Repetitive transcranial magnetic stimulation (rTMS) is a technique for stimulating brain activity using a transient magnetic field to induce an electrical current in the brain producing depolarization of focal groups of brain cells. TMS is a protocol approved by the U.S. Food and Drug Administration in routine clinical practice as a treatment for depression. A major limitation of rTMS is the large amount of time taken for a standard protocol (38 min a day for 20–30 working days). The optimal type and duration of TMS are still uncertain, as is the optimal strategy for continuing or changing the type of rTMS if there is a poor initial response.The trial aims to assess whether a 1-week compressed course of left dorsolateral prefrontal (L DLPFC) 5 Hz accelerated rTMS (aTMS) treatment is as effective as an established 4-week course of non-accelerated rTMS and if additional 5 Hz L DLPFC aTMS treatments will be efficacious in non-responders as compared to 1 Hz right DLPFC aTMS treatment.A randomized, single-blind, delayed-start trial was planned to commence in Jan 2020. A total of 60 patients will be enrolled from the Institute of Mental Health Singapore within a 2-year period and randomized into the early or delayed-start phase of the trial. The primary outcome of the trial is the improvement of Montgomery-Asberg Depression Rating scale at the end of the active treatment phase.If this study protocol proves to be effective, the findings of this trial will be updated to the College of Psychiatrists, Academy of Medicine Singapore, as well as published in a peer-reviewed journal to enhance local and international TMS treatment guidelines.NCT03941106ClinicalTrials.gov ID:  Depression confers a large burden of disease because of its early onset, high prevalence, and the profound disability involved . In SingElectroconvulsive therapy (ECT) is considered to be one of the most effective treatment modalities yielding an 80–90% response rate in patients with the acute phase of depression . Althoug. TMS shows that TMS is a highly cost-effective option compared to ECT for non-psychotic depression [There is now established evidence that rTMS is effective in treating depression –13 with pression .Although intermittent theta-burst stimulation (iTBS), a FDA-approved novel form of magnetic stimulation is undergoing intensive study as an alternative approach of rTMS –24, the Furthermore, the original FDA-approved protocol for rTMS, which to date has the largest evidence base, is the 10Hz L DLPFC protocol. This involves one session in each working day and patients need to come for the treatment for continuously 4 weeks. For those patients with no or poor response, additional 2 weeks of treatment by rTMS will be supplemented. Therefore, a standard course of unilateral TMS is about 38 min a day for 20–30 working days, substantially limiting the capacity of TMS services to meet the treatment needs of all patients. rTMS requires a considerable time commitment from both patients and clinicians and is of limited utility for patients who do not live within convenient traveling distance from treatment centers. The time needed before treatment response in some patients also makes rTMS unsuitable for some acutely suicidal patients. Accelerating the administration of rTMS (aTMS) by administering multiple sessions of rTMS over a shorter period of time has been shown to potentially be as efficacious as standard rTMS therapy –43. ThesThe primary aim of this study is to test the symptomatic efficacy of aTMS trial protocol. The 2nd aim is to compare the symptomatic efficacy of the early-start aTMS treatment group and delayed-start aTMS treatment group in patients with a DSM 5 Major Depressive Episode. The exploratory aim is to test whether the non-responders to a 1-week course of aTMS will have a higher chance of response to a change in aTMS than repeated the same aTMS modality (from 5 Hz L DLPFC aTMS to 1 Hz R DLPFC aTMS).We hypothesize that accelerated 5 Hz L DLPFC rTMS is effective for the treatment of depressive symptoms with regard to reducing the MADRS scores from baseline to end week 3 in the overall study sample.Participants will be recruited from inpatients and outpatients at the Institute of Mental Health (Singapore) by the research team psychiatrists and referrals from other psychiatrists at the Institute of Mental Health.Age ≥21 years.DSM-5 diagnosis of current major depressive disorder.Montgomery-Asberg Depression Rating Scale score of 20 or more.Able to give informed consent.DSM-5 psychotic disorder.Drug or alcohol abuse or dependence (preceding 3 months).Inadequate response to ECT (current episode of depression).Rapid clinical response required, e.g., high suicide risk.Significant neurological disorder, which may pose increased risks with TMS, e.g., epilepsy.Metal in the cranium, skull defects, pacemaker, cochlear implant, medication pump, or other electronic devices.Pregnancy.The following clinical data will be collected at baseline: age, gender, ethnicity, duration of index episode, type of depression , number of failed antidepressant treatments, number of previous depressive episodes, age at the first depressive episode, and family history of affective disorder will be noted. Full informed written consent will be obtained from all participants.In phase 1, subjects were randomly assigned either to the early-start group (immediate active treatment) or the delayed-start group , every weekday for 1 week. We will use a Magventure X100 TMS Therapy System to conduct TMS stimulation. The first treatment session will take approximately 3 h as participants will also be familiarized with the TMS equipment/procedure and resting motor threshold (RMT) will be determined at this session. We use visual assessment of the respective dorsal interosseous muscle contraction (5 out of 10 stimulations) induced by TMS to determine RMT. Subsequent sessions are expected to last about 2 h each. Stimulation will be applied at the left prefrontal cortex (F3 according to the EEG 10/20 system). L DLPFC aTMS will be delivered at 120% of the RMT, as measured at the initial session. Each TMS train will last for 8 s and have a 12-s intertrain interval. Each session will have 75 trains. Thus, it will be taking about 25 min for each session which includes setting up time.aTMS will be conducted in a room with facilities for managing seizures. Participants and experimenters will be offered earplugs during the sessions. Sessions will be done by staff trained and credentialed in TMS according to the Singapore College of Psychiatrists guidelines for TMS. Staff will also be trained for initial seizure management.Patients in the delayed-start group will be treated as usual during the delayed week and will receive aTMS treatment at 1 week after recruitment. Subjects in the delayed-start group and early-start group will both be monitored with weekly outcome assessments. Subjects in the delayed start arm who still fulfill the criteria for study entry at the end of the delayed start phase will continue on to the active treatment phase of the study. No change in psychiatric medication will be allowed during this period.Q-LES-Q-SF), assessed at baseline and end of week 2. MoCA is a brief cognitive instrument recommended for screening for cognitive impairment (CI) in patients. Several studies have compared the discriminant abilities of the MoCA and the Mini-Mental State Examination (MMSE) for screening post-stroke CI, and most studies have demonstrated that the MoCA is superior or equivalent to the MMSE for the detection of CI after stroke. Furthermore, the MoCA has been reported to be sensitive to changes in acute temporary CI after mild stroke/TIA, whereas the MMSE is reportedly not [A blinded, the trained rater will assess mood each week, from baseline to 2 weeks after treatment, using the Montgomery-Asberg Depression Rating Scale (MADRS) scale. MADRS scale is more sensitive to change in patients’ mental condition than Hamilton depression rating scale . The priedly not . TherefoAfter aTMS treatment, all subjects will then be followed up once a week for 2 weeks and subjects who achieve remission will exit the study.Subjects who have not remitted from the 1st active treatment will enter phase 2 treatment. The second randomization will be stratified into three groups based on whether the participant showed <25% improvement in MADRS scores, between 25% and ≤50% improvement in MADRS scores or >50% improvement in MADRS scores. After that, participants in each group will be randomized (in permuted blocks) to either L DLPFC or R DLPFC treatment. The purpose of patient stratification basing on MADRS score before randomization is a method of permuted block randomization, which is a way to randomly allocate a participant to a treatment group, while maintaining a balance across treatment groups. Each “block” has a specified number of randomly ordered treatment assignments depending on the response to the initial aTMS treatment. Randomization will be generated by study team members who are not involved in the treatment. Treatment assignment is determined by treating doctors. Patients, their caregivers, nurse clinicians, and doctors who do the treatment and outcome assessments will be blinded to the randomization.Subjects who entered phase 2 will receive another 1 week of 5 Hz L DLPFC aTMS (same protocol as above) or aTMS consisting of right DLPFC at 1Hz, 4 treatment sessions a day (each session consisting of 3000 pulses for 12000 pulses a day), every weekday for 1 week. These patients will also be followed up with weekly assessments for 2 weeks after the end of the second round of aTMS. Therefore, participants who receive a total of 1 week of daily aTMS would have received 60000 pulses while those who received a total of 2 weeks of daily aTMS would have received 120000 pulses.We will use the Magventure X100 to carry out low-frequency R DLPFC. 