samples with test result bias<±7.5%.After the cervix uteri were fully exposed with the aid of a vaginal speculum, a specially designed cervical brush (known as a cytobrush) gently scoured the cervix uteri through clockwise 5 rotations to obtain the cervical secretions and cells needed for further testing. The samples were placed in a cell preservation solution and stored at 2–8 °C until HPV DNA extraction and genotyping could be performed. The HPV genotype testing of the samples was completed within a week.The transfer of samples is the responsibility of the nursing staff, the hospital is equipped with a portable thermostat to facilitate the transfer of special specimens, HPV typing test samples belong to this kind of special specimens.Our process was that the clinician taked the samples and placed the samples in the refrigerator of the department (2–8 °C), and then the nurse placed the samples in a portable thermostat (2–8 °C), finally the samples were put in the laboratory refrigerator. Since 2008, our hospital began to carry out HPV typing detection. This study retrospectively analyzed all patient data in the existing database that were complete and available.This part of methodology has been elaborated in my previous articles  and willAnalyses were done with Python (version 3.6.1), R Software (version 3.5.1) and Excel (version 2011). Descriptive statistical analysis was performed on the distribution of HPV genotypes using indicators such as frequency and prevalence. Chi square testing was used to test for the differences in HPV prevalence between simple infection and multiple infection in different age groups, time groups, and ethnic groups. Chi square tests were also conducted to test for differences in HPV prevalence among HR HPV infection, LR HPV infection and Mixed HPV infection in different age groups, time groups, and ethnic groups..Through nearly a decade of surveillance, a total of 37,722 objects’ samples were collected and detected by HPV genotype. The genotype test showed 5287 samples were HPV positive, so the overall prevalence of HPV in the study population was 14.02% for all typesThe details of genotypes distribution of HR-HPV and LR-HPV were shown in Table As shown in Table P < 0.05), while there were no statistical differences in the distribution of single and multiple infections (P > 0.05), relevant data could be seen in the Table Overall, 37,722 women (age 13–89 years) were included in this study. All the participants were divided into eight groups ranging from ≤25 years, 26–30 years, 31–35 years, 36–40 years, 41–45 years, 46–50 years, 51–55 years, and ≥ 56 years. Among the eight age groups, there were significant differences in the distribution of pure HR, pure LR and mixed HPV infection , however, the HPV prevalence dropped drastically after the first peak, maintained a plateau for 10 years before reaching the second peak (15.45%) at 36–40 age group, then followed by a moderately decline, finally reached the bottom at ≥56 age group (11.52%) , while there were no statistical differences in the distribution of single and multiple infections (P > 0.05) , pure HR, LR, and mixed HPV infection (P < 0.05) , Eastern Europe 21.4%), Latin America (16.1%), and Southeastern Asia (14%) successively . It was .4%, Lati.0%, EastIn this research, HPV 16 (3.79%), 52 (2.47%), 58 (1.76%), 53 (1.35%), and 31 (0.72%) were the five most common HPV genotypes among females while HPV 16, 52, 58, 33, 18 were regarded as the most prevalent genotypes in China . NeverthInformation on the distribution of HPV infection at different ages was critical for the design of age-specific prophylactic HPV vaccines. Regarding the HPV prevalence in different age groups, there were similarities and differences in scholars’ research. What many studies had in common was that the first peak appeared at a younger age group . In some regions, a modest second peak was observed at age >40 years or >45 years or >55 years or >60 years. In some regions, this second peak was not observed. To sum up, age-specific HPV distribution presented as either a bimodal curve (including the “U” curve) or a unimodal distribution skewed to the left , 22, 26.International correlation between the prevalence of HR-HPV infection in the general population and its cervical cancer burden has been proven . It is wIn this study, from 2009 to 2014, the HPV infection rate maintained at a high level (20.62% in 2009) and reached peak in 2014 (29.80%), which might be attributed to the change of cervical cancer screening strategy. In 2011 , the recThe results of this study showed that HPVl6 and 52 were common infection genotypes in Chinese population, which was consistent with the results of most cross-sectional studies in China –29. HoweThe major strengths of this study were the large number of patients, in addition, the study was a decade long, so we could see a yearly trend of HPV infection in Xinjiang. However, several limitations should be acknowledged. First, the data of HPV infection were not further confirmed with cytopathology and histopathology, resulting in the effectiveness of HPV screening could not be accurately assessed. Especially, we didn’t have any additional information from the women testing that the HR-HPV was positive but the liquid-based cytology was negative, hindering a more comprehensive evaluation of the role what HPV testing played in screening in this population. Furthermore, a follow-up study should be done to track changes in genotype, cervical pathology and cytology as there was a close relationship between cervical carcinoma and long-term persistent HR-HPV infections. Finally, the results relied on samples referred to our laboratory for diagnostic purposes. Thus, these data cannot be considered nationally representative because the Xinjiang women included in the study might not be a representative sample of all the China women living in the country.In conclusion, the most significant findings in this survey consisting 37,722 samples in Xinjiang Province are as follows: (1) the prevalence of HPV infection showed variations by age, nationality and year. (2) HPV 16, 52, 58, 53, 31 were always the major genotypes, although the rank varied according to the age, nationality and year; particular attention should be paid to the high prevalence of non-vaccine genotypes (e.g. HPV53). (3) Although the prevalence of HPV had decreased in recent years, the HR-HPV infection and the changes of the main infection type cannot be ignored . Our results would provide guidance for primary screening and vaccination program for cervical cancer.


