Resuscitation at birth of infants with Congenital Diaphragmatic Hernia (CDH) remains highly challenging because of severe failure of cardiorespiratory adaptation at birth. Usually, the umbilical cord is clamped immediately after birth. Delaying cord clamping while the resuscitation maneuvers are started may: (1) facilitate blood transfer from placenta to baby to augment circulatory blood volume; (2) avoid loss of venous return and decrease in left ventricle filling caused by immediate cord clamping; (3) prevent initial hypoxemia because of sustained uteroplacental gas exchange after birth when the cord is intact. The aim of this trial is to evaluate the efficacy of intact cord resuscitation compared to immediate cord clamping on cardiorespiratory adaptation at birth in infants with isolated CDH. The Congenital Hernia Intact Cord (CHIC) trial is a prospective multicenter open-label randomized controlled trial in two balanced parallel groups. Participants are randomized either immediate cord clamping (the cord will be clamped within the first 15 s after birth) or to intact cord resuscitation group . The primary end-point is the number of infants with APGAR score <4 at 1 min or <7 at 5 min. One hundred eighty participants are expected for this trial. To our knowledge, CHIC is the first study randomized controlled trial evaluating intact cord resuscitation on newborn infant with congenital diaphragmatic hernia. Better cardiorespiratory adaptation is expected when the resuscitation maneuvers are started while the cord is still connected to the placenta.  Congenital Diaphragmatic Hernia (CDH) is a rare disease (1/3000 pregnancies) caused by diaphragmatic defect with ascension of the abdominal content into the thoracic cavity. Pulmonary consequences of CDH present a broad spectrum of severity . FailureThe CDH EURO Consortium proposes intubating newborn infants with CDH immediately after birth to limit PPHN . TreatmeTraditionally, the umbilical cord is clamped and cut immediately after birth. Clamping the umbilical cord immediately increases systemic peripheral resistance, resulting in an increase in arterial pressure (afterload). However, as the placental circulation receives 30–50% of fetal cardiac output, cord clamping transiently reduces venous return (by 30–50%), which, combined with the increase in afterload, decreases cardiac output . FollowiIn 2015, the International Liaison Committee on Resuscitation (ILCOR) recommended that the cord should not be cut for at least 1 to 3 min after birth in infants not requiring resuscitation . This reThe ex-utero intrapartum treatment (EXIT) procedure has been proposed during a variety of surgical procedures, mainly cervical mass resection, performed at birth to secure the fetal airway or ensure successful transition to postnatal environment . The aimWe have shown in three previous studies in normal newborn lambs and in newborn lambs with PPHN that blood gases did not change 30 min after birth despite lack of breathing compared to the blood gases obtained before birth ,14,15.We also evaluated the safety, feasibility and impacts of intact cord resuscitation (ICR) on cardiorespiratory adaptation at birth in newborn infants with CDH in a prospective pilot study . Resuscitation before cord clamping was possible for all infants in the ICR group. The cord was clamped at 7 ± 3 min after birth. No increase in maternal or neonatal adverse events was observed during the period of ICR. A multicenter randomized clinical study is required to confirm the benefit of intact cord resuscitation in CDH infants on cardiorespiratory adaptation at birth. To know whether intact cord resuscitation improves initial cardiorespiratory adaptation at birth is a major issue. The primary endpoint is the rate of infants with APGAR score < 4 at 1 min or <7 at 5 min.This primary objective is to show the efficacy of intact cord resuscitation compared to immediate cord clamping on cardiorespiratory adaptation at birth in full term newborn infants with isolated CDH. APGAR score is used to assess cardiorespiratory adaptation at birth. APGAR score is based on clinical assessment of color, heart rate, grimace, muscle tone, and respiratory effort. It is used worldwide by neonatal caregivers, both as a measure of the infant’s clinical status as well as a measure of the infant’s response to resuscitation. APGAR score can be assessed during resuscitation maneuvers, including intubation. Slight inter-observer variation may exist in intubated baby mainly due to lack of standardization of scoring respiratory effort states. In order to limit inter-observer variability, we will standardize respiratory effort scoring as previously reported . In ordeAPGAR is universally recognized as a major prognostic variable of neonatal outcome . More sp-Frequency of postpartum hemorrhage (PPH) defined by blood loss ≥ 500 mL;-Frequency of severe PPH, defined by measured blood loss ≥ 1000 mL;-Blood loss volume at 15 min after birth;-Total postpartum blood loss volume ;To ensure maternal safety of the procedure, blood loss will be carefully monitored after birth. A graduated collector bag for blood, placed under the woman’s buttocks just after delivery of the child, will be used systematically to measure the blood lost through the vagina in the immediate postpartum period. This bag will be left in place at least for 15 min. The following maternal safety endpoints will be assessed:-Frequency of infants with the need for epinephrine administration and/or fluid resuscitation;-Frequency of infants with the need for chest compressions;-Pre-ductal SpO2, and heart rate at 1, 5, and 10 min after birth: a pulse oxymeter sensor will be placed at the right hand as soon as possible (within the first minute after birth), which then will be connected to a pulse oxymeter; -Blood gases and plasma lactate concentration at one hour after birth (H1): these quantitative variables can be considered as objective markers of early cardiorespiratory adaptation at birth. -Blood gases, pre- and postductal SpO2, lactate, FiO2 set to obtain preductal SpO2 90–94%, ventilatory parameters , heart rate, blood pressure, and urine output at H1, H24, H48, H72, D7, D28;-Volume of fluid resuscitation during the first 24 h;-Frequency of infants with the need for vasoactive drugs during the first 24 h;-Frequency of infants with the need for pulmonary vasodilator during the first 24 h;-Hemoglobin concentration at H24;-Echocardiographic parameters  at H6, H24, H48, D7, D28;To assess the effect of intact cord resuscitation compared to immediate cord clamping on cardiorespiratory adaptation of infants after birth, the following secondary endpoints will be assessed:-Infant mortality rate at 90-day after birth;-○mechanical ventilation free-days , ○extracorporeal membranous oxygenation free-days ○pulmonary vasodilator treatment free-days ,○O2 supplementation free-days ,○parenteral nutrition free-days  ○Total duration of hospitalization,Infant morbidity outcomes assessed within the first 90 days after birth:To assess the effect of intact cord resuscitation compared to immediate cord clamping on infants’ mortality and morbidity, we choose to assess the number of free-days from medical support in order to address the effect of mortality on the assessment of morbidity. Because of the randomization process, we can assume that the two groups will be similar in term of severity. In the hypothesis that intact cord resuscitation decreases the mortality, it means that more severe CDH infant may survive, which may in turn increase the apparent morbidity. In case of death, the number of free-days from medical support is zero, which will not modify the number of free-days from medical support in the surviving population. Therefore, the following secondary end-points will be assessed:-Number of refusals to participating with the protocol: the reasons for refusal will be recorded ; -Anxiety and depression level assessed by Hospital Anxiety and Depression Scale, HADS. HADS is an auto-questionnaire translated in French, including seven questions to assess anxiety and seven questions to assess depression. HADS questionnaire lasts 3 to 6 min, and is mostly well accepted. Both parents will be requested to answer the questionnaire within the first 3 days after birth, in a calm and neutral room at the maternity ward. Psychologist in charge of the parents will be informed of the results of the test to adapt family support if required; -Semi-structured interviews will be proposed by a psychologist to the parents whatever the issue, at the end of the study period (90 ± 7 days after birth) to assess their personal experience of the resuscitating period at birth, including both early/delayed cord clamping and close/remote resuscitation maneuvers. The interviews tape recordings will be transcribed and then analyzed using thematic coding. The anonymized data will be independently coded by three researchers and compared for consistency of interpretation. The themes that emerged following the final coding will be used for a qualitative analysis of the parental verbatim;To assess parental acceptability and psychological impact of starting resuscitation while the cord is intact as compared to the immediate cord clamping group:The secondary objectives of the studies are as follows:This is a prospective multicenter open-label randomized controlled trial in two balanced parallel groups. Antenatal diagnosis of CDHNo severe additional malformation or chromosomal diseasesFull term No inclusion in another antenatal trialWritten informed consents from the parentsThe fetuses which required Fetoscopic Endoluminal Tracheal Occlusion (FETO procedure) will also be included in the study.Preterm birth before 37 weeks gestational ageOther severe malformation(s) or chromosomal diseasesNeed for emergency cesarean section (“red code”)Twin pregnancyPostnatal diagnosis of additional severe malformation(s) or chromosomal diseasesAfter assessing the inclusion and exclusion criteria, the parents will be informed of the study protocol and the intact cord resuscitation procedure . The written parental consents are requested before 37 weeks gestational age. A web-based randomization with electronic case-report form (eCRF) system is performed at admission for delivery at the maternity ward. Prenatal o/e LHR on ultrasounds and lung volumetry on fetal MRI allow to classify patients in according the severity of the CDH  ,21. RandThe newborn infant should be intubated routinely without bag and mask ventilation, with an endotracheal tube 3.5 mm;Ventilation in the delivery room should be done with a peak pressure below 25 cmH2O, and an initial FiO2 = 1;The goal of treatment in the delivery room is achieving heart rate > 120 beats/min and increasing preductal SpO2 or achieving acceptable preductal SpO2 targets between 80 and 95%;An oro- or nasogastric tube with continuous or intermittent suction should be placed;Group 1: Immediate cord clamping