1 Hz R DLPFC aTMS will take about 4 h a day. Stimulation will be delivered at the right prefrontal cortex (F4 according to the EEG 10/20 system). R DLPFC aTMS will be delivered at 120% of the RMT, as measured at the initial session. Each session will have 300 trains which is equivalent to a consensus protocol of delivering 3000 pulses per session over the L DLPFC .Similar to the outcome assessment during phase 1, mood improvement and subject QoL will be assessed every week during acute treatment by a trained rater  using the MADRS scale until 2 weeks after the initiation of 2nd round of treatment.In summary, the primary outcome of this study will be a mean change in the MADRS score from baseline to the end of 4 weeks after treatment initiation. The primary endpoint of the trial will be the MADRS score <11 at any time points of follow-up after initiation of rTMS treatment which indicates the patients have achieved symptoms remission hence can exit from the study.See Fig. t test or chi-square test wherever appropriate. The effect size of aTMS will be assessed by the proportion of response rate, which was defined as “Response: ≥50% MADRS score decrease from baseline” and “Non-response: <50% MADRS score decrease from baseline”. “Remission” is defined by score of 10 or less on MADRS score. Changes in mood and other ratings from baseline to each follow-up time point will be assessed with paired t test followed by repeated-measures analysis of variance (ANOVA). Statistical tests will be two-tailed.Clinical, demographic characteristics and mood, cognitive, and QoL scores will be tested for any differences at baseline between the two randomized groups using Some participants have reported mild headache after TMS—this has not been problematic and has required mild analgesia at the most.The TMS device emits a clicking noise when it delivers the stimulation. There have been reports of hearing impairment in animals (permanent) and human participants (temporary) when TMS was given without the use of earplugs. Participants and experimenters will be provided with earplugs and/or protective headphones during TMS in this study.n=11). Most of these have occurred with stimulus parameters outside current recommended safety guidelines [.Seizures: Only a very small number of accidental seizures have been reported worldwide with TMS  to treat depression in pregnant women , 49. FurMood switching: there are 6 case reports of TMS causing a mood switch into mania in participants with bipolar disorder . The risParticipants will undergo periodic observer-rated mood assessments during the study to check for any side effects or mood deterioration, including the emergence or exacerbation of psychiatric symptoms. In addition, before and after each TMS session, participants will be asked about any side effects or adverse events experienced. Responses will be documented, and spontaneous reports of the side effects will also be noted. An adverse event will be defined as any unfavorable medical change occurring that is accompanied by functional or clinical impairment. Participants who deteriorate to an unacceptable level during the trial  will be withdrawn from the trial and appropriate clinical measures for their further treatment will be taken, including notification of their own treating doctor, referral to crisis team/community health center/public or private hospital.Based on our center’s TMS results to date, patients start TMS treatment with an average MADRS score of 25.4 (SD 7.3), which decreases significantly to 18.4 (SD 9.9) after 2 weeks of treatment with standard rTMS and to 14.1 (SD 10.7) after 4 weeks of treatment. Participants enrolled in the study would be expected to have similar improvements in MADRS with aTMS treatment.This study is powered to test the primary aim, i.e., a significant reduction in MADRS scores from baseline to end week 3, in the overall study sample. Based on the MADRS scores cited above for baseline (25.4 ± 7.3) and end week 4 (14.1 ± 10.7) time points, detecting a similar significant change after a course of aTMS would require a sample of 12 participants . However, given the lack of data on the efficacy of aTMS in an Asian population, and to give a better estimate of response and remission rates, we anticipated that 60% of those recruited will drop out before the end of the trial with an unequal allocation ratio of 2:1, we thus aim to recruit 40 subjects in the delayed start arm and 20 subjects in the immediate start arm for a total of 60 subjects.No prior data exist on the comparative effects of delayed response with immediate treatment of aTMS (or TMS), we will also explore the aim that non-responders to a 1-week course of aTMS will have a higher chance of response to a change in aTMS from L 5 Hz DLPFC TMS to R 1 Hz DLPFC for depression. This study will produce pilot data for 2nd and exploratory aim, informing the power analysis for a future clinical trial if results reject a null hypothesis.This research proposal both addresses important practical and scientific considerations in depression, a major international  and locaIn addition, there is the scientific question of how to optimize TMS treatment after initial poor response which has been poorly investigated thus far. There is insufficient evidence on the optimal duration of TMS treatment for patients with good response. The results of this study will be of significant interest to the wider research community looking into the optimization of TMS treatment in depression.Moreover, the results of this study have significant potential to affect local and international routine clinical practice of TMS for depression. Potentially, all restructured hospitals and more private psychiatrists could be offering TMS treatment for depression and having evidence that it can be accelerated will significantly increase adoption of this novel treatment for depression. It will also improve patient care as patients can potentially respond in 1–2 weeks rather than the 4–6 weeks of standard TMS care today. This increased speed of response will not only relieve suffering and distress but improve patient productivity by reducing time spent on treatment and accelerate their return to work or societal duties.This trial has several strengths. The delayed start phase will decrease the likelihood of a significant placebo response by subjects who respond to increased attention in a clinical trial. The psychological response elicited by placebos is very specific depending on various factors such as the patient’s expectations, feelings, beliefs, volition, and hope for improvement , 53. GroWe proposed a 2-week follow-up period after patients have been treated with compact 20 sessions of rTMS. Several open-label large-scale RCTs have shown that some patients with standard treatment of rTMS experienced a delayed response and required 4–6 weeks of stimulation before showing adequate response , 59–61. The proposed study is an open-label single-blinded study and will have the limitations of a lack of a control group in which patients will receive sham-operated magnetic stimulation of the brain and a control group to minimize the expectancy bias by raters. TMS is now a standard treatment for depression , 63. DepIn summary, this trial is expected to clarify the clinical advantages of compressed TMS treatment vs. the standard treatment protocol of TMS. If this trial reveals the appropriate protocol of aTMS, this result can be published to enhance local and international TMS treatment guidelines.