Recurrent Respiratory Papillomatosis (RRP) is a rare disease of the aerodigestive tract caused by the Human Papilloma Virus (HPV) that manifests as profoundly altered phonatory and upper respiratory anatomy. Current therapies are primarily symptomatic; enhanced insight regarding disease-specific biology of RRP is critical to improved therapeutics for this challenging population. Multiplex PCR was performed on oral rinses collected from twenty-three patients with adult-onset RRP every three months for one year. Twenty-two (95.6%) subjects had an initial HPV positive oral rinse. Of those subjects, 77.2% had an additional positive oral rinse over 12 months. A subset of rinses were then compared to tissue samples in the same patient employing HPViewer to determine HPV subtype concordance. Multiple HPV copies (60–787 per human cell) were detected in RRP tissue in each patient, but a single dominant HPV was found in individual samples. These data confirm persistent oral HPV infection in the majority of patients with RRP. In addition, three novel HPV6 isolates were found and identical HPV strains, at very low levels, were identified in oral rinses in two patients suggesting potential HPV subtype concordance. Finally, somatic heteroplasmic mtDNA mutations were observed in RRP tissue with 1.8 mutations per sample and two nonsynonymous variants. These data provide foundational insight into both the underlying pathophysiology of RRP, but also potential targets for intervention in this challenging patient cohort. Our laboratory previously reported a much higher rate of oral HPV infection in patients with RRP compared to the general population3. Specifically, greater than 90% of patients with RRP lesions were HPV positive in the oral cavity. Based on these preliminary findings, we hypothesized that HPV DNA was likely released from RRP lesions into the oral cavity, and as such, patients with RRP maintain HPV positivity in the oral cavity beyond the reported clearance interval of approximately 7 months in healthy adults2.Recurrent Respiratory Papillomatosis (RRP) is a rare disease characterized by recurrent papillomatous lesions of the upper aerodigestive tract in adults and children caused by the Human Papilloma Virus (HPV). Current therapies are limited beyond symptomatic, operative management potentially related to our collective lack of understanding regarding disease-specific biology of HPV in RRP in spite of increased insight regarding the role of HPV in the pathogenesis of malignancy. Oral HPV infection has been strongly associated with oropharyngeal squamous cell carcinoma5. Specifically, metagenomics allows for characterization of the entire HPV genome and interactions between the HPV and host genomes6. In the current study, we employed a novel method of HPV detection using metagenomic data to analyze specimens containing HPV DNA isolated from oral rinse and laryngeal tissue samples from patients with RRP. We also sought to provide additional insight regarding potential mechanisms underlying RRP persistence/recurrence. In that regard, the role of aberrant mitochondrial function in cancer has been of particular interest for decades7. Specifically, mitochondrial DNA (mtDNA) mutations have been reported in renal adenocarcinoma, colon cancer cells, head and neck tumors, astrocytic tumors, thyroid tumors, breast tumors, ovarian tumors, prostate and bladder cancer, neuroblastomas, and oncocytomas14. mtDNA mutations have been considered essential factors for tumorigenicity17. Reactive oxygen species generated from mitochondria intensify the tumorigenic phenotype and induce additional genome mutations18. To date, mtDNA mutations have not been described in RRP and could potentially provide additional insight regarding the pathophysiology of RRP with the ultimate goal of identifying novel therapeutics.The HPV subtype(s) identified in RRP lesions and oral DNA must be consistent to further this hypothesis. In the current study, we sought to provide foundational insight into these issues employing novel sequencing technology. Significant advances in genome sequencing allow for disease-specific HPV genomic data and metagenomic sequencing of human tissues allow for expanded and enhanced HPV detection methods for all HPV subtypes (182 types) as HPV sequences can be identified across the whole genome rather than just L1Twenty-three patients had multiple oral rinse specimens for analysis with a maximum of five specimens per subject; positivity was determined qualitatively via consensus across investigators Fig. . Twenty Shotgun sequencing of both oral rinse and tissue samples was performed on a subset of patients who underwent operative resection of RRP lesions and oral rinse acquisition within a reasonable interval, as described in the