with transfer to the resuscitation roomThe infants with CDH will be included in the study and randomized in one of the two groups. Severity subgroups is considered as a stratified factor in randomization. A dynamic randomization procedure using the Pocock and Simon minimization method is used . WhateveIn the immediate cord clamping group, the cord will be clamped within the first 20 s after birth and the infant will be transferred to the resuscitation room. The newborn infant will be intubated and mechanically ventilated as quickly as possible on the resuscitation table as recommended in the Programme National de Soins. After cardiorespiratory stabilization , the infant will be transferred to the neonatal intensive care unit (NICU). Oxytocin is infused to the mother as recommended in the local protocol .Group 2: Intact cord resuscitation with resuscitation maneuvers performed on a dedicated trolley placed close to the motherIn the intact cord resuscitation group, the umbilical cord will be kept intact during the initial phase of the resuscitation. The infant will be placed on a specifically designed compact trolley with a warmed platform, suitable for commencing resuscitation between the mother’s legs in case of vaginal birth or near the operating table beside the mother in case of cesarean section. This trolley will be fully equipped for resuscitation, including a suction device, gas flowmeter/blender, ventilator, and monitoring system. Its height can be adjusted to position the infant close to the maternal perineum. The infant will be intubated and mechanically ventilated on this trolley; special care will be taken to prevent stretching, compression, or kinking of the cord. The cord will be clamped after cardiorespiratory stabilization will be obtained  or in case of spontaneous placental expulsion. Oxytocin is infused to the mother just after cord clamping. The infant will be then transferred to the NICU. See Except the timing of cord clamping, the national guidelines will be applied during the course of the prenatal and postnatal management.Here is a photograph of the trolley  :Some differences with usual care during implementation of the protocol are as follows:In both groups, although APGAR score is routinely assessed in every baby at 1 and 5 mn, special care will be taken to optimize scoring taken into account the fact that most infants with CDH are intubated at 5 min. APGAR score can be assessed during resuscitation maneuvers, including intubation. Slight inter-observer variation may exist in intubated baby mainly due to lack of standardization of scoring respiratory effort states . In ordean infant who is apneic and requires intubation and ventilation should receive the minimum value of 0 for respiratory effort;an infant who requires artificial ventilation at birth due to irregular or shallow ventilation should score 1;To assess whether an artificially ventilated infant is apnoeic or not, ventilation should be stopped briefly, when possible, to check for the presence of spontaneous respiratory movements .Preductal SpO2 and heart rate are routinely assessed in every baby with CDH. They will be recorded at 1, 5 and 10 min after birth in both groups.Blood gases and lactate concentration are routinely assessed in every baby with CDH. These parameters will be recorded at H1, H12, H24, H48, H72, D7, D28, in both groups. Compared with usual care, no additional blood samples are required for the protocol;In both groups, although noninvasive echocardiography is routinely assessed in every baby with CDH, left and right mean blood flow velocities, pulmonary artery pressure will be recorded at H6, H24, H48, D7, D28. Compared with usual care, no additional echocardiography is required for the protocol;Acceptability by the parents and psychological impact of intact cord resuscitation will be assessed using a questionnaire and a semi-directive interview.The healthcare providers  in charge of the baby or the mother may stop the procedure—i.e., clamp the cord—if they judge it is required for the safety of the patient. The reason for stopping the procedure and the timing at cord clamping will be recorded. In that case, the patient is not excluded from the study and the variable is recorded as planned in the protocol. The patient data are analyzed according to intention to treat principle.The follow-up of the infants is planned till the age of 3 months (90 days), corresponding to the last visit. If the patient is transferred in another hospital, the variable will be recorded till the age of 3 months.The primary objective is to show the superiority of intact cord resuscitation  compared to immediate cord clamping (control group) to improve cardiorespiratory adaptation at birth of CDH infant. The primary endpoint is defined as the rate of infants with low APGAR score, defined by a APGAR score < 4 at 1 min or <7 at 5 min of life. On the basis of literature, we expected that 37% of CDH infant in control arm will have a low APGAR score. To show a 50% relative reduction , with a type I error of 5% (two-sided test), and a power of 80%, we calculated that 90 subjects (mother/infant) per group are needed, which means a total of 180 subjects.Statistical analyses will be independently performed by the Biostatistics Department of University of Lille under the responsibility of Professor Alain Duhamel. For the data analysis, statisticians will be unaware of the treatment group allocation. Data will be analyzed using the SAS software  and all statistical tests will be performed with a two-tailed alpha risk of 0.05. All analyses will be performed in all randomized patients based on their original group of randomizations, according to the intention-to-treat principle. No interim analysis will be performed. A detailed statistical analysis plan will be written and finalized prior to the database lock. Baseline characteristics will be described for each group. Quantitative variables will be expressed as mean (standard deviation), or median (interquartile range) for non-Gaussian distribution. Categorical variables will be expressed as frequencies and percentages. Normality of distribution will be assessed graphically and using the Shapiro–Wilk test.The Data and Safety Monitoring Board (DSMB) is an independent consultative board asked to express an opinion to the sponsor of the study on the benefit/risk ratio and the management of the clinical trial.A DSMB will be set up, composed of the following members, at least: two pediatricians, two obstetricians, one anesthetist, and one methodologist.Its members will not participate in the study and will be independent from the investigation centers. They will be nominated by the sponsor of the study and will participate as volunteers in the respect of confidentiality of the data. -If the number of Serious Adverse Event (SAE) within the first 24 h after birth is twice higher in the “intact cord resuscitation” group after the inclusion of 30 patients,-If the DSMB judges that a reported SAE caused by the procedure requires to stop the study,-If inclusion rate is less than 25% of the total inclusion objectives, 24 months after starting the inclusion in the study. The board will meet after the inclusion of 30, 90 and 150 patients, to address those points, but it could be summoned on other circumstances:-If a mother’s death occurs within three months after she gave birth to her child,-At the request of any PI involved in the study.The DSMB will analyze the management of the study as well as its benefit/risk exposure and might recommend to stop the study in certain circumstances:Presently, 11 centers are participating to the study: two additional centers are expected to include CDH infants. The first center started enrolling patients for 6 months. At the present time, 10 patients have been recruited. For now, parental consents have been obtained in all cases. Recruitment is expected to be completed by December 2023.Resuscitation at birth of infants with congenital diaphragmatic hernia remains highly challenging because of severe failure of cardiorespiratory adaptation at birth.The traditional approach in the delivery room is immediate cord clamping followed by intubation. Initiating resuscitation prior to umbilical cord clamping (UCC) may support this transition, to avoid the loss of venous return and decrease in left ventricle filling caused by cord clamping, ideally increase in pulmonary blood flow should precede cord clamping. This would enable pulmonary venous return to replace umbilical venous return as the primary source for left ventricle preload and to minimize swings in left ventricular output caused by cord clamping. In infants with CDH, the cord is clamped while the pulmonary blood flow is still low. Evidence indicates that utero-placental gas exchange continues after birth when the cord is intact. A previous experimental study in newborn lambs showed that heart rates and right ventricle output were markedly decreased within 120 s of cord clamping. In contrast, if umbilical cord clamping was delayed until after ventilation had commenced, these large changes in heart rate, arterial pressures and flows were greatly reduced, resulting in a much more stable cardiovascular transition after birth ,26. We hPreliminary study has demonstrated the feasibility of intact cord resuscitation. Starting mechanical ventilation of the infant newborn with congenital diaphragmatic hernia before clamping the cord allowed a potential benefit to improved blood pressure at 1 h of life compared to those resuscitated traditionally .p < 0.05). Mean blood pressure was significantly higher in the intact cord resuscitation than in the immediate cord clamp group at H1 (52 ± 7 vs. 42 ± 7 mmHg), H6 (47 ± 3 vs. 40 ± 5 mmHg) and H12 (44 ± 2 vs. 39 ± 3 mmHg) (p < 0.05). Therefore, commencing resuscitation and initiating ventilation while the infant is still attached to the placenta is feasible in infants with CDH. The procedure is safe, and may support the cardiorespiratory transition at birth in infants with CDH [Lefebvre et al. demonstrated that Intact cord resuscitation was associated with higher APGAR scores at 1 and 5 min after birth. The pH was higher and the plasma lactate concentration was significantly lower at one hour after birth in the intact cord resuscitation than in the immediate cord clamp group (pH = 7.17 ± 0.1 vs. 7.08 ± 0.2; lactate = 3.6 ± 2 vs. 6.6 ± 4 mmol/L, with CDH .To our knowledge, CHIC trial is the first prospective multicenter randomized controlled trial to assess the impact of delayed cord clamping during resuscitation of newborn infant with congenital diaphragmatic hernia. Future publication on the primary and secondary results of the CHIC trial study will make a substantial contribution to this important orientation of research and the results will provide clear evidence on the impact of delayed cord clamping during resuscitation of the newborn infant with congenital diaphragmatic hernia.