The skin barrier is important in the pathogenesis of atopic dermatitis and stratum corneum lipids have a critical role. Skin surface lipids have been largely overlooked but also contribute to barrier function. An untargeted approach was used to compare the skin surface lipids from atopic and non-atopic West Highland White terrier dogs (WHWT).The primary hypothesis was that a difference in the lipidome would exist. The secondary hypothesis was that affected and unaffected skin lipids would differ.This prospective, cross-sectional, case-controlled study included thirty-nine privately owned WHWTs. Dogs were assigned to one of four disease status groups based on strict criteria. Samples for lipid analysis were collected from the skin surface of unaffected and affected sites. Lipid analysis was by untargeted liquid chromatography/mass spectrometry and utilised lipid identification software packages. Principle component analysis (PCA) and partial least-squares discriminant analysis (sPLS-DA) statistical methods analysed the association between the relative lipid abundance and disease status and affected and unaffected skin.Samples for lipid analysis found 421 lipid soluble features of which ten lipids were positively identified. Statistical analysis could not distinguish between non-atopic and atopic dogs but did reveal a statistically significant difference in the lipid profiles from affected and non-affected skin irrespective of disease status.A large array of unidentified lipids from the skin surface were found with a difference between affected and unaffected skin unrelated to disease status. Investigation into the lipidome of the skin surface is an emerging area of research with clinical and therapeutic applications. There is evidence an impaired skin barrier plays a role in the pathogenesis Marsella  which maThe primary aim of this study was to investigate the skin surface lipids in healthy non-atopic and atopic dogs, utilising liquid chromatography-mass spectrometry (LC-MS). This study was conducted using a group of privately-owned West Highland White terrier dogs (WHWT). The WHWT is a breed that is recognised to be predisposed to developing AD .2.1.Forty-three WHWTs were initially enrolled in the study with an age range of four months to 15 years. Dogs were recruited from the West Highland White & Scottish Terrier Club (38 dogs) and from the Animal Dermatology Clinic (five dogs). A list of 50 dog owners who were members of the West Highland White & Scottish Terrier Club was provided by the club secretary. Forty-four WHWT club members were contacted based on their geographical location and there were 22 responses from owners willing to allow their dog(s) to be a part of the study. Several owners had more than one of their dogs included in the study. Owners were advised not to bathe the dogs for at least seven days prior to day of examination. All other current medications including topical, systemic medications and immunotherapy were permitted and their use recorded.2.2.Each dog was allocated to one of four skin disease status categories: 1. Apparently Healthy/Not Atopic (HNA) 2. Atopic dermatitis (AD) 3. Likely Atopic dermatitis (LAD) 4. Other skin disease/Not Atopic (ONA). Allocation to one of these 4 categories was determined based on strict criteria which are outlined in The combination of these criteria was applied to designate each dog to a disease status group. When identifying pruritic individuals, more emphasis was placed on abnormal levels of pruritic behaviours identified in the survey than the PVAS score. A PVAS of up to 1.9 was the highest score allowed in the HNA group but only if there was no abnormal pruritic behaviour revealed in the survey.2.3.Sample sites were kept as consistent as possible between dogs. The medial aspect of the hindleg (MHL) was chosen as the preferred site because it is relatively sparsely haired in the WHWT and provided a large enough area for the samples to be collected comfortably. Samples were collected from the MHL in all dogs. If the MHL was affected, then an unaffected site was chosen in order of preference, where possible, from the ventral abdomen, lateral chest, axilla or dorsum. Affected skin was defined as skin exhibiting erythema with or without other primary or secondary lesions including alopecia, papules, pustules, epidermal collarettes, excoriations, crusts, lichenification, hyperpigmentation and seborrhoea. If the skin to be sampled was in a haired area, then the hair was clipped from the site using electric clippers taking care not to disturb the skin surface.2.4.To collect the lipid sample, a flexible plastic board (12 × 14 cm) with a central rectangular hole (2 × 4 cm) was pressed to the surface of the skin. A cotton-tipped applicator whetted with acetone and n-hexane (1:1 v/v) was firmly rolled over the exposed area of skin to cover the entire surface once for 24.5 +/− 4.5 seconds. The cotton-tipped applicator was then placed back into the solvent into a screw-top plastic tube and stored at −20 degrees Celsius until analysis.2.5.3: MeOH: H2O, v/v/v, 65:33:2), containing phosphoethanolamine  as the internal standard  to 99% solvent B . The MS1 data (used for quantification) was collected at 70,000 mass resolution while the MS2 data was collected at 35,000 resolutions. Lipid concentrations were calculated by using the known amount of standard in each sample and calculating the concentrations of peaks detected relative to the amount of standard added. While not accounting for the relative differences of ionisation efficiency between the different lipids detected, it does provide an estimate of the relative abundance for comparison between the different samples. The average coefficient of variation for standard detection was 19%. The LC-MS was performed under contract at AgResearch, Palmerston North, New Zealand. Data files were converted to mzXML format using the file converter ProteoWizard. Non-targeted peak detection was performed using the R package XCMS. The resulting peak area matrix data files were de-isotoped using a custom script written in R version 2.15.0 . The data was analysed using the lipid identification software package LipidSearch . A further number were tentatively identified in positive ion mode using Lipidblast. Data are reported as relative abundances (mol%), whereby 100% corresponds to the total amount of lipids present.Mass spectrometric analysis was carried out on a Q-Exactive Mass Spectrometer  using positive and negative electrospray ionisation and collection of MS2 spectra from m/z 200–2000. Samples were injected onto an Acquity CSH C18 column . Lipids eluted over a 15 min gradient from 85% solvent A , and partial least-squares discriminant analysis (sPLS-DA). PCA was used to provide an unconstrained visualisation of the lipid variation between samples. sPLS-DA is an effective tool for determining which quantitative variables are informative in a discriminant analysis. The relative abundances of lipid moieties were used to discriminate disease status and the most useful lipids for predicting disease status were extracted. All analysis and plotting were performed using the prcomp and sPLS-DA functions of the statistical software R  were present in the skin samples from the 39 dogs. After removal of highly correlated (>0.99 correlation) lipids the total was 392. There were 10 lipids positively identified with perfect alignment using the lipid identification software package LipidSearch . These 14.This was an explorative study using an untargeted approach which sought to identify differences in the lipid profiles of the skin surface between WHWTs with and without AD, and between affected and unaffected skin. We detected 392 lipids, the majority of which could not be readily identified. The lipids found included ceramides, which were different to those that have been previously identified as being important in canine SC and were in relatively low abundance  . Lipids https://osf.io/7v9yk/).It is possible that these are lipids produced by the sebaceous glands or corneocytes, but they could also be structural cellular lipids, or even microbially derived lipids or degradation products. It was beyond the scope of this explorative study to identify lipids that did not match with the published databases and it is clear that further chemical characterisation will be required. Given that some or all of them could be pathophysiologically relevant, further targeted lipidomic techniques are indicated to characterise and quantify these lipids. To facilitate further research, we have provided the full data set (Supporting information 3) and it has also been registered with the Open Science Framework  which was a limitation for the statistical analysis. There were also only 5/39 dogs under 12 months of age, and the groups were not age or gender matched. It is not known how these variables may have impacted the lipid profiles obtained but it is known from human studies that skin lipids vary with age and gender (Ghadially et al. 5.Investigation into the lipidome of the skin surface in health and disease is an emerging area of research which could have clinical and therapeutic applications. In this study, an untargeted approach revealed a vast array of lipids present on the surface of the skin in WHWTs, the majority of which could not be identified. There was no overall pattern in the skin lipid profile that distinguished between WHWTs with and without AD. However, there was a pattern that distinguished affected from unaffected skin and this was irrespective of the disease status of the dog. This difference may be due to consistent changes to the skin lipids induced by inflammation or infection however it remains to be seen what the key pathophysiological changes are, and the identity and functional significance of the lipids associated with this difference.


Acute febrile patients presenting at hospitals in Douala, Cameroon between July and December 2020, were screened for dengue infections using real time RT-PCR on fragments of the 5’ and 3’ UTR genomic regions. In total, 12.8% (41/320) of cases examined were positive for dengue. Dengue virus 3 (DENV-3) was the most common serotype found (68.3%), followed by DENV-2 (19.5%) and DENV-1 (4.9%). Co-infections of DENV-3 and DENV-2 were found in 3 cases. Jaundice and headache were the most frequent clinical signs associated with infection and 56% (23/41) of the cases were co-infections with malaria. Phylogenetic analysis of the envelope gene identified DENV-1 as belonging to genotype V, DENV-2 to genotype II and DENV-3 to genotype III. The simultaneous occurrence of three serotypes in Douala reveals dengue as a serious public health threat for Cameroon and highlights the need for further epidemiological studies in the major cities of this region. Acute febrile patients presenting at hospitals in Douala, Cameroon between July and December 2020, were screened for dengue infections by Polymerase chain reaction. In total, 12.8% (41/320) of cases examined were infected by dengue virus. Dengue virus 3 (DENV-3) was the most common serotype found (68.3%), followed by DENV-2 (19.5%) and DENV-1 (4.9%). Co-infections of DENV-3 and DENV-2 were found in 3 cases. Jaundice and headache were the most frequent clinical signs associated with infection and 56% (23/41) of the cases were co-infections with malaria. The simultaneous occurrence of three serotypes in Douala reveals dengue as a serious public health threat for Cameroon and highlights the need for further epidemiological studies in the major cities of this region. Flavivirus (Flaviviridae family). DENV genome is a positive sense, non-segmented, single stranded RNA of ~10.7kb which comprise an open reading frame encoding for three structural proteins and seven non-structural (NS) proteins in the order 5’ C-preM/M-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 3’  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Marilia