Methods. HPV6 was detected in four RRP lesions and HPV11 was detected in the remaining two lesions. Copy number of HPV genomes in the lesion specimens ranged from 60–787 per human cell. According to the alignments of HPV reads to their prototype genomes, HPV in RRP specimens were different with the prototype HPV in genome level and a single dominant HPV strain in each individual sample. HPV reads found by sequencing rinses from two patients were identical to HPV sequenced from RRP tissue from those same patients Fig. . Only twAfter alignment of HPV reads to HPV Reference genomes with Bowtie2, the genome of dominant HPV type was reconstructed, if all base pairs of the genome were covered by metagenomic reads more than 10 times. The genomes of three different HPV6 strains and one HPV11 strain were rebuilt from the four RRP tissue samples  with at least 273 copies of HPV genome per human cell Table . The phyhttps://www.mitomap.org/MITOMAP).mtDNA mutations were observed in the tissue samples and were identified as nucleotide changes in mtDNA compared to mtDNA in matched oral rinse samples to avoid the germline mutations. Somatic mtDNA mutations were observed in six RRP tissue specimens with 1.8 mutations per sample; all were heteroplasmic  or Proline (P) is conserved across multiple species. Of note, both of these amino acids have nonpolar side chains, but the mutated T has a polar side chain; this COI mutation may also lead to dysfunction. These hypotheses warrant further investigation.Since both nonsynonymous mutations, A336T in COI and G215D in ND5, have not been reported previously, we hypothesize that healthy subjects will not present with these mutations; however, further investigation is certainly warranted. In addition, the connection between RRP pathophysiology and these mutations remains unclear. However, at position 215 of ND5, Glycine (G) is conserved across multiple species: 3. In addition, the current study did not include analysis of control rinses; our data in patients with RRP were qualitatively compared to impressive, previously-published results regarding the prevalence of oral HPV infection in the United States28. Conversely, the sensitivity of shotgun sequencing analysis for HPV DNA was limited by the overabundance of human DNA in our specimens. Although these two methods are both useful, each carries non-trivial limitations with regard to data yield. Additionally, the technique is prone to false-negatives due to saturation by competing HPV DNA in small amounts29. Regardless, these data provide insightful and foundational information regarding this particularly morbid disease process.The current study is not without limitations. As with previous studies employing qualitative PCR, the longitudinal data are limited by diminished capacity to capture multiple concurrent HPV subtypesThe current study was approved by the Institutional Review Board at the New York University School of Medicine; informed consent was obtained from all participants. In addition, all methods in the current study were performed in accordance with all relevant guidelines and regulations. Oral rinses were collected from 23 subjects with pathology confirmed adult-onset RRP  every 90 days over 12 months. Subjects ranged from 30 to 80 years with a mean age of 49 years (SD = 13 years). Tissue blocks were analyzed from a subset of six of subjects  based on close temporal proximity between the acquisition of both oral rinse and tissue samples during operative resection of disease as a component of standard clinical care. The interval between oral rinse and tissue acquisition ranged from 0 to 18 weeks with a mean of 5.7 weeks.g. DNA in the cell-free supernatant was precipitated with the isopropanol/glycogen solution and pelleted for 10 minutes at 2000 g, as previously described3. The pellet was resuspended with 200 µL DNA Hydration Solution (Qiagen) and DNA was extracted using the Gentra Pure-gene Buccal Cell Kit (Qiagen), according to the manufacturers’ protocol, modified slightly. Rinse samples were spun for 10 minutes at 3,200 g and after transferring the supernatant to a isopropanol/glycogen solution, samples were mixed by inverting 50 times and spun again for 10 minutes at 2,000 g. DNA hydration solution (200 mL) was added to the pellet.Subjects were instructed to swish 5 mL of Scope® mouthwash without gargling for one minute. Samples were then sealed and stored for no more than one week at 4 °C prior to DNA extraction. For DNA extraction, samples were spun for 10 minutes at 3200 PCR was performed using the biotinylated PGMGY09/11 consensus primer system  to detect a broad spectrum of HPV genotypes. Briefly, 10pmols of the PGMY09/11 and 2.5pmols of the PCO4/GH2O sequence were employed per reaction. For each reaction, 4 mM MgCl2, 400 µM dNTP, 2.5 