Background: Adequate information and knowledge about COVID-19 has been shown to induce the confidence and positive performance among healthcare workers (HCWs). Therefore, assessing the relationship between confidence in knowledge and associated factors among HCWs is vital in the fight against COVID-19. This paper investigates factors associated with HCWs' confidence in their overall knowledge about COVID-19 in South Africa in the early stages of the epidemic.Methods: Data utilized in this paper were from an online survey conducted among HCWs using a structured questionnaire on a data free online platform. The study population were all the medical fraternity in South Africa including medical and nurse practitioners as well as other healthcare professionals. Bivariate and multivariate logistic regression models were performed to examine the factors associated with confidence in HCWs' overall knowledge about COVID-19.Results: Overall, just below half (47.4%) of respondents indicated that they had confidence in their overall knowledge about COVID-19. Increased odds of having confidence in the knowledge about COVID-19 were significantly associated with being male , having a doctorate degree , being satisfied with the information about COVID-19 guidelines , having received training in 6–8 areas  and having received training in 9–11 areas , and having already treated COVID-19 patients . Those who were highly concerned with the levels of training of HCWs  had decreased odds of having confidence in their overall knowledge about COVID-19.Conclusion: This study sheds light on the importance of capacitating HCWs with knowledge and adequate relevant training as part of infection prevention control measures during pandemics. Future training and information sharing should be sensitive to knowledge gaps by age, gender, qualifications, professional categories, and experience. Severe Acute Respiratory Syndrome Coronavirus 2 or SARS-CoV-2 is the virus responsible for the illness now commonly referred to as “corona virus” disease 2019 (COVID-19). This novel coronavirus was first reported in December 2019 with cases emanating from Wuhan city in the Hubei Province of China . ApproxiCOVID-19 transmission occurs through droplets, bioaerosols, fomites , 6, it aRecent studies on HCWs' knowledge, attitude, and practice about COVID-19 reported generally, sufficiently high knowledge, positive attitudes and good practices across several countries –17. In aAcquisition of adequate knowledge has a positive effect on confidence where higher levels of knowledge on personal protective equipment (PPE) were significantly positively correlated to the HCWs confidence in PPE . HoweverData utilized in this paper were from an online survey conducted from 11th April to 7th May 2020 using a structured questionnaire. The study population was all HCWs aged 18 years and older from all provinces in South Africa. These included medical and nursing practitioners as well as other types of HCWs. An online survey was utilized instead of the face-to-face method as it was administered during lock down level 5 in South Africa and physical distancing was advised.via mainstream media channels, including social media, email, radio and television broadcasters, and on local websites; an extensive network of strategic partners in the South African government, science councils, higher education, HCWs' professional councils, healthcare sector partners, non-profit organizations, the private sector, and faith-based and community organizations. These communication alerts included an invitation with the survey link, which when clicked on, directed potential respondents to the online survey. Both a data-free link and a standard internet link to the survey were distributed.A data-free mobile messaging platform was utilized to facilitate participation in the online survey by HCWs. Additionally, the study used a comprehensive communication strategy and communication alerts to publicize the call for participation were widely distributed The primary outcome variable was confidence in overall knowledge about COVID-19 based on the question, “Do you feel confident in your overall knowledge about COVID-19?” with response options being 1 = yes, 2 = no, and 3 = unsure. These were categorized into a dichotomized primary outcome with 1 = yes and 0 = no (no and unsure).Explanatory variables were socio-demographic variables such as sex , age group in years , population group according to Statistics South Africa (Stats SA) designations . Risk perception was assessed using the question “How would you rate your personal risk of contracting COVID-19 in the workplace?” with response options being 1 = extremely high risk, 2 = high risk, 3 = moderate risk, 4 = low risk, and 5 = very low risk. These responses were recoded into three categories namely 1 = low risk (very low risk and low risk), 2 = moderate risk and 3 = high risk (extremely high risk and high risk). Concern among HCWs about their levels of training was explored using the question “What is your level of concern regarding each of the following? Levels of training of HCWs about COVID-19” with response options of 1 = not concerned at all, 2 = moderately concerned, 3 = highly concerned, and 4 = extremely concerned. These were recoded into three categories, 1 = not concerned, 2 = moderately concerned, 3 = highly concerned (highly concerned and extremely concerned). Having received training in managing COVID-19 was assessed using the following question, “Have you received any formal training about the management of COVID-19 ?” with 11 areas of training being (1) Screening people for COVID-19; (2) Referring individuals for testing; (3) The tests that should be done to make the diagnosis; (4) Case definitions; (5) Protocol for workplace infection control; (6) Isolation procedures for patients; (7) Treatment guidelines; (8) Staff transportation; (9) Patients transportation; (10) Visitor policies; and (11) Declaring patients as recovered, with each having response options of 1 = yes, 2 = no, and 3 = n/a. The responses to these 11 items were used to create a received training areas sum score ranging from 1 to 11, where 1 was assigned to each correct response. These were further recorded into four groups, 1 = score of 2 or less training areas, 2 = 3–5 training areas, 3 = 6–8 training areas, and 4 = 9–11 training areas. Having treated COVID-19 patient was assessed with the question “Have you treated/provided care for a patient diagnosed with COVID-19?” with binary responses of yes or no.There was no readily available and compiled central database of the total South African HCWs population by demographic characteristics. Therefore, the sampling frame was estimated using information that was readily available from two databases, namely the Health Professions Council of South Africa (HPCSA), and the South African Nursing Council (SANC).Evidence from other studies has shown that re-weighted online samples can produce response patterns that are statistically similar to population characteristics. Therefore, the survey data were then benchmarked (weighted) to the distribution of South Africa's estimated HCWs population by age, sex, population group, and province using the estimated sampling frame database. Benchmarking was aimed at correcting for potential bias that may result due to the convenient sampling. Benchmarking ensure that the views and perceptions of overrepresented sectors of the HCWs population are scaled downwards and those of less represented are illuminated. This in turn enabled us to increase generalizability to the national sample of HCWs.p < 0.0001).Statistical analysis was performed using Stata version 15.0  using dap < 0.05 was considered statistically significant.Descriptive statistics with unweighted frequencies and weighted percentages are presented. Differences in confidence in overall knowledge about COVID-19 across the socio-demographic variables were compared using 95% confidence intervals (CIs) and the Chi-square-test. The association between confidence in overall knowledge about COVID-19 and potential explanatory variables was assessed using bivariate logistic regression models. All statistically significant variables from these bivariate logistic regression models were entered into the final multivariate logistic regression model to examine the factors associated with confidence in overall knowledge about COVID-19. Crude and adjusted odds ratios (aOR) with 95% confidence intervals and a The study sample used for this paper was 5,530 respondents, based on the primary outcome variable of confidence in overall knowledge about COVID-19. After benchmarking, females accounted for 76.7% and Black Africans constituted 58.5% of the sample . About 2Respondents who were satisfied with information about COVID-19 guidelines being filtered to them from their workplace structures (73.6%), those with low perceived risk of contracting COVID-19 (59.9%), those who were not concerned with levels of training of HCWs (78.6%), and those who said they had treated COVID-19 patients (56.6%) were significantly more confident in their overall knowledge about COVID-19 than their counterparts.p < 0.05] were significantly more likely to be confident in their overall knowledge about COVID-19 than their female counterparts. Those with doctorate degrees  were significantly more likely to be confident in their overall knowledge about COVID-19 than those with diplomas/occupational certificates.p < 0.001] were significantly more likely to be confident in their overall knowledge about COVID-19 than those who were not. Healthcare workers who were highly concerned with their levels of training  were significantly less likely to be confident in their overall knowledge about COVID-19 than those who were not concerned at all.Respondents who were satisfied with the information about COVID-19 guidelines being filtered to them from their workplace structures  and those who received training in 9–11 areas  were significantly more likely to be confident in their overall knowledge about COVID-19 than those who received training in two or fewer training areas. This suggests confidence was boosted among those who had received a wider scope of training than those with limited scope. Those who reported that they had already treated COVID-19 patients  were significantly more likely to be confident in their overall knowledge about COVID-19 than those who had not treated such patients, suggesting that direct hands on experience with patient care and management also boosted confidence.Those who indicated they received training in 6–8 training areas [aOR = 2.54 (1.89–3.43), The COVID-19 pandemic has presented HCWs globally with enormous challenges in their work environments. The management of this infectious disease in a hospital setting is critical to the recovery of patients found to present with severe symptoms as well as patient care on an outpatient basis to manage recovery and prevent progression to severe disease. The present study's objective was to investigate factors associated with healthcare workers' confidence in their overall knowledge about COVID-19 as the epidemic had newly emerged in South Africa. This study found that less than half of the participating HCWs reported having confidence in their overall knowledge about COVID-19 This finding must be viewed in the context of the time frame that the study was done, which was just as cases started to increase in wave 1 of the emergence of the epidemic in South Africa and can be considered to be a baseline of HCWs confidence in these areas.It is expected that knowledge about this novel virus is time sensitive in