Sá CarvalhoAssociate EditorPLOS Neglected Tropical DiseasesAmy MorrisonDeputy EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: The study aims to characterize the contribution of DENV to acute febrile illness in Douala, Cameroon. The authors obtained 320 samples from symptomatic patients and were able to detect DENV in 41 samples. The positive samples were serotyped by RT-qPCR and subsequently submitted to envelope gene sequencing. Maximum likelihood analysis was performed to identify the genotypes of DENV1, DENV2 and DENV3 circulating in Duoala, Cameroon. The authors concluded that the simultaneous occurrence of three DENV serotypes in Douala reveals that the virus is a serious public health threat for Cameroon.Reviewer #2: The objectives are clear and well delineated for the study. The design is appropriate, however the sample size was low to compose four public hospitals. Even being from a poor country with a lack of surveillance. The low number of samples that were sequenced are unrepresentative. It is known that DENV1 is well established in the Aedes aegypti mosquito on several continents. Currently, dengue viruses have originated lineages within a genotype and this is very important to show the variability, especially for dengue viruses 1 and 2. It is important to emphasize that there are already many methodologies for obtaining low-cost and sensitive sequences that allow a broad analysis.The target population is in agreement with the proposed objectives. Statistical tests are adequate, as well as molecular analysis. Ethical aspects are reported in the text.Reviewer #3: (No Response)--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: Some inconsistencies were observed in Results section; therefore, authors should clarify or rectify their statement in the text:Table 2: in the age column, the sum of positive dengue patients is not 41:- 6 to 15 age line: the sum of positive and negative dengue cases is 48, but only 7 positive and 40 negative cases are listed- 25 to 45 age line: the sum of positive and negative dengue cases is 103, but only 16 positive and 86 negative cases are listedLines 159-160: the number of dengue and malaria co-infections is inconsistent: 176 cases were co-infections with malaria, but only 41 patients tested positive for dengue. Is that accurate?Lines 177, 179, 185, 187 and 190: the names of the figures should be changed to Figure 2, 3 and 4 instead of Figure 2a, 2b and 2c. Figure 2, Figure 3 and Figure 4: ML trees were inferred with a number of dengue sequences different from the mentioned number of sequences generated in this study, for example: the authors generated 2 DENV1 sequences, but the ML tree shows only 1 sequence. The same was observed for DENV2 and DENV3. The authors should include all sequences generated in this study for phylogenetic analysis or make it clear why only some sequences were used.Line 193: The authors mentioned that DENV3 strains from Cameroon might have evolved from South Africa strains. For clear understanding, the authors should provide the names of the countries they are referring to.Line 226: the ages of the patients co-infected with DENV2 and DENV3 are inconsistent with the information presented on f the Table 3.Reviewer #2: The study proposed to determine the DENV serotypes and genotypes of dengue cases in four public hospitals, however a low number of positives was obtained by molecular biology. With regard to genetic sequencing, few sample sequences were obtained, this may be related to the sequencing methodology for genomic analysis used to determine the genotypes. There is an important reference in the study that malaria was found in more than 70% of cases and people are co-infected with two denv serotypes, such as denv2 and denv3.The results are clear, with a lot of clinical information, which demonstrate good accuracy in data collection. This is relevant epidemiological information, as there is little data of this type in the study area, as endemic malaria makes it difficult to detect dengue cases. Studies of this type should be strengthened, as the clinical manifestations of dengue have caused serious conditions, such as dengue hemorrhagic fever and neurological alterations.Figures are of good quality, but the table 2 needs improvement in presentation, I suggest a subdivision within it, to separate population demographic characteristics from clinical manifestations.Reviewer #3: (No Response)--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: The conclusions provided by the authors are sounded, but little is discussed about the limitations of the study. The authors discussed that their results are relevant for public health in Africa and how the monitoring of virus could help preventing dengue outbreaks.Reviewer #2: The conclusion reports on the importance of arbovirus surveillance in urban areas in Africa, which is really important for public health. To improve viral surveillance, it is necessary to change the study algorithm, in order to have a closer phylogenetic profile of the genotypes, a greater number of sequences must be obtained, including using more sensitive methods, such as next-generation sequencing or at least larger partial sequences by Sanger.At no time were the limitations of the study reported, such as difficulties with methodologies. Phylogenetic analyzes are in agreement and provided important and reliable information, but for a more detailed study of genotypes it is necessary to increase the fragment size, as well as the number of sequences.Reviewer #3: (No Response)--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: (No Response)Reviewer #2: Minor Revision: Review table 2, with division of demographic information from clinics.Reviewer #3: (No Response)--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: Some inconsistencies were observed in Results section and related tables; therefore, authors should clarify or rectify their statement in the text.Reviewer #2: Strength of the study:1- Contribute to the knowledge of the situation of arboviruses in urban areas in Africa;2- Identify circulating serotypes and genotypes;3- Propose DENV surveillance in the study area, as three circulating serotypes were detected.Reviewer #3: The article proposed by SIMO TCHETGNA et al., acute febrile patients were presenting at hospitals between July and December 2020 for monitoring the circulation and co-circulation of DENV in Douala, Cameroon. Still, some details in the study queue should be taken into account:The quantity of dataset samples is not representative of the phylogenetic inference analyses. I recommend the authors check the phylogenetic signal of the dataset before carrying out the phylogenetic reconstructions, considering that it is only the envelope region of the virus, requiring a more extensive set of data to represent the cohesive statements of the cohesive statements the analyses. It would be interesting for the authors to check the introduction of serotypes. It is not common to have three dengue serotypes circulating in a small region of a country. What were the criteria used for the sequencing of the samples, considering that of the 41 positives, only nine samples were sequenced, only 21% were sequenced? Was there sequencing of co-infection samples between serotypes 2 and 3? 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Read more information on sharing protocols at  16 Sep 2021AttachmentResponses to the reviewers.docxSubmitted filename: Click here for additional data file. 28 Sep 2021Dear Dr SIMO TCHETGNA,We are pleased to inform you that your manuscript 'Concurrent circulation of dengue serotype 1, 2 and 3 among acute febrile patients in Cameroon' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.Best regards,Marilia Sá CarvalhoAssociate EditorPLOS Neglected Tropical DiseasesAmy MorrisonDeputy EditorPLOS Neglected Tropical Diseases*********************************************************** 21 Oct 2021Dear Dr SIMO TCHETGNA,We are delighted to inform you that your manuscript, "Concurrent circulation of dengue serotype 1, 2 and 3 among acute febrile patients in Cameroon," has been formally accepted for publication in PLOS Neglected Tropical Diseases.We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. 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Following an implementation plan based on dynamic dialogue between researchers and clinicians, this study implemented an evidence-based patient education program (tested in an RCT) into routine care at a clinical transplant center. The aim of this study was to investigate renal recipients’ knowledge and self-efficacy during first year the after the intervention was provided in an everyday life setting.The study has a longitudinal design. The sample consisted of 196 renal recipients. Measurement points were 5 days (baseline), 2 months (T1), 6 months (T2), and one-year post transplantation (T3). Outcome measures were post-transplant knowledge, self-efficacy, and self-perceived general health.No statistically significant changes were found from baseline to T1, T2, and T3. Participants’ levels of knowledge and self-efficacy were high prior to the education program and did not change throughout the first year post transplantation.Renal recipients self-efficacy and insight in post-transplant aspects seem to be more robust when admitted to the hospital for transplantation compared to baseline observations in the RCT study. This may explain why the implemented educational intervention did not lead to the same positive increase in outcome measures as in the RCT. This study supports that replicating clinical interventions in real-life settings may provide different results compared to results from RCT’s. In order to gain a complete picture of the impacts of an implemented intervention, it is vital also to evaluate results after implementing findings from RCT-studies into everyday practice. In recent years, considerable improvements have been made in the field of transplantation. However, shorter hospital stays combined with fewer follow-up appointments may have imposed greater demands on kidney recipients in terms of coping with life after transplantation –5. ProviA randomized trial performed at a Norwegian hospital in 2012 tested the effect of a new education programme that emphasized individualizing and tailoring the educational content to each patient’s needs . The proHealth-care professionals have shown increased interest in facilitating the implementation of research results from academic settings to clinical practice. Such interest has been described using various concepts and with models using various names , 18. YetThis study is the fourth in a row within a broader implementation project aiming to examine the implementation of the new education program, from different angles and in different phases utilizing different study designs , 27–29. The study was conducted at the only transplant centre in Norway. Annually, it performs about 270 kidney transplants and employs approximately 100 nurses within a surgical unit, medical unit, and outpatient clinic. Patients are discharged from the hospital approximately one week post-transplantation. After discharge, patients attend the outpatient department unit medical controls for about 8–10 weeks. Except for patients living close to the centre, renal transplant recipients stay at the patient hotel situated close to the hospital.The main differences between conventional patient education used at the transplant centre and the evidence-based patient education programme were the emphasis on individual adaptation, the method of knowledge transfer, the later timing of the education program and the extended number of training sessions.To ensure individualisation, the evidence-based patient education programme utilized the Academic Detailing method . This mePreviously at the centre, patient education had been provided during the 7–10 days of hospitalization while the new