mL of GC enhancer, and 7.5U of Amplitaq Gold DNA Polymerase were used per 50 mL reaction. Subsequently, 8 µL of DNA plus water were used per reaction with RNAse-free water as a loading control. PCR was performed using an Eppendorf Mastercycler  with the following settings: 50 °C for 2 minutes, 95 °C for 10 minutes and 40 cycles of 95 °C for 1 minute, 55 °C for 1 minute, and 72 °C for 1 minute. This protocol was followed by a final extension at 72 °C for 5 minutes and then 4 °C indefinitely. PCR products were then analyzed on a 2% agarose gel; distinct bands and smears were considered positive results for HPV DNA if clear expression of β-actin was observed.All RRP tissue specimens were obtained during intraoperative, endoscopic resection and were received as formalin-fixed paraffin blocks. The diagnosis of RRP was made by standard histopathological examination. Blocks were sectioned at 20 µm and total genomic DNA was extracted using BiOstic FFPE Tissue DNA Isolation Kit .30 and the number of high quality reads per sample was from 2,926,186 to 91,833,436. All high-quality reads were aligned to the human genome (GRCh38) to obtain average coverage across all 22 autosomal chromosomes. A HPV profile with HPV coverage for each sample was generated by HPViewer. With two copies of human autosomal DNA per cell, copy number of HPV per human cell was defined as:Paired-end metagenomic sequencing was performed to determine concordance of HPV subtype between RRP specimens and oral rinse employing the Illumina HiSeq 2500 platform  with 100 ng template DNA per sample and 12 samples per lane. Sequencing reads were trimmed by TRIMMOMATIC31. SAMTools/1.332, Picard/2.6.0, and GATK/3.633 were then applied to de-duplicate and re-align insertion/deletions and to recalibrate base quality score. Single nucleotide polymorphisms (SNP) and small insertions and deletions (INDEL) were identified by GATK HaplotypeCaller. Based on SNP and INDEL information, complete genomes were constructed for those four isolates, three strains of HPV6, and one strain of HPV11.Only four specimens had sufficient sequencing depth to reconstruct the local HPV genome. The entire HPV genome was covered over 10 times by short DNA reads from the four samples; the HPV genome covered by the reads from the other two samples were lower than five, limiting the ability to reconstruct the local genome. Thus, shotgun sequencing data from three HPV6-positive specimen samples were aligned to the entire HPV6 reference genome and the data from the HPV11-positive sample was aligned to the entire HPV11 reference genome via Bowtie234. GenBank accessions of those 189 genomes are HG793809 to HG793938 and FR751320 to FR751338, HE599226 to HE599246 and HE962026 to HE962032, and JN252314 to JN252323 and non-prototypic HPV6a (accession no. L41216) and HPV6vc (accession no. AF092932) genomes. Because of the circular structure of HPV, the genomes were linearized at the first ATG of the E6 ORF. The HPV11 phylogenetic tree was built from 79 HPV11 complete genomes including one genome found in this study, one reference genome from PaVE, and 77 genomes from GenBank. GenBank accession of those 77 genomes are LN833161 to LN833190 and EU918768, FR872717, N870021, FN870022, and FN907957 to FN907964, JQ773408 to JQ773412, HE574701 to HE574705, JN644141 and HE611258 to HE611274, HE962023 to HE962025, and HE962365 to HE962368. All genomes were linearized at the first ATG of the E6 ORF.Four HPV genomes in the RRP lesions were constructed. The HPV6 phylogenetic tree was built from 193 HPV6 complete genomes including novel genomes identified in this study, one reference genome from PaVE, and 189 genomes from GenBank35, respectively. RAxML 8.2.9 was employed to build maximum likelihood trees under a GTRCAT substitution model with 1,000 bootstrapping replicates36. MrBayes-3.2.6 was employed to construct Bayesian trees with 10,000,000 generations for Markov Chain Monte Carlo runs under a General Time Reversible model (GTR) with gamma-shaped distribution rate variation and a proportion of invariable sites37. Phylogenetic trees with a midpoint rooted were visualized by FigTree v1.4.338. MEGA7 was used to compare the number of DNA difference between close strains39.All HPV6 genomes and HPV11 genomes were aligned using MUSCLE3.8.3140. SAMTools/1.3, Picard/2.6.0, and GATK/3.6 were then applied to de-duplicate and re-align insertion/deletions and to recalibrate base quality score. MuTect2 was used to identify somatic mtDNA mutations from RRP tissue compared to oral rinse mtDNA to eliminate germline SNP. Annotation of SNPs was conducted by ANNOVAR (version 2017Jul16)41.All high-quality reads were aligned to the human mitochondrial genome (NC_012920) by Burrows-Wheeler Aligner (BWA/0.7.7 bwa-mem)Supplementary Info-Figure 1