the local healthcare setting and it is dependent on several factors including receiving timely information from the global setting as well as the local setting. Previous studies reporting on the H1N1 pandemic, found that overall knowledge about that virus had also been lower during the initial stages of the pandemic in April 2009 but this improved around August 2009 . The curThe present study also found that those with college degrees and certificate qualifications were found to have higher level of overall confidence in their knowledge of COVID-19 compared to those without degrees and certificates. Studies have shown that self-confidence has a strong relationship with academic achievement but this strength is partially dependent on the societal context .The present study also found that male HCWs had higher levels of confidence in their overall knowledge of COVID-19 compared to female HCWs Recent studies have reported that society tends to readily accept men as confident but only accepts women as confident if there is an element of prosocial orientation . TherefoHealthcare workers in the Western Cape province had higher levels of confidence in overall knowledge about COVID-19 than HCWs in other provinces. The Western Cape had experienced higher infection and transmission rates compared to other provinces in the country at the initial and progressive stages of the pandemic . This coThe study had strengths and some limitations. The strength of the study was the relatively large sample size of HCWs who participated on the study, representing all major professional categories in South Africa. In addition, the strength of this study is that it utilized an online survey methodology which provided real-time results as the COVID-19 pandemic was unfolding during the period when the country was under the highest lockdown level which restricted movement of people and rendered it difficult to reach frontline HCWs through face-to-face contact. A shortcoming of the study is inadequate measure for knowledge of COVID-19 among HCWs. The questionnaire did not have a fully encompassing knowledge about COVID-19 indicator that could be measured in this paper. A previous study  had founHowever, the fact that HCWs who received training in more areas on COVID-19 management were more likely to be confident in their knowledge about COVID-19 than those who received training in fewer areas showed that knowledge (acquired through training) leads to confidence in HCWs' overall knowledge about COVID-19. Participation through online platforms could be biased to HCWs who had access to technology and internet as well as the time and motivation to do so. To mitigate the impact of this limitation, the survey data were then benchmarked to the distribution of South Africa's estimated HCWs population by age, sex, population group, and province using the estimated sampling frame database. One of the limitations of the study could have been the fact that the study was conducted during the first wave in South Africa and there is a possibility that the confidence levels among HCWs could have increased after the second wave. Based on the above mention strengths and limitations, the authors believe that the findings from this study contribute to the COVID-19 research with evidence from the developing countries.Less than half of the HCWs in South Africa had confidence in their overall knowledge about COVID-19 during the early stages of the pandemic and this is expected to improve over time as the pandemic develops and HCWs receive more formal and informal training, knowledge and experience. This study sheds light on the importance of capacitating HCWs with knowledge and adequate training as part of infection prevention control measures for epidemic preparedness. Intensified future training activities for HCWs should be inclusive and sensitive to knowledge gaps by age, gender, qualifications, professional categories, and levels of experience.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Sciences Research Council Research Ethics Committee (REC) Protocol Number: REC 5/3/20. The patients/participants provided their written informed consent to participate in this study.PR conceived the study. TMa, TMo, SS, and PN conceptualized the paper. TMo conducted the data analysis. TMa and TMo drafted the manuscript. ND, RS, IN, SJ, BT, MMo, MMa, KZ, and PR contributed to designing the analyses, interpreting results, and made substantial contributions to the manuscript. All authors read and approved the final manuscript before submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Contact mixing plays a key role in the spread of COVID-19. Thus, mobility restrictions of varying degrees up to and including nationwide lockdowns have been implemented in over 200 countries. To appropriately target the timing, location, and severity of measures intended to encourage social distancing at a country level, it is essential to predict when and where outbreaks will occur, and how widespread they will be.We analyze aggregated, anonymized health data and cell phone mobility data from Israel. We develop predictive models for daily new cases and the test positivity rate over the next 7 days for different geographic regions in Israel. We evaluate model goodness of fit using root mean squared error (RMSE). We use these predictions in a five-tier categorization scheme to predict the severity of COVID-19 in each region over the next week. We measure magnitude accuracy (MA), the extent to which the correct severity tier is predicted.Models using mobility data outperformed models that did not use mobility data, reducing RMSE by 17.3% when predicting new cases and by 10.2% when predicting the test positivity rate. The best set of predictors for new cases consisted of 1-day lag of past 7-day average new cases, along with a measure of internal movement within a region. The best set of predictors for the test positivity rate consisted of 3-days lag of past 7-day average test positivity rate, along with the same measure of internal movement. Using these predictors, RMSE was 4.812 cases per 100,000 people when predicting new cases and 0.79% when predicting the test positivity rate. MA in predicting new cases was 0.775, and accuracy of prediction to within one tier was 1.0. MA in predicting the test positivity rate was 0.820, and accuracy to within one tier was 0.998.Using anonymized, macro-level data human mobility data along with health data aids predictions of when and where COVID-19 outbreaks are likely to occur. Our method provides a useful tool for government decision makers, particularly in the post-vaccination era, when focused interventions are needed to contain COVID-19 outbreaks while mitigating the collateral damage from more global restrictions. The global spread of SARS-CoV-2, the virus that causes COVID-19, has brought about the worst public health crisis in a generation. As of April 2021, there have been nearly 150 million COVID-19 cases and more than 3.1 million COVID-19 deaths . While lTo appropriately target the timing, location, and severity of such measures, it is essential to be able to predict when and where outbreaks will occur and how widespread they will be. Recent work has focused on using past health data to forecast accumulated COVID-19 cases and deaths using exponential smoothing models , 11, autOne potentially useful input to such forecasts is human mobility data in the form of cell phone data , 20. PasSeveral studies have incorporated aggregated and anonymized mobile phone geolocation data into epidemic models to analyze the spread of COVID-19. One study used mobile phone data indicating “time spent at home” in an SEIR model , demonstDue to constant updates in measures and changes in individuals’ behavior , the use of an epidemic model for prediction necessitates calibration to realized data at each stage of the epidemic in each region. Because we do not model transmission explicitly but instead use a model that learns from past health and mobility data, our approach to prediction is distinct from that of past work. Furthermore, our methods are generalizable to a wide range of applications as they can be used to predict outbreaks of other communicable diseases such as influenza, measles, and SARS.Our work adds to the literature on how human mobility drives disease transmission and is the first to use mobility data directly to predict daily new cases and the COVID-19 test positivity rate in each region of a country over time. We find that aggregated, anonymized human mobility data improves predictions of new COVID-19 cases and the test positivity rate, and classifications of outbreak severity, beyond predictions made using only past health data. Our forecasts are especially useful in the post-vaccination era as they can help determine when and where outbreaks will occur, and how severe they will be.Using aggregated, anonymized health data and cell phone mobility data, we forecast next 7-day averages of new cases of COVID-19 and the COVID-19 test positivity rate for different geographic regions of Israel from March to December 2020. We combine these forecasts with a categorization system to predict the severity of COVID-19 in each region one week in advance.Our work is motivated by the link between mobility and epidemic severity. We used publicly available, daily data on accumulated COVID-19 cases, active cases, tests, and recoveries from the Israeli Ministry of Health from February 1, 2020 to January 7, 2021 . IndividWe mapped the 1,642 statistical regions to 16 districts  covering all of Israel. The movement patterns take the form of origin-destination (OD) data representing daily and hourly numbers of trips between pairs of regions. For privacy considerations, if fewer than 50 individuals were moving from one region to another in a given hour, the number of reported individuals was set to 1. We replaced these 1’s with a more realistic value of 7 in our analysis . BecauseWe mapped the 2,630 TAZ regions to the 16 districts for analysis.For each district, we obtained 2017 data on the age distribution, the population size, and socioeconomic score from the Israeli Central Bureau of Statistics (CBS). The socioeconomic score is an integer value between 1 and 10, with 1 reflecting the most impoverished regions and 10 reflecting the wealthiest regions. The socioeconomic score is calculated by the CBS from demography, education, employment, and standard of living. Lastly, we calculated the median age for each district from the age distribution.We use 7-day average lagged health features to reflect COVID-19 incidence in each district over the past week. In addition to these solely health-based predictors, we combine health and mobility data to develop predictors reflecting the spread of COVID-19 between and within districts.Lag features. Due to the availability of daily updated data, when predicting values for the next week, our models can see the previous days’ realized values for incidence and the test positivity rate. Thus, we introduce lag features, which allow pure time series problems to be converted into supervised learning problems. For example, a 1-day lag would have the label of day X − 1 input as a predictor for predicting on day X. Similarly, a 6-day lag would have the labels of days  as six different predictors for predicting on day X. We test 1-day, 3-day, and 6-day lagged features for each prediction task.Pressure score. The Pressure Score for a district reflects potential cases imported into that district over the past n days. This metric has been used in the literature to predict the spatial spread of cholera with mobile phone data . Th. Th35]. We