evidence-based patient education programme was provided during 7 weeks of post-transplant outpatient care. The programme consisted of five one-to-one educational sessions and main teaching focus were immunosuppressive medication, organ rejection, and lifestyle. With each of those themes, content was contextualized and adapted based on each patient’s needs. Each session lasted from 40 to 60 min and was provided by the research nurse in the RCT study.Some events during the last years should be made explicit at they might have impacted on the patient education situation in our study context. Since the RCT study was performed in 2012, the standard written information previous handed out to the patients when attending to the hospital has been digitalized and made available online. Another factor related to dissemination of information was that the transplant centre had participated in a documentary program shown on one of the main Norwegian TV channels. In this documentary of 10 episodes, patients shared their experiences of living with kidney disease and going through a transplantation.Routines for follow-up care and the structure and content of the education program for renal transplant recipients were comparable to the current study.The implementation of the new education program was inspired by the Formative Evaluation Consultation And Systems Technique (FORECAST) approach which emphasizes collaboration and mutual influence between the project stakeholders and research teams; it provides advice for continuous evaluation throughout the implementation . We estaIn order to promote mutual understanding of the new patient education programme the implementation team developed educational sessions for the transplant nurses that would be providing the new education program to the patients. This included an introductory lecture concerning pedagogical theory related to tailored education and the aspects of the academic detailing tailoring method . FurtherThis study has longitudinal design. As with the reference study, the primary outcome was post-transplant knowledge ; the secOur measurement points were at baseline (5 days post-transplantation), T1 (2 months post-transplantation), and T2 (6 months post-transplantation). All measure points in the current study were in accordance with the measurement points in the reference study. In both study settings, standard care of patients was treatment in hospital during the first week after the kidney transplantation. The standard follow-up program for the patients at the transplant centre had not changed since the RCT study was conducted, therefor the measure point at baseline was assumed to be comparable to the reference RCT study. To capture longer-term changes, the present study included in addition a measurement point 1 year post-transplantation (T3). The study was approved by the Data Protection Office at Oslo University Hospital (2014/5573). Written informed consent was obtained from each participant prior to study procedures. All participants were informed that their data would be anonymous and treated with confidentiality. They were informed about publication plans. They were told they could withdraw from the study at any time without any consequences to them.Participants were recruited by trained nursing staff at transplant centre in Norway within the first days after kidney transplantation. The criteria for inclusion were being above 18 years of age, being able to participate in post-transplantation patient education, and being able to read Norwegian sufficiently to complete the questionnaire. Based om the sample size calculation from the reference study, it was estimated that a sample of around 200 was required.Among the 357 patients who received kidney transplants between February 2016 and August 2017, 111 were for administrative reasons (holidays or busy days at the transplant clinic) not invited; 29 did not meet the inclusion criteria (25 were unable to read Norwegian). Of the 217 patients asked to participate in the study by trained nursing staff, 199 agreed. Of those 199, three were excluded . Ultimately, 196 patients were included.As with the reference study, we applied the Knowledge Questionnaire for Renal Recipients  and we mThe Knowledge Questionnaire for Renal Recipients was developed in Norway; it focuses on medication, rejection symptoms, and lifestyle . Owing tThe General Self-Efficacy Scale (GSE) contains 10 statements reflecting an individual’s belief in their ability to respond to novel or difficult situations . Each stThe single item to assess self-perceived evaluation of total health was posed as follows: ‘In general how would you rate your health?’ The response categories were ‘excellent’, ‘very good’, ‘good’, ‘fair’, and ‘poor`. A score above 5 indicates lower evaluation of health . The quet test for each outcome measure from baseline to T1, T2, and T3. We investigated statistically significant differences in levels of knowledge in terms of gender, age, educational level, and duration of kidney disease using univariate regression analysis.We used IBM SPSS® statistics for Windows version 25 ) for the statistical analyses. We performed a paired sample Most respondents were men (67.7 %) and living with someone (73.5 %). Their mean age was 56 years . Before transplantation, 50 % received social security, 36.6 % were employed, and 13.5 % were students, unemployed, or full-time home-maker. Almost one-fifth (19 %) of participants reported having completed over 4 years of university or college education. The available baseline demographics for the participants in the implementation study and those in the reference study appear in Table We categorized the underlying kidney disease according to the annual report of the US Scientific Registry of Transplant Recipients and the Organ Procurement and Transplantation Network . The prot test results for each outcome measure from baseline to T1, T2, and T3 are presented in Table We observed no statistically significant changes in levels of post-transplant knowledge (primary outcome) or self-efficacy (secondary outcome) from baseline to 7 weeks, 6 months, or 1 year post-transplantation. Furthermore, we found no statistically significant differences in post-transplant knowledge according to gender, age, education level, or duration of kidney disease at any of the measurement points. The overall health decreased significantly from baseline to 7 weeks post-transplantation; however, no significant changes were evident from baseline to 6 months or 1 year post-transplantation. The descriptive information and paired sample n = 171). The scores for the knowledge questionnaire in both study populations at baseline appear in Table At baseline, the mean for correct post-transplant knowledge answers in the experimental group in the reference study was 58.5 % (n = 78); mean for correct post-transplant knowledge answers in the implementation study was 72.1 % (n = 71). By contrast, the participants in the implementation study showed an increase only from 71 to 73.5 % (n = 157).At T1, participants in the experimental group in the reference study showed an increase in the mean for correct post-transplant knowledge answers from 58.5 to 65 % (n = 160). Self-efficacy score in the experimental group at baseline was 28.7 (SD 3.7) and significantly increased to 30.8 (SD 4.2) after 7 weeks and 34 (SD 4.3) after 6 months. In the implementation study, self-efficacy score was 32.32 (SD) and did not significantly change throughout the first year after the transplantation.At T2, the experimental group in the reference study showed an increase in correct answers of up to 70 % (i = 64). Participants in the implementation study demonstrated a stable mean of 71 % correct answers . One explanation for this could be the recent years advances in kidney transplantation treatment. Better surgical technics and post-operative anaesthetic management care might have led to a faster post-transplantation recovery for the current study population –43. ThisA more general factor explaining the difference in post-transplantation findings between the reference and implementation study groups could be the shift in health education needs. The rapid proliferation of and access to health information on the Internet have created new possibilities related to health information seeking. Patients have become increasingly comfortable using the Internet as a prime source of basic health information. In the context of transplantation, learning material has been made available online and through TV programs. Through social network platforms such as Facebook, patients are increasingly sharing their disease experiences . Shared It is also necessary to reflect on the implementation process and the challenge of finding an appropriate balance between fidelity to the original intervention and adaptation . The impAnother aspects explaining a possible lack of intervention impact is that in the original RCT study, the educational intervention was provided by the research nurse while the implementation study it was provided by the transplant nurses. This might have made a difference in terms of commitment to the intervention and consistency of deliver. On the other hand, a survey of transplant nurses perceptions of the implemented education program demonstrated that they felt confident and had enough knowledge about the new program and were motivated for using it in their nursing practices .This follow-up study is the fourth in a series that presents results from within a broader implementation project, which follows the implementation of a new education programme for kidney recipients , 27–29. Another interesting finding to discuss why the patients did not get more knowledgeable confident over time. This is surprising, as one could have expected that life experience throughout the first-year post-transplant would have led to higher competences. Other studies have found that the effect of patient education wanes over time as distance from the intervention widens . If thisA limitation of this study is the large number of patients who were not approached for participation, which was due to less focus on including patients during busy periods and holidays. Due to ethical constrains, we were unable to gather information about participants who were not included in the sample; thus, we do not know if they deviated considerably from the study sample. However, except for the lower number of post-operative complications and less living donor transplant, the demographical and clinical characteristics of participants in the implementation study were comparable to the sample in the reference RCT study.In conclusion, there are several possible explanations for why we did not observe the same changes in the implementation study as with the reference RCT. This study highlights the complexity of determining the success of educational programmes in a shifting clinical setting. Renal recipients’ self-efficacy and insight in post-transplant aspects seem to be more robust when admitted to the hospital for transplantation and this may be one of the explanation of why the implemented educational intervention did not lead to the same positive increase in outcome measures as in the RCT. This study supports that replicating clinical interventions in real-life settings may provide different results compared to results from RCT’s. In order to gain a complete picture of the impacts of an implemented intervention, it is vital also to evaluate results after implementing findings from RCT-studies into everyday practice.Future efforts should focus on obtaining insights into implementation and post-implementation outcomes: this study has demonstrated that evidence-based educational interventions may not always work as expected in a real-life setting. To gain a more complete picture of the impact of an implemented intervention, it will be necessary to apply complementary study designs.