use magnitude accuracy (MA) to evaluate the performance of our predictions when classified into severity tiers. All magnitude accuracy metrics reflect performance on the 7 test districts through November and December . We measr score) over two periods: the period for which we have actual mobility data  as well as the period of the entire dataset . The extended mobility period includes the month of December and uses perturbed estimates of mobility during that month. To inform the selection of predictors for our models, we explored correlations within the health data and correlations between the health data and the non-COVID-related data such as mobility and socioeconomic data  with equal ability, rather than specializing for one task. Therefore, we weighted the evaluation periods equally when selecting predictors. Models using mobility data outperformed models that did not use mobility data, reducing RMSE by 17.3% when predicting new cases and by 10.2% when predicting the test positivity rate .The best set of predictors for new cases comprised 1-day lag of past 7-day average incidence and the Excess Internal Movement Score. This model had an RMSE of 6.825 new cases per day on the validation set and 4.812 new cases per day on the test set . Fig 3 sThe best set of predictors for the test positivity rate comprised 3-days lag of past 7-day average test positivity rate and the Excess Internal Movement Score. This model had an RMSE of 1.02% and 0.79% on the validation and testing districts, respectively.Magnitude accuracy for the prediction of new cases and the test positivity rate is shown in Confusion matrices are shown in We accurately predicted 38 of 57 district-date instances where next week truly fell into Tier 5, and 38 of the 41 district-dates that were predicted to be in Tier 5 were truly in Tier 5, leading this tiering system to have a sensitivity of 0.67 and a positive predictive value of 0.93 for new cases being in the most severe tier.The overall proportion of days for which the predicted and actual average new cases fall in the same severity tier is 0.775, which is significantly higher than would be achieved by random guessing (.20). The tier-specific classification accuracies are 0.86, 0.89, 0.65, 0.73, 0.67, for Tiers 1–5 . MisclasMagnitude accuracy using the test positivity rate tiers is 0.820 when using mobility data . The tieOur tiers were selected based on test positivity rates that occurred between April to October. In November and December, most districts had lower daily test positivity rates. The higher test positivity rate at the beginning of the pandemic is indicative of the limited test supply at that time. No district-date pairs truly fell into Tier 5 during our testing period.During this period, 58% of district-date pairs in our test districts fell into Tier 1, pictured by the dark blue squares at the top left corner of In today’s interconnected world, human mobility data in the form of cell phone data can provide valuable insight into human behavior. We have shown that aggregate and anonymized cell phone mobility data can be used to improve the prediction of COVID-19 outbreaks, measured as daily new cases and the test positivity rate. Our best model’s predictors consisted of 1-day lag of past 7-day average new cases or 3-days lag of the test positivity rate, respectively, along with the Excess Internal Movement Score. Our forecasting approach allows for early identification of regions that may develop outbreaks in the coming week, potentially allowing policy measures to be targeted to the regions where they would be the most effective. Additionally, we found that a balanced tiering system for categorizing outbreak severity (based on fractiles of previously observed data) allows for more accurate prediction than an unbalanced tiering system.Our analysis has several limitations. We assumed that both the mobility and the health data were relatively accurate estimates of the true amounts of travel and prevalence of COVID-19 in a region, respectively. If the health data for a given district is skewed due to selection bias in who receives tests, forecasts for other districts would be affected through the mobility data. Districts were included in our dataset only when one statistical region within the district reported at least 15 accumulated cases, tests, and recoveries. Each time a statistical region started to be documented in the health dataset, our dataset experienced an increase in the number of cases that may not reflect an actual outbreak. Future work could develop methods to impute these missing values with constraints based on the total number of reported cases on a day. Smoother data would aid predictions of actual outbreaks as models would be less likely to overfit to random noise in the dataset. Our analysis predicts new cases based on information about known cases and does not take into account cases that were never detected . Future work could develop methods for adjusting predictions to accurately account for undetected cases.Our models predict new cases more accurately than the test positivity rate. This is because the daily changing sample sizes make it hard to consider the test positivity rate as a consistent stochastic process or to draw conclusions based on the test positivity rate’s patterns. For example, an increase in the daily test positivity rate does not necessarily indicate a worsening situation in a region if the sample size on this date is relatively small, and vice versa. Further research could investigate the use of predictors that take into account sample size, if known.Cell phone mobility data of the type we used may not be available in other settings. Future work could use Google Community Mobility data, available for most countries online . This daOur work shows that anonymized, aggregated human mobility data can improve the prediction of when and where COVID-19 outbreaks are likely to occur. We merged anonymized country-level data from different sources  to develop a simple and accurate method for predicting weekly new COVID-19 cases and the test positivity rate. Accurate prediction of outbreak severity allows for better allocation of resources  at a district level. Our method provides a useful tool for government decision makers, particularly in the post-vaccination era, when focused interventions are needed to contain COVID-19 outbreaks while mitigating the collateral damage from more global restrictions. The methods we have developed can also be applied to predict outbreaks of other communicable diseases such as influenza, measles, and SARS.S1 Fig(TIF)Click here for additional data file.S2 FigNew cases and the Pressure and Internal Movement Scores are per 100,000 people.(TIF)Click here for additional data file.S3 FigThe Pressure and Internal Movement Scores are per 100,000 people.(TIF)Click here for additional data file.S4 FigThe set of predictors that minimized average RMSE over each month is shown as a dark blue diamond and is near the bottom left corner of each plot.(TIF)Click here for additional data file.S5 FigTiers are defined by (a) new cases and (b) test positivity rate that are positive from November 1 to December 31, 2020.(TIF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S1 Table(PNG)Click here for additional data file.S2 Table(PNG)Click here for additional data file.


We show that sex chromosome changes are very frequent in Lake Tanganyika cichlids and repeatedly involve the same chromosomes. Sex is a fundamental trait determined by environmental and/or genetic factors, including sex chromosomes. Sex chromosomes are studied in species scattered across the tree of life, yet little is known about tempo and mode of sex chromosome evolution among closely related species. Here, we examine sex chromosome evolution in the adaptive radiation of cichlid fishes in Lake Tanganyika. Through the analysis of male and female genomes from 244 cichlid taxa  and of 396 multitissue transcriptomes from 66 taxa, we identify signatures of sex chromosomes in 79 taxa, involving 12 linkage groups. We find that Tanganyikan cichlids have the highest rates of sex chromosome turnover and heterogamety transitions known to date. We show that sex chromosome recruitment is not at random. Moreover convergently emerged sex chromosomes in cichlids support the “limited options” hypothesis of sex chromosome evolution. Sex chromosomes—referred to as Z and W in female- and X and Y in male-heterogametic sex determination (SD) systems—define, through their properties and combinations, the sex of an individual  , a model system for the evolution of sex chromosomes  could contribute to the differentiation of sex chromosomes  (Oreochromis niloticus). This species features the most frequent diploid number of 44 chromosomes of African cichlids (The genomic locations of inferred sex-linked regions refer to the 23 linkage groups (LGs) of the version of the reference genome used in all analyses, the one of the phylogenetically equidistant outgroup to the cichlid species of the LT radiation, the Nile tilapia , which we applied to the larger  cichlid tribes from LT only, aimed at testing for the presence of sex chromosomes shared among several species of their respective tribes followed by the inspection of all individual genotypes in regions that showed sex association in the GWAS at the tribe level. We thus identified an XY heterogametic SD system on LG19 in Haplochromini/Tropheini . This reference genome was sequenced from a homozygous clonal XX female with LG1 being the sex chromosome and was chosen in the original study , applying the settings ReadPosRankSum < −0.5, MQRankSum < −0.5, FS < 20.0, QD > 2.0, MQ > 20.0, and placing tribe-specific thresholds on minimum and maximum read depths to account for varying sample sizes . For the tribes Lamprologini, Tropheini/Haplochromini, Ectodini, and Limnochromini, we further applied InbreedingCoeff > −0.8.In addition to the variant file containing all species derived from Ronco We normalized indels with BCFtools’s norm function, excluded monomorphic sites, and masked SNPs around indels depending on the size of the indel: For indels with a size of 1 base pair (bp), 2 bp were masked on both sides, and 3, 5, and 10 bp were masked for indels with sizes of 3 bp, 4 to 5 bp, and >5 bp, respectively. We then masked individual genotypes with VCFtools (v.0.1.14) . We performed approaches 1 and 2 at the tribe level, the taxonomic rank above genus but below family, which, in the case of the LT radiation, groups monophyletic clades that are between 6.2 and 9.7 million years old. Note that LT cichlid genera in contrast to tribes are not always monophyletic. The tribes comprise between 1 (Boulengerochromini) and ~100 (Lamprologini) species. Approaches 1 and 2 have been designed to detect signatures of sex chromosomes shared across species within tribes, which we presumed to likely exist because of the close relatedness of the species.P value) > 3; extraction of the top 100 most significantly sex-associated SNPs revealed same clustering and no further sex-associated region because those SNPs were scattered across the genome] of all individuals analyzed by GWAS were clustered and visualized with the R package Pheatmap  in R (v.3.5.2). We inspected these genotype clusterings further for grouping by sex to (i) infer which species drove the global pattern observed in the GWAS and (ii) with which heterogametic SD system.For approach 1 (figs. S1 and S2), the phased sets of variants for tribes with at least 10 species  were each transformed into bim and bed format with PLINK (v.1.90b)  XY if the observed XY value was greater than the expected one and the