There are several surgical options for the management of pilonidal disease, including midline and off midline closure, but prospective studies are rare. The study hypothesis was that Karydakis flap surgery would result in shorter wound healing and fewer recurrences than excision of pilonidal sinus and suture in the midline.A randomized clinical trial was conducted in two hospitals in Sweden between 2006 and 2015 to compare excision and suture in the midline with Karydakis flap surgery. Adult patients with a chronic pilonidal sinus disease were randomized 1:1 at the outpatient clinic without blinding. Power calculation based on recurrence of 2 per cent for Karydakis flap and 10 per cent for excision and primary closure in the midline required 400 patients with 90 per cent statistical power at 5 per cent significance assuming 10 per cent loss during follow-up. Participants were followed up until complete wound healing; late follow-up after 6–13 years was performed by telephone by two blinded assessors. The two co-primary outcomes were time to complete wound healing and recurrence rate.P < 0.001). There were five recurrences in each group, after a median follow-up of 11 years (P = 0.753).The study was terminated early at a planned interim analysis due slow recruitment and a significant difference in primary outcome. In total, 125 patients were randomized, of whom 116 were available for the present analysis. Median wound healing time was 49 days  32 to 66) for excision with suture in the midline and 14 days (95 per cent c.i. 12 to 20) for Karydakis flap surgery Registration number: NCT00412659 ( Two surgical techniques for pilonidal sinus disease were studied in an expertise-based randomized controlled trial with long term follow-up. Karydakis flap surgery for pilonidal sinus disease leads to a shorter wound healing time than excision and suture in the midline, while the recurrence rate is similar. Without effective treatment, it causes a painful lump or secretion of pus through one or several skin openings in the intergluteal area; the condition can persist for years3.Chronic pilonidal sinus disease mostly affects young adults and is more common in men5. Meta-analyses indicate advantages for flap techniques6–8 such as the Karydakis flap procedure, which is an advancement flap known to be one of the simplest off-midline procedures9. Despite this, in many countries, the most common technique is excision of the pilonidal sinus and tension-free primary suture in the midline10. According to one comparative study11, the Karydakis technique had fewer complications with fewer recurrences than midline closure. The only randomized controlled trial (RCT) to compare these techniques reported no significant differences in postoperative complication and recurrence rates after Karydakis surgery compared with primary excision and tension-free midline closure, while wound healing was not reported12. The present RCT was conducted to test if Karydakis flap surgery results in shorter wound healing time and fewer recurrences than excision and tension-free primary suture in the midline.Several surgical techniques are used to treat pilonidal sinus disease, but there is a lack of consensus regarding best surgical practiceThis was a prospective, pragmatic, expertise-based, randomized controlled clinical trial.Inclusion criteria were adult patients aged 18 years of age or older with primary or once-recurrent pilonidal disease considered for surgical treatment. Patients were included if at least 4 weeks had elapsed since any acute incision for a pilonidal abscess.Patients considered for only minimally invasive procedures  were excluded. Patients were informed and asked for consent to participate by the attending surgeon who assigned participants at the outpatient clinic. Approval from the Regional Ethical Review Board of Umeå University was obtained in January 2006 (registration number 05-178M).9, with minor modification. A symmetric elliptical excision was centred 2 cm lateral to the midline at the side with the most affected tissue or secondary fistula openings in the skin of the buttocks . All affected pilonidal sinus disease tissue was removed with some normal tissue, enabling a lateral shift of the final closed wound. A 2-cm wide and 1-cm-thick skin flap was mobilized from the contralateral side to cover the skin defect and create a new natal cleft, with normal skin in the midline . A modification of the original technique consisted of using a continuous 2-0 monofilament polydioxanone suture (PDS II) for the two deep layers of suture lines to secure the flap in its new position. A compression dressing made of a polyester fibre pad was placed on the closed wound.All patients received antibiotic prophylaxis with a single dose of 1 g metronidazole and 160 mg sulfamethoxazole combined with 800 mg trimethoprim as an intravenous injection prior to surgery. One gram of paracetamol and a standard dose of a non-steroidal anti-inflammatory drug were given as premedication. All operations were performed under general anaesthesia. The skin was shaved in the wound area to promote wound healing both in the operating room and later during wound care if healing complications occurred. In both interventions, surgery started and ended with a 20 ml injection dose of a local anaesthetic subcutaneously in the wound area. The standard intervention arm involved excision of all tissue affected with pilonidal sinus disease with a symmetric elliptical incision centred in the midline and subcutaneous incisions performed lateral enough to allow tension-free midline closure and primary suture of the wound. A gauze roll sutured on top of the wound was used as a compression wound dressing. The experimental intervention arm was Karydakis flap surgeryIn both interventions, incisions were made using diathermy, and bleeding was controlled by diathermy coagulation. No dye was used to indicate the pilonidal sinus cavity or tracks and no drains were used.®  or left open. Open wounds were covered with an absorbing pad. Wound dressings were changed daily, or more often if required, to keep the outer surface dry. Rinsing of infected open wounds at home with a shower twice daily was encouraged. Antibiotics were prescribed based on the surgeons’ judgement.All patients were discharged the same day with simple analgesia prescribed for 2 weeks and invited to follow-up visits until complete wound healing. Return visits were recorded by the case report forms for the study and the hospitals’ computerized patient record system. Routinely, the compression gauze roll or fibre pad was removed on day 3 or 4, and a thin wound dressing was applied. Skin sutures were removed on days 11 to 14 (not on weekends). Nurses responsible for follow-up of wound healing were instructed on standard wound care according to study protocol, case report forms detailing wound healing issues, and documenting when complete wound healing occurred. Impaired wound healing involving infections led to consultation with the attending surgeon for wound care instructions. An infected wound was sufficiently opened to drain any infected wound cavities, followed by washing out with saline. Subcutaneous wound cavities were either packed with SorbactSpecific surgeons were involved in each intervention arm, but no other adjustments to routine surgical practice were made. The surgeons involved at the two hospitals chose one of the two methods and performed only their selected procedure. Three surgeons chose to perform the excision with primary suture in the midline and another three performed the Karydakis flap surgery. All surgeons involved were specialists in general surgery and had more than 10 years of surgical experience.A detailed illustrated manual (in Swedish) for both methods  and a viCo-primary outcomes were time to complete wound healing and recurrence rates. Wound healing was evaluated at the outpatient clinic at 1- to 2-week intervals until complete wound healing was documented. Complete wound healing was defined as a dry scar requiring no further care or dressing. Nurses responsible for postoperative wound care and registration were not involved in the surgery. The time elapsed from surgery to the day of suture removal was defined as the shortest possible wound healing time if no wound complications occurred within 30 days.Recurrence was evaluated by a surgeon, who was not involved in the surgery, at the 3-, 12-, and 36-month follow-up visits, and with a telephone interview at the end of the study. In addition to the planned follow-up visits, all patients were instructed to contact the operating centre if any problems with the wound or scar occurred. The telephone interview was structured and conducted according to a specific data form by a surgeon or research nurse. Both were blinded regarding the intervention performed. If a wound opened within 30 days of surgery, it was considered a wound healing complication with delayed healing, affecting wound healing time. Recurrence of pilonidal sinus disease (31 days or later) was defined as a later opening of a healed wound, as well as any openings in the scar, nearby skin, or formation of a painful subcutaneous mass in the intergluteal area.13. Open postoperative wounds included a minor wound opening to total dehiscence, with or without purulent infection. Measures of postoperative open wound length, width, and depth were recorded, as well as details of wound care, at each visit. The largest measurements of the open wounds during follow-up were used for the report.Secondary outcomes were postoperative wound complications that were