observed ZW value smaller than the expected one and as (ii) ZW if the observed ZW value was greater than the expected one and the observed XY value smaller than the expected one. If both the observed XY and ZW values were larger than the expected value, then a window was declared ambiguous and not further considered. If both observed XY and observed ZW values were equal or smaller than the expected values, then a window was declared nosex and not considered further. Fisher’s exact test and Pearson’s χ2P values of XY and ZW windows were plotted jointly  and with an overlay of the calculated XY-ZW difference for each window normalized by dividing the obtained value through the number of species analyzed. We inspected the obtained plots for the presence of LG-wide or regional shifts in XY-ZW difference and outliers from the expected XY or ZW sites. We also calculated and visualized the XY-ZW difference in each window at the species level. To assess a false discovery threshold, we permutated the observed data within each tribe 100 times by randomly assigning the SNPs to the observed genomic positions. We recalculated the XY-ZW difference per window and the expected values. We assessed, from each permutation, the highest absolute XY-ZW difference of a window and the smallest P value for XY/ZW sites. The largest absolute XY-ZW difference normalized by species number across all permutations within each tribe was then used as minimal threshold to define the sex-linked regions in the observed data. The lowest P value of all XY/ZW windows across all permutations was −log10 = 5.04 (obtained in the tribe Haplochromini/Tropheini), which corresponds to a false discovery rate (FDR) ~ 4 after Bonferroni correction. To minimize the possibility of false positives after a comparison of all observed data across all tribes, we lastly retained only drastic XY/ZW outlier regions that, in addition of exceeding the tribe-wise threshold of XY-ZW difference derived from each tribe’s permutation, also had a −log10 > 20 (corresponding to a FDR of 2.30 × 10−26 after Bonferroni correction).We applied approach 2 to tribes that contain more than a single species , again under the assumption that closely related species of the same lineage might share a sex chromosome: We here tested for an accumulation of sites with sex-specific alleles, referred to as XY and ZW sites depending on the heterogametic sex (figs. S3 and S4), under the assumption that a sex chromosomal region will show an accumulation of sex-specific alleles due to linkage caused by suppressed recombination. To this end, we subset the unphased, filtered sets of variants per tribe and included only species for which we had individuals of both sexes (table S1). We then removed indels and sites with more than 20% missing data and more than two alleles with VCFtools (v.0.1.14). We loaded the resulting files into R (v.3.5.0) with VCFR (v.1.8.0.9)  is annotated with 26 protocadherin tandem gene copies. We suspect that this array of similar genes affects mapping and hence masked this region from our sex chromosomal call. Furthermore, it has previously been shown that LG03 is a sex chromosome in https://github.com/droglenc/FSA). We inspected resulting dendrograms for grouping by sex rather than species and the boxplots of species-specific XY-ZW difference for support by increased absolute XY-ZW difference. Because of the reduced sample size per species and to avoid false-positive signals, we made final calls for sex linkage on LGs and heterogametic status based on the outlier regions only if at least two species within a tribe shared the same signature of sex linkage (table S2).Because we applied approach 2 on the tribe level, we next needed to identify how many and which species were responsible for the sex chromosome signals detected within a tribe, i.e., identify the sex chromosomes on the species level from this approach. To this aim, we visualized, per window and for all specimens analyzed by this approach, species level XY-ZW differences in the outlier regions and clustered individual genotypes  with divisive hierarchical clustering based on a pairwise dissimilarities matrix of Gower’s distances calculated with the R package FSA (v.0.8.30) . We classified SNPs as (i) XY if they had zero read counts in all females and a minimum of one count in at least two males and as (ii) ZW if they had zero counts in all males and a minimum of one count in at least two females. Next, we assessed the density of these XY and ZW SNPs in 10-kb nonoverlapping windows .For approach 3, the identification of sex-linked regions and heterogametic system on the species level by species-specific association tests (fig. S6), we pooled tissue-specific transcriptomes of brain, gonad, and gills into one transcriptome per individual and quality-filtered and trimmed these with Trimmomatic (v.0.33) , then we defined the heterogametic system as the distribution (XY or ZW) with the higher total amount of SNPs. As a last step, we quantified XY or ZW SNPs of outlier windows (depending on the previously defined heterogametic system) per reference LG (corrected by chromosome length) and defined as potential sex chromosome the LG(s) with a number of SNPs higher than the 75th percentile value +3 times the interquartile range. To keep only the most extreme outliers and to further avoid false positives, we kept only the LG(s) with a number of SNPs higher than the standard deviation for this final call. In species for which a heterogametic system was identified, we further visualized all SNPs of the outlier windows of that system along the genome for illustrative purposes .We then calculated the difference between XY and ZW SNPs per 10-kb window and kept only outlier windows . We defined these outlier windows as windows with a difference of XY-ZW SNPs greater than the 75th percentile value +1.5 times the interquartile range. We then compared the distribution of XY and ZW SNPs in all outlier windows with a paired two-sided Mann-Whitney test . If the two distributions were significantly different from each other  per species from sex-association in GWAS (approach 1), the sex-specific allele test (approach 2), and species-specific sex-differentiated site accumulations identified via the allele differences test on the basis of transcriptomes (approach 3). For approaches 1 and 2, which at first resulted in tribe-level identification of sex chromosomes , we eventually made sex chromosome calls on the species level as follows: We required the same sex-linked region to be present in at least two species of a tribe to base a sex chromosomal call on WGS data only. This might underestimate the presence of sex chromosomes in our dataset but further reduces the number of false positives. On the basis of approach 3, which includes more individuals per species and was run on the species level, we could confirm the larger sex-differentiated regions identified by approaches 1 and 2. However, we failed to detect some of the rather small sex-linked regions with approach 3, such as the narrow ~5-kb region in Lamprologini, which we think is due to a combination of the low number of genes present in these regions, limiting signal to nonexpressed regions, and probably low levels of expression of these genes in adults. In these cases, approach 3 either returned no sex chromosomal signal for the species in question or supported the heterogametic system identified with the WGS data but showed no clear overrepresentation of sex-linked SNPs on a particular LG. Only in Eretmodini, transcriptome, and WGS data were conflicting to some extent in the sense that RNA data indeed supported LG10, which was also identified in approach 2, and additionally identified LG15 as sex-linked regions  but failed to detect LG07 (identified by approach 2). The latter was thus excluded from the stringent dataset. For species, in which approaches 1 and/or 2 identified a sex chromosome but approach 3 returned no signal, we relied on the genomic data only . Approach 3 not only allowed us to largely confirm sex chromosomes shared across species identified by approaches 2 and 3 but also characterized species-specific sex chromosomes that we would not call/identify otherwise [note that similar sample sizes and transcriptome approaches have previously been used to identify sex chromosomes, e.g., . For these species, the presence or absence of genetic SD would need to be determined in the future. We placed the sex chromosome identities onto a time-calibrated phylogeny of LT cichlids . In addition to the reconstructed turnover points, we here added a turnover on the terminal branch leading to To test whether gene content or chromosome size drives the observed pattern of sex chromosome recruitment in LT cichlids, we randomly picked 30 times  a window of 10 kb of the reference genome and attributed the LG containing this window as sex chromosome to a species. We simulated this operation 10,000 times and counted how many times each LG was recruited in each simulation. We then counted in how many simulations nine or more LGs that were not recruited, as this was the observed pattern. We then tested for an association of the number of sex chromosome turnovers leading to the tips of each tribe with the number of species investigated in each tribe with a pGLS using the R package ape (v.5.2) (Oryzias latipes). We derived information on sex chromosomes from Hilgers and Schwarzer , as these were not included in the phylogeny and no other comprehensive time-calibrated tree comprising these species was available to us.We then ran the same two analyses for ricefishes (Adrianichthyidae), which, to the best of our knowledge, are the only fish family with detailed data on sex chromosomes with synteny inference based on a comparison to a common reference genome (http://treeofsex.org/). We used the data for all Tree of Sex species that were also included in the recent comprehensive ray-finned fish phylogeny (www.fishbase.org) and Eschmeyer’s Catalog of Fishes Online Database  and corrected the names accordingly. This allowed us to map SD data for 472 species from the Tree of Sex database onto the phylogeny. We further added published data for cichlids [ and in O. indermauri . Our ancestral state reconstruction suggested an independent origin of the LG19 SD system in Perissodini, Tropheus, and O. indermauri. To further investigate the hypothesis of convergence, we extracted all SNPs from LG19 that were XY in at least one of the species with a positionally overlapping XY system from a variant call file containing all species investigated in the present study  and other haplochromine species . We only kept variants with less than 10% missing data. We next extracted the two haplotype sequences of each individual for all variants in FASTA format. Assuming that the variant phasing with Beagle was not error free across whole chromosomes, we inspected the haplotypes and corrected the phasing for the eight LG19-XY species. We did this so that for sites where an XY male was heterozygous while the corresponding XX female was homozygous, the allele of the male shared with the female was designated as haplotype 1  and the other allele as haplotype 2 . We then inferred a phylogenetic tree by maximum likelihood with IQ-TREE (v.1.7-beta12)  .et al. (www.ensembl.org). Because this Nile tilapia genome is a different genome release from the reference genome used by us, we searched the NCBI database for the obtained Ensembl gene IDs and translated them to the assembly version that we used with the NCBI Genome Remapping Service. Candidate genes for SD included genes previously identified through a literature search .