categorized by two methods, open (or opened) postoperative wounds (yes or no), and separately with the Southampton Wound Assessment ScaleAdditional secondary outcomes, according to the study protocol , will be reported separately.The randomization sequence was computer-generated using a random number technique and concealed. After informed consent was obtained, allocation to a study arm was provided online from a secure web-based registration and randomization application. Participants were randomly assigned to either the control or experimental arm with a 1:1 allocation. Permuted blocks were used, and randomization stratified by centre. The size of the permuted blocks varied by chance within predefined concealed limits. The permuted block sizes were not disclosed to the participating clinics. The entire process of registration, randomization, and allocation was completed on the study homepage, with coding performed by a system developer independent of the remainder of the study recruitment, conduct, and analysis. The centre-specific allocation sequences were stored on a secure server, accessible only by the above system developer at Information and Communication Technology Services and System Development, Umeå University, Sweden.https://www.norrlandskirurgi.se/Randomisera/Blanketter/Psin/PSIN_Studieprotokoll_V8.pdf.The original study protocol in Swedish can be downloaded from Analyses were performed with the intention-to-treat principle. Median wound healing time and 95 per cent confidence interval (c. i.) was calculated with survival analysis, using time to event  and allowing censoring (loss to follow-up). The log-rank test was used to compare wound healing time between the groups and the result was visualized with a Kaplan–Meier failure graph. Differences in proportions between groups were tested with Fisher’s two-sided exact test. Study groups were also compared with the Southampton Wound Assessment Scale, treating this variable on an ordinal scale, using the Mann–Whitney U test.15 and 10 per cent for excision and primary closure in the midline16. A requirement of 400 patients was estimated to achieve 90 per cent statistical power at a 5 per cent significance level (with an assumed 10 per cent loss to follow-up). According to the literature at study inception, no data existed on differences in wound healing time, providing no further information for a power calculation. An interim analysis was planned to take place after the first 100 operations. To balance for multiple testing, a Benjamini–Hochberg procedure with a 5 per cent false-discovery rate was used to determine statistical significance for the two co-primary endpoints in the study.The power calculation was based on reported figures for recurrence: 2 per cent for Karydakis flap surgeryP value was less than 0.05. Data analysis was performed in STATA .All tests were two-sided, and test results were considered significant if the Fig. 3. Of the 116 patients, 103 were men. Patient characteristics are presented in Table 1. Median wound healing time was 51 days (95 per cent c.i. 34 to 66) for excision with primary suture in the midline and 14 (95 per cent c.i. 12 to 20) days for Karydakis flap surgery, calculated with the survival analysis concept. This difference was statistically significant (P < 0.001).The surgical department at Umeå University Hospital, in Umeå, Sweden, and the surgical department at Sunderby Hospital, Luleå, Sweden, participated in the study from September 2006 to March 2020 (first randomization to last long-term follow-up). An independent Professor of Surgery  recommended early termination of the study based on the results of a planned interim analysis of the first 100 patients recruited. The preliminary results suggested a significant difference in wound healing time between the groups. Guidance was provided by the Regional Ethical Review Board, which agreed to early termination of the study. Further inclusion of new patients was stopped in September 2015, when 125 patients had been enrolled and randomized. Of these, 116 could be analysed, with 56 and 60 patients in each group. Seventy-three patients were operated on at Sunderby hospital and 43 at Umeå University Hospital. The intention was to include consecutive cohorts at each hospital. However, 43 patients were treated by visiting surgeons, specialists, or residents not involved in the study and not assessed for eligibility in the inclusion period from September 2006 to September 2015. A CONSORT diagram of the study is presented in Fig. 4 and results are summarised in Table 2. Median operation time was 31.5 min (95 per cent c. i. 25 to 36) for excision and suture in the midline group, and 60 min (95% c.i. 50 to 69) for Karydakis flap surgery (P < 0.001).Wound healing time for the two different operations is illustrated in a Kaplan–Meier (failure) graph in P = 0.753). Recurrences were registered at postoperative day 88, 535, 803, 894, and 3245 for the excision and suture in the midline group and at postoperative day 91, 1033, 1142, 1688, and 3547 for the Karydakis flap group. The estimated frequency of recurrences for both groups by time is illustrated in Fig. 5. One recurrence occurred within 3 months in each group.Median follow-up time was 3940.5 (range 11 to 5069) days. Successful follow-up as per protocol for a minimum of 3 years if no recurrence was achieved in 90 of 116 patients. There were 10 recurrences, of which five occurred in each group . Wound healing categorized according to the Southampton Wound Assessment Scale is presented in Table 3. There were more disturbances of wound healing in the excision and suture in the midline group than in the Karydakis flap surgery group (P < 0.001).Patients with open postoperative wounds were more common in the excision and suture in the midline group  seems to be more appropriate for all techniques. This study did not show any significant difference in recurrence rate, in contrast to a previous study (2.5 versus 11.0 per cent), favouring the Karydakis flap operation, possibly due to shorter follow-up time (mean 30.4 months)11. In contrast to another randomized trial12, the present study revealed significantly more postoperative wound complications associated with a longer wound healing time in the excision and primary suture in midline group. Similar results have been found elsewhere20, raising questions about why midline wounds are associated with dehiscence or surgical site infections. Tension in the suture line is a possible cause22, but it has not been proven whether small excisions in the midline would produce more tension than large off-midline wounds. The theory of pressure changes in the subcutaneous tissue of the natal cleft when the patient stands up from a sitting position has been proposed as a primary cause of pilonidal sinus disease23. This mechanism might also result in postoperative midline wound complications.The wound healing time after Karydakis flap surgery in this study is in keeping with the literatureKarydakis flap surgery is more complex than the excision and primary tension-free suture in the midline, clearly demonstrated by procedure time, which was approximately twice as long as the excision with primary suture in the midline technique. One of the negative aspects of Karydakis flap surgery is the long vertical scar it produces and a minimum wound length of 12 cm. In combination with the relatively large wound area under the flap, the authors consider the operation unsuitable for local anaesthesia alone, in contrast to many other less extensive techniques. The present follow-up time is long (6 to 13 years) and must be considered a strength, although there were some dropouts during the follow-up period. While the results from the present study can be generalized for a young male population with manifest pilonidal sinus disease, these outcomes may not be applicable in children or females.There are limitations to this study. The study was terminated early, partly owing to slow recruitment, and also as a result of a planned interim analysis, which demonstrated a significant different in the primary endpoint. The study was only partly blinded, indicating that only the assessors of late recurrences were blinded to the type of operation performed. The assessors of early results (nurses at the outpatient clinic) were impossible to blind for type of operation but were not involved in the surgery or the analyses of the results. Another limitation was recognized regarding the planned follow-up by visits to the clinic 3, 12, and 36 months after surgery. This strategy largely failed, resulting in incomplete follow-up data. To compensate for this, a structured telephone interview was performed. The study did not include all consecutive patients, and some were not operated on by the preselected surgeons providing the standardized study treatment options, but they were analysed according to the intention-to-treat principle.This was a pragmatic study in that no other adjustments to routine surgical practice were made, making the results easily applicable. The six surgeons in the study were experienced general surgeons but did not claim to be subspecialists in pilonidal sinus disease surgery, which may affect the generalizability of the results.versus 62 per cent), the present study implies more research on wound healing and prevention of postoperative complications after surgery for pilonidal sinus disease is required.As the proportion of patients with complication-free postoperative wound healing was very low in both groups (29 per cent zrac007_Supplementary_DataClick here for additional data file.


Next-generation sequencing is a promising approach for the detection of causal variants within previously identified quantitative trait loci. Because of the costs of re-sequencing experiments, this application is currently mainly restricted to subsets of animals from already genotyped populations. Imputation from a lower to a higher marker density could represent a useful complementary approach. An analysis of the literature shows that several strategies are available to select animals for re-sequencing. This study demonstrates an animal selection workflow under practical conditions. Our approach considers different data sources and limited resources such as budget and availability of sampling material. The workflow combines previously described approaches and makes use of genotype and pedigree information from a Landrace and Large White population. Genotypes were phased and haplotypes were accurately estimated with AlphaPhase. Then, AlphaSeqOpt was used to optimize selection of animals for re-sequencing, reflecting the existing diversity of haplotypes. AlphaSeqOpt and ENDOG were used to select individuals based on pedigree information and by taking into account key animals that represent the genetic diversity of the populations. After the best selection criteria were determined, a subset of 57 animals was selected for subsequent re-sequencing. In order to evaluate and assess the advantage of this procedure, imputation accuracy was assessed by setting a set of single nucleotide polymorphism (SNP) chip genotypes to missing. Accuracy values were compared to those of alternative selection scenarios and the results showed the clear benefits of a targeted selection within this practical-driven approach. Especially imputation of low-frequency markers benefits from the combined approach described here. Accuracy was increased by up to 12% compared to a randomized or exclusively haplotype-based selection of sequencing candidates. Next-generation sequencing (NGS) experiments provide data that are of increasing importance in animal genetics with the potential to improve knowledge of the genetic background of complex traits with societal and economical interest . In addUntil now, many of the genetic analyses in pigs are based on single nucleotide polymorphism (SNP) genotype data from chip arrays e.g., , 4). How. How4]).Available NGS data are used to enhance information content of routinely phenotyped and genotyped animals, especially in terms of imputation from a lower to a higher marker density. An efficient approach is to select a highly informative and representative subset of animals for re-sequencing and to use it as reference panel for imputation of a population that is genotyped at a lower marker density . SeveraIn a simulated data set, five approaches to select candidates for re-sequencing were discussed by Druet et al. . ResultsNGS data and imputed whole-genome sequence data are assumed to include causative variants and to enable their detection e.g., , 14, 15), 1514, 1The aim of this study was to evaluate alternative strategies for the choice of animals for re-sequencing in the maternal Landrace (LR) and Large White (LW) pig lines. Selection was conducted by applying several theoretical concepts and approaches under practical conditions. Selection steps were built on existing pedigree and real SNP genotype information within the pigFit project which focuses on piglet survival and immunocompetence , 21. PerThe aim of the project ’pigFit-Molecular genetic and immunological analysis of survival and postnatal growth of piglets’ is to identify biological relevant regions associated with survivability, health and immune traits in piglets and growing pigs. Within this project, phenotyping and genotyping were performed in the two maternal pig lines LR and LW. Blood and tissue samples were collected from piglets and their biological dams. SNP genotype data, based on the PorcineSNP60v2 BeadChip , were used to conduct the animal selection process. In total, SNP genotype information for 944 LR and 800 LW animals that were genetically linked to the pigFit data set was available. Before starting the analysis, breed-specific quality control was performed for SNPs on autosomes. Markers showing a high linkage disequilibrium , as recommended for a 60k SNP density [Haplotype-based selection aims at maximizing the diversity of haplotypes with or without weighting the haplotype frequency . Our hap density . Hickey  density  demonstrIn general, the aim of the H approach is to cover the maximum proportion of haplotype diversity within the population. The results obtained with simulated data led to the assumption that these animals will enable accurate imputation from chip genotype data to sequence data .To carry out the selection step, we used the AlphaSeqOpt-Method 1 . In a nuGeneralCoreLength used in the AlphaPhase software. Since the H approach aimed at covering a large amount of haplotypes, the threshold for haplotype frequency in the population was set to 0 to take all the haplotypes into account, regardless of their frequency. In both lines, the availability of sample material was a key limiting factor, particularly in previous generations. Thus, the target number of animals to select for re-sequencing was set to 100. Animals were added to the candidate set in an iterative manner. Although the AlphaSeqOpt software includes an additional approach, Method 2 [The settings for the AlphaSeqOpt software were modified according to the porcine genome structure with 18 autosomes and to the chip structure with a varying number of SNPs per chromosome. The call rate of SNPs and individuals had to exceed 90% to be included in the construction of the haplotype library. Information on the number of cores and the core length was adapted to the Method 2 , this meOptimisationMethod was set to Pedigree. A subset of 100 candidates from the genotyped animals was selected based on pedigree relationships [One of the strategies discussed by Druet et al.  maximizeionships , 25.Ancestors (P2) were identified as the animals that best explained the genetic variability of both populations considering their unbalanced use in breeding [The ENDOG v.4.8 software  was usedbreeding , 27.and at least by one of the P approaches Step 1: Animals that were selected by the H approach Step 2: Animals that were selected in both P approaches and both parents, sire and dam, were identified as important ancestors (P2)Step 3: Animals that were selected by the H approach and one parent was identified as an important ancestor (P2)Step 4: Animals that were selected by the H approach As the starting point for the C approach, all animals from the H, P1, and P2 approaches were taken into account to select animals for re-sequencing. According to the available budget, the target number of animals was set to 57, divided into 28 LR and 29 LW animals. The C approach is practical-driven and aims at optimizing imputation accuracy of common and low-frequency variants by combining haplotype- and pedigree-based criteria. The findings shown by Druet et al.  providedcombined sample) was compared to the set of the top candidates from the H approach (haplotype sample) and a set of randomly selected animals in a 30-fold repetition (random sample). Genotype data in the reference panel contained information on all markers that passed quality control .Evaluation of alternative selection strategies was performed based on the 60k SNP genotype data in a 0, 1, 2 coding as obtained from the PorcineSNP60v2 BeadChip . The workflow for the validation is shown in Fig. 1) or half of the number of SNPs (LD2) to missing. Imputation was performed from LD to HD for each of the reference panels combined sample, haplotype sample, and random sample, using FImpute v3 [The remaining animals  were allocated to the target panel. The assessed imputation scenarios are shown in Fig. mpute v3 . ImputatThe H approach resulted in 100 candidates per breed that maximized the diversity of haplotypes across the population. As shown in Fig. The P1 and H approaches are based on the same animals originating from the most recent generations. Within the P1 approach, generations 3 and 4 (relative to the most recent generation being generation 1) comprised about two thirds of the 100 candidates in each breed see Fig. .The P2 approach included 17 generations of pedigree for both populations. In total, 148 LR and 117 LW animals were selected based on pedigree relationships. Selected LR animals were from 13 of the 17 generations, with the largest number from generations 6 to 8  and all MAF classes . Imputation using the random sample  and haplotype sample  as reference panel had a lower mean accuracy.Imputation accuracy was calculated to evaluate and quantify the performance of the selected animals for re-sequencing in a practical data set based on 60k SNP genotypes. The detailed results are in Table Assessment of the imputation accuracy was performed by imputing masked genotypes from the PorcineSNP60v2 BeadChip  in a 20-fold repetition and reflect the accuracy of imputation to a HD density in these populations. However, this study cannot make a conclusive statement regarding the accuracy for imputation to sequence data.1) led to higher accuracies compared to imputation of 50% of the SNP set (LD2). Exceptions were found for the random sample in LR and the MAF class >5% in the combined sample in LR.Overall, imputation of 10,000 masked SNPs . This may be due to the prioritization of haplotype diversity in the selection process for the combined sample and to the imputation to 60k level. However, in terms of low-frequency variants, imputation to the sequence level remains a major challenge, especially with a small number of sequenced animals.The strategy for selecting animals is very important, especially to enable an accurate imputation of low-frequency variants . As expeIn addition, our study observed differences in imputation accuracy between LR and LW. For low-frequency (1–3%) and higher-frequency markers (>5%), imputation performance was better in LW. In addition to breed-specific genetic aspects, one possible explanation is the larger number of animals within the reference panel for LW. It is important to underline that both nucleus populations are genetically distinct and differ in their breeding objectives to a considerable extent. Moreover, genotyped animals were assumed to be representative samples of both nucleus populations.combined sample as reference panel for LR .Compared to random selection, targeted selection approaches achieved up to 12.4% higher imputation accuracy using the In general, imputation accuracy varied a lot between replicates of randomly selected reference panels. This emphasizes the importance of the structure of reference panels for re-sequencing experiments, especially when resources are limited . Our res1 in LR. Nevertheless, an increase of about 2.4 to 8.1% was found with low-frequency markers of the C over the H approach.Assuming the availability of sample material from all prioritized candidates, the H approach was tested as an alternative approach, by considering haplotype-based criteria, only. It should be noted that the C approach is a heuristic, practical-driven approach and its results differ slightly from those using exclusively the H approach. For markers with a higher MAF (>5%), we observed no improvement in imputation accuracy of the C approach compared to the H approach. The latter achieved an even slightly higher accuracy for LDTargeted selection using the C approach with haplotype- and pedigree-based selection criteria, as described in this paper, shows that it is superior compared to alternative approaches to select animals for a re-sequencing experiment using data from the LR and LW populations investigated here.The respective characteristics of the LR and LW populations are common for commercial maternal nucleus breeding populations. Moreover, we had to consider the practical constraints  within our C approach. Against this background, our practical-driven approach and its results could be considered for similar situations in pig breeding using the data of nucleus herds.combined sample will be used to supplement the pigFit project in future investigations. Making use of highly informative genotype data, genome-wide association studies (GWAS) that use highly informative genotypes are a highly efficient approach to map loci and to detect QTL and candidate genes for complex traits . This as .Targeted animal selection for re-sequencing is recommended. Taking into account all available information on population structure, when selecting animals for re-sequencing, including pedigree information and additional SNP genotype information, increased the imputation accuracy of masked SNP chip genotypes, measured as the correlation between true and imputed genotypes, by an average of around 7.3 to 12.4% for low-frequency markers and up to 5.3% for markers with a MAF >5%. The combined use of pedigree- and genotype-based approaches in practical data sets is particularly suitable for improving imputation accuracies for low-frequency markers.