The aim of this descriptive article is to share the experience in Ege University, Turkey with favipiravir in the treatment of severe SARS-CoV-2 pneumonia.  This retrospective descriptive study included patients diagnosed with COVID-19 who presented with or developed severe pneumonia. Forty patients who completed a full course (at least 5 days) of favipiravir were included in the study. At baseline, 30 (75%) patients required treatment for respiratory distress. Thirty-three patients (82.5%) were discharged from the hospital with full recovery, 6 patients (15%) died and 1 case (2.5%) was still at the intensive care unit (ICU) when this paper was written. This study provides relevant information for the treatment of COVID-19, suggesting that favipiravir was associated with significant clinical and laboratory improvements in the majority of the patients, is a safe drug with no serious side effects and would merit further investigation. The large-scale pandemic due to the severe acute respiratory syndrome coronavirus (SARS-CoV-2), which started in November 2019 in Wuhan, China has caused 4,425,485 confirmed cases of coronavirus infectious disease (COVID-19) in 216 countries, and 302,059 confirmed deaths as of May 16th, 2020. Republic of Turkey Ministry of Health (2020). Guidelines for the Management of Adults with COVID-19 [online]. Website https://covid19bilgi.saglik.gov.tr/depo/rehberler/COVID-19_Rehberi.pdf [accessed 01 May 2020].SARS-CoV-2 is reported to be transmitted from human to human by droplets and to cause lower respiratory tract infection, which may result in severe respiratory distress and death [1]. Treatment of COVID-19 is challenging as there is no proven treatment strategy yet. Small-scale, randomized controlled trials and observational cohort studies are available for several drugs with controversial results. Chloroquine/hydroxychloroquine, azithromycine, lopinavir/ritonavir, favipiravir, and remdesivir, are some of which have been shown to have in vitro activity against MERS-CoV, SARS-CoV, other coronaviruses and various other viruses. These drugs are among those that are most promising, but with controversial results in clinical studies that are difficult to interpret [2–17].While it is difficult to design a treatment approach without proven benefit of a drug or a treatment regimen, the severity of the situation calls for urgent action. Considering the emergency state we are all in, every piece of data counts and contributes to the knowledge and understanding of the pandemic, helping us shape our approach to treating the patients. The aim of this descriptive article is to share the experience in Ege University, Turkey with favipiravir in the treatment of severe SARS CoV-2 pneumonia by providing real-world data while waiting for the solid evidence to build up.This retrospective descriptive study included patients diagnosed with COVID-19 who were hospitalized in the Department of Clinical Microbiology and Infectious Diseases and the Department of Chest Diseases, at Ege University Faculty of Medicine from March 23rd to April 26th 2020. The diagnosis was made by a positive nucleic acid amplification test (NAAT) from nasopharyngeal swabs  and radiologic findings in line with COVID-19 pneumonia [19]. Patients who presented with or developed severe pneumonia [tachypnea (> 30 breaths/min) and/or hypoxia (SpO2 < 90% on room air) and/or bilateral diffuse ground glass infiltrations] with no response to first-line treatment with HCQ (± azithromycin) and started favipiravir were included. The treatment approach was in line with the recommendations of the Guidelines for the Management of Adults with COVID-19 produced by the Turkish Ministry of Health.1Favipiravir 200 mg tablets were administered orally starting with a loading dose of 1600 mg bid, followed by 600 mg bid daily for 5 to 7 days. For each patient, demographic characteristics, underlying conditions, clinical signs and symptoms, radiologic and laboratory findings, oxygen requirement, and adjunctive treatments used were recorded. Response to favipiravir treatment was assessed by comparison of baseline and day 4 of treatment in terms of the level of respiratory distress and laboratory parameters . Oxygen therapy was classified as oxygen delivery via face mask, continuous positive airway pressure (CPAP) and invasive mechanical ventilation (IMV). Respiratory distress level and chest X-ray/high resolution computed tomography (HRCT) findings were evaluated at baseline prior to initiation of favipiravir and on day 4 of the treatment. Patients who did not require oxygen therapy before or after favipiravir treatment were defined as the “ambient air” group. Patients whose oxygen requirement did not change from baseline until day 4 were classified as the “no change” group. Patients whose oxygen requirement reduced from baseline to day 4 were grouped as “improvement”, and those whose oxygen requirement increased were defined as “impairment”. Treatment was considered successful for patients who were discharged from the hospital with no symptoms, improvement in chest radiology, and no respiratory distress. In addition, adverse events and serious adverse events that developed during favipiravir treatment were recorded. Data was analyzed using SPSS v.18.0 . Numbers and percentages were used for categorical variables. Mean values with standard deviations, and median values with minimum and maximum values were calculated for continuous variables. Continuous variables were tested for agreement with normal distribution. Since all variables did not show normal distribution in graphical analyses and normalization tests, and considering the size of the study population, nonparametric methods were preferred for comparisons. The Wilcoxon signed ranks test was used to analyze the change in repeating measurements. A type-1 error α was determined as 0.05 and was tested. A value of p < 0.05 was considered statistically significant and was represented with a box pilot graphic.Out of a total of 559 patients admitted to the hospital, 40 patients who completed a full course (at least 5 days) of the drug during the study period were included in the study. More than half (58%) of the patients were male. The mean age was 55.58 ± 13.49 (26–79 years). All patients were confirmed COVID-19 cases with positive nasopharyngeal swabs. Two thirds of the patients had at least one underlying disease, and the most common underlying conditions were cardiovascular disease and diabetes mellitus. The most common clinical symptoms were fever and cough; almost all (95%) patients had more than one symptom. Baseline demographic characteristics, comorbidities, clinical signs and symptoms, as well as respiratory distress levels are shown in Table 1.Twenty (50%) patients were managed in the intensive care unit (ICU); 5 patients were admitted directly to the ICU at presentation and 15 patients were transferred to the ICU from the clinic ward due to clinical worsening. The mean duration of stay at the ICU was 9.31 (1–19) days. At baseline, 30 (75%) patients required treatment for respiratory distress, Table1. While 9 patients (22.5%) maintained respiration at ambient air before and after favipiravir treatment, oxygen requirement increased in 13 (32.5%) patients, decreased in 12 (30%) and did not change in 6  (15%) on day 4 of favipiravir treatment. The change in oxygen requirement after favipiravir treatment did not differ according to age or sex (p > 0.05). On day 4 of favipiravir treatment, radiologic findings improved or did not deteriorate in 42.5% of the cases, and deteriorated in 42.5%; no radiologic follow-up was available in 15%. Table 2 includes laboratory findings before and 24 h after the termination of favipiravir treatment. C-reactive protein, procalcitonin, LDH and d-dimer levels were elevated before favipiravir treatment; while CRP, procalcitonin and LDH levels decreased significantly, d-dimer levels continued to increase after favipiravir treatment. Figure 1 shows the distribution of statistically significant laboratory results.Adverse events developed in 5 patients (13%) during favipiravir treatment. All five had mild to moderate elevations in hepatic enzymes. Three of these cases also reported nausea and one case developed neutropenia. Adverse events were resolved spontaneously, no patient developed a serious adverse event and no patient discontinued favipiravir due to adverse events. While 17 patients required no adjunctive treatment, 23 patients received various combinations of favipiravir with tocilizumab, corticosteroid, intravenous immunoglobulin (IVIG), and plasma transfer. Low molecular weight heparin (LMWH) was administered to 34 patients (85%). Thirty-three patients (82.5%) were discharged from the hospital with full recovery, 6 patients (15%) died and 1 case (2.5%) was still at the ICU when this paper was written. Of those who died, 4 were older than 65 years and the remaining two were 52 and 64 years old. All patients who died had started favipiravir when they were already in the ICU, had at least one serious comorbidity excluding one 64-year old case, and had received various combinations of tocilizumab (n = 5), corticosteroid (n = 5), and plasma transfer (n = 1). Figure 2 summarizes the method of treatment for respiratory distress before and after favipiravir treatment, and the outcome of each patient. Overall 30/40 patients were screened with nasopharyngeal swabs after the completion of favipiravir treatment before or after discharge at different time points. Twenty-seven patients were negative. Of the remaining 3 patients with positive results, 1 recovered and was discharged from the hospital without a negative result, 2 died before a negative result.This study is the first from Turkey and one of the few globally on favipiravir use in the treatment of COVID-19 patients with severe pneumonia. Despite its retrospective nature, it may aid in providing insight into whether favipiravir deserves further investigation and may be an option for treating COVID-19 patients by presenting real world data. COVID-19, an acute respiratory condition due to SARS-CoV-2 has spread rapidly resulting in a pandemic with devastating effects within a few months since November 2019. Unlike its predecessor SARS, asymptomatic cases are present, the viral load peaks earlier and commonly before the patient is symptomatic, transmission rate is higher, and attack rates are variable in different geographic regions in SARS-CoV-2 infection, which makes containment difficult and creates an emergency for rapid action [18–23]. Although more than 80% of the patients with COVID-19 have mild pneumonia, which usually resolves rapidly, 13%–18% of the cases develop severe pneumonia and 4%–9% are reported to be critically ill . Individuals with diabetes and hypertension were reported to have a higher likelihood of developing severe infection  and a higher case fatality rate . The analysis of our cohort including severe COVID-19 cases revealed a relatively high age distribution and a high rate of comorbidities with hypertension and diabetes being the most common, in line with the results of other cohorts . The outcome of severe COVID-19 is poor, mostly requiring hospitalization at the ICU and mechanical ventilation [26]. A major challenge in the COVID-19 pandemic is the lack of evidence on reliable treatment options. Although various agents are under investigation in ongoing clinical trials, the urgency of the situation has led scientists to use empirical treatments or drugs that have been investigated for other viruses in the past. Chloroquine and its derivative HQ, which are antimalarial drugs were among the most studied and recommended molecules for the treatment of COVID-19 based on earlier reports on their in vitro antiviral effects on SARS virus [27] and their antiinflammatory and immunomodulator effects [5]. Several clinical studies were undertaken to study the clinical success of CQ/HQ as monotherapy or coadministered with azithromycine with conflicting results . A metaanalysis showed that although HCQ treatment compared to standard treatment had some clinical benefits, there was no difference in virologic cure, clinical progression or death rates [28].Similarly, clinical studies reported controversial results with LPV/r . A systematic review reported that no specific conclusion could be drawn for the efficacy of LPV/r in COVID-19 [31]. Favipiravir was shown to inhibit a wide array of RNA viruses such as the influenza virus, arenavirus, bunyavirus, flavivirus, and filoviruses [32] and to improve survival in patients with Ebola virus infection . It was considered as a potential candidate for the treatment of COVID-19 and the urgency of the situation required its premature use without in vitro and animal studies [35]. Favipiravir was studied in a few randomized controlled studies with promising results. It was reported to significantly shorten the time to viral clearance and alleviate the symptoms of pneumonia, thereby improving the chest imaging compared to LPV/r [36]. It also had a significantly higher recovery rate compared to umifenovir, and shortened the time with fever and respiratory symptoms even in patients with hypertension and diabetes, albeit with no statistically significant difference between the two drugs in terms of requirement for oxygen support and noninvasive mechanical ventilation [37]. Favipiravir did not become a favored drug globally and was recommended and used only in Japan, China and Turkey during the pandemic [38].1 Since the beginning of the epidemic the Guidelines for the Management of Adults with COVID-19 produced by the Turkish Ministry of Health has suggested the addition of favipiravir or LPV/r in COVID-19 patients who do not respond to first-line treatment with HCQ and develop severe pneumonia, and direct initiation of favipiravir or LPV/r ± HCQ to those that present with severe pneumonia.1 Th treatment approach in the Ege University was to initiate favipiravir in severe cases after, during or in addition to HQ treatment. The main reason to prefer favipiravir over LPV/r was previous experience with LPV/r in people living with HIV in terms of severe adverse events sometimes leading to permanent discontinuation of the drug. Despite its retrospective design the results of our study suggest a high rate of improvement in severe COVID-19 patients. More than 80% of the patients, including several who received noninvasive and invasive mechanical ventilation were discharged from the hospital with full recovery. The most notable finding was an early response in approximately a third of the patients in terms of reduced oxygen requirement with further improvement within time. In addition, all cases but one that required no oxygen support at baseline maintained their status after favipiravir treatment. The favorable outcome in the majority of the cases resulting with full recovery was promising, although several patients recovered at later stages. The poorest response was among those who were already on IMV or CPAP when favipiravir was initiated suggesting that early initiation of the drug before or right after respiratory distress or radiologic deterioration develops might be most beneficial. Favipiravir treatment did not seem to have a major effect on the resolution of radiologic findings at early stages in contrast to the findings of Cai et al. This may be attributed to the severity of lung involvement in our cases mostly requiring oxygen support whereas those with severe pneumonia were excluded in the study by Cai et al. [36]. Favipiravir proved to have a favorable safety profile both for COVID-19 and influenza treatment at various dosing regimens and compared to umifenovir and LPV/r, the most common adverse events being elevations in liver enzymes and bilirubin as well as gastrointestinal disturbances, and elevations in uric acid . Our cohort confirms the safety of the drug even in severe pneumonia cases with multiple comorbidities using other adjunctive therapies. There was no serious adverse event that resulted with discontinuation of the drug.The severity of the COVID-19 pneumonia is associated with a cytokine storm with increased levels of proinflammatory cytokines and ferritin causing severe inflammation and hypoxia [40–42]. In addition, the development of thromboembolic events worsens the situation, sometimes even leading to death. Elevated d-dimer level was suggested to be an independent risk factor for death [43]. Thus, adjunctive treatment approaches in addition to antiviral treatment to alleviate the inflammation such as anticytokinic biological agents, corticosteroids and IVIG in addition to anticoagulants, and especially LMWH were recommended . The antiinflammatory effects of CQ/HCQ and azithromycine have also been suggested to contribute to the management of inflammation [5]. The analysis of our patients reveal increased levels of inflammation markers, ferritin, and d-dimer, which are in line with the severity of the condition. Various adjunctive treatments were used to address the inflammation and coagulopathy in our patients. While inflammation markers reduced significantly after the relevant treatment approach, high d-dimer levels maintained despite the use of heparin and this may be related to the course of the disease. Tang et al. reported that elevated d-dimer levels were significantly associated with higher mortality and although nonsignificant, 28-day mortality was lower in heparin users compared to nonusers. Timely and correct use of corticosteroids in conjunction with ventilator support may be life-saving in severe COVID-19 patients by preventing ARDS . It was not possible to analyze the association of inflammation and coagulation markers to patient outcome due to the small size of the study group. This study has several limitations:(1) This is not a randomized controlled study and its level of evidence is low.(2) The study group was too small to make detailed statistical analyses.(3) It was difficult to define the role of favipiravir in the recovery of the patients because many additional adjunctive treatments were administered.(4) Patients were not followed-up for the duration of time required for viral clearance.However, despite these limitations, this study provides relevant information for the treatment of COVID-19, suggesting that favipiravir was associated with significant clinical and laboratory improvements in the majority of the patients and is a safe drug with no serious side effects. Currently there are several completed and ongoing clinical trials studying favipiravir [47] and in a time of emergency when drugs found ineffective in randomized clinical trials [48] are authorized for emergency use by drug agencies it would merit further investigation.2,3The study was approved by the Ege University Ethical Board . Informed consent was submitted by all subjects when they were enrolled.


Innate lymphoid cells (ILCs) are mostly tissue resident lymphocytes that are preferentially enriched in barrier tissues such as the skin. Although they lack the expression of somatically rearranged antigen receptors present on T and B cells, ILCs partake in multiple immune pathways by regulating tissue inflammation and potentiating adaptive immunity. Emerging evidence indicates that ILCs play a critical role in the control of melanoma, a type of skin malignancy thought to trigger immunity mediated mainly by adaptive immune responses. Here, we compile our current understanding of ILCs with regard to their role as the first line of defence against melanoma development and progression. We also discuss areas that merit further investigation. We envisage that the possibility to harness therapeutic potential of ILCs might benefit patients suffering from skin malignancies such as melanoma. The family of innate lymphoid cells (ILCs) comprises a heterogeneous population of immune cells harboring pleiotropic functions. Based on the expression of signature cytokines and an assembly of transcription factors, they have been divided into five subsets, namely natural killer (NK) cells, group 1 ILCs (ILC1s), ILC2s, ILC3s and lymphoid tissue inducer (LTi) cells .The heterogeneity and diversity of ILCs might further increase owing to their plastic potential –6. For eSimilarly to NK cells, ILC1s require IL-15 for their development. Additionally, they are both activated by IL-12, IL-18 and IL-15 , 15. WheIn contrast to NK cells, which circulate in the body and are particularly detected in the peripheral blood, the remaining ILC subsets are mostly tissue resident and preferentially enriched in barrier tissues such as the skin. The involvement of NK cells in antitumor immunity is unquestionable –22. TheiThe skin is the largest organ of the body . It prov+ and CD8+ T cells, γδT cells, B cells, macrophages, basophils, eosinophils, mast cells and NK cells (+ cells in mice (Gata3 and Il5) in subcutis, whereas ILCs in epidermis were found to predominantly express genes associated with ILC3s/LTi cells (e.g. Rorc and Lta) but also ILC2s (e.g. Il13 and Il2). On the other hand, ILCs in dermis shared resemblance with ILCs from epidermis and subcutis  . An analThe involvement of ILC1s and ILC3s in skin homeostasis requires further investigation. It should be noted, however, that ILC3s might play a critical role in the maintenance of tolerance towards skin microbiota . NeverthThe skin has evolved precise and orderly mechanisms to close breaches to its integrity in a process known as the wound healing response. Human ILC2s isolated from the skin have been typified by an increased gene expression of amphiregulin when compared with those purified from blood. Since amphiregulin aids tissue repair, it has been suggested that ILC2s in the skin are involved in wound healing response . This no-/-Il17a mice  involved in the healing response through the regulation of epidermal proliferation and macrophage recruitment into dermis  melanoma patients has recently been found to negatively correlate with overall patient survival  and granzyme B (GrB) or by the engagement with death receptors that initiate caspase cascade. Portrayed as the first line of defence, NK cells have been viewed as innate immune cells that operate at an earlier stage than T and B cells. However, involvement of NK cells in T cell-mediated immunity has also been demonstrated , 51. Recand CCL5 . A broad+TRAIL+ ILC1s  of melanomas expressing IL-12. It has therefore been suggested that NKp46+ ILC3s might play a significant antitumor role in the presence of IL-12 , has been found to inhibit the growth of B16F10 melanoma tumors. It has also been demonstrated that the tumor-suppressing activity of this combined therapy occurs independently of adaptive immunity and NK cells, but is mediated mulation . Additioof IL-12 .In another study, it has also been revealed that the tissue microenvironment shapes the phenotype of ILC3s. Whereas ILC3s isolated from the spleen have been able to suppress the growth of B16F10 melanoma tumors expressing IL-12, intestinal ILC3s have been found ineffective. Interestingly, transcriptome analysis has revealed mutually exclusive gene expression signatures between the splenic and intestinal ILC3s regarding (but not limited to) leukocyte adhesion and activation. Increased frequencies of leukocytes have been observed in B16F10 melanomas engineered to express IL-12 and co-injected with splenic ILC3s when compared with tumors co-injected with intestinal ILC3s .The involvement of LTi cells in melanoma immunosurveillance remains to be determined. However, it is tempting to hypothesize that LTi cells might contribute to the formation of tertiary lymphoid structures often observed in human melanoma .The involvement of NK cells in antitumor immunity is unquestionable, however, the role and function of other members of the family of ILCs have only recently gained attention. Given the preferential enrichment of ILCs in the skin, a deeper understanding of their contribution to the development and progression of skin malignancies is required. Indeed, a growing body of evidence indicates that ILCs play a critical role in the control of melanoma Figure 1In order to improve our understanding of ILCs with regard to their role in skin malignancies, it seems imperative to determine how ILCs regulate healthy skin homeostasis. An increasing degree of heterogeneity among ILCs also necessitates their separate assessment in epidermis, dermis and subcutis. Furthermore, studying the mechanisms by which ILCs communicate with other non-hematopoietic parenchymal cells such as keratinocytes, fibroblasts and adipocytes might help to better understand the extent of their involvement in the homeostatic and pathological states Figure 1in vitro models might prove useful during analysis of interactions between ILCs and malignant cells, since culture in 3D has been suggested to affect the expression of molecules involved in melanoma recognition  to MW and by a Grant-in-Aid for Scientific Research (A) (20H00511) from the Japan Society for the Promotion of Science, to SK.We thank Tetsuro Kobayashi for discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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