The aim of this study was to determine the prevalence of infection, genotypes and risk factors for human papillomavirus (HPV) among men who have sex with men (MSM) and transgender women living with and without HIV in Pakistan. Anal infection with HPV is very common worldwide among MSM, particularly among MSM living with HIV. The high prevalence of HIV among MSM and male-to-female transgendered individuals in Pakistan is a significant health concern since access to screening and health-seeking is often delayed in this stigmatised key population.This cross-sectional study was conducted between March 2016 and November 2017.This study recruited MSM and transgender-women who self-reported to have had anal sex in the last 6 months, and were at least 18 years of age, from the sexual health and antiretroviral therapy centres. Structured questionnaires were administered, and blood samples were obtained to confirm HIV status. Anal swabs were collected for HPV-DNA detection and typing.The primary outcome was the prevalence of ‘HPV-DNA infection’. The prevalence ratios (PR) were calculated using Cox proportional hazard model algorithms to analyse the association between exposure variables and HPV-infection.2p≤0.001). Likewise, 28.9% of participants living with HIV were infected with two or more than two types of HPV as compared with 18.8% participants without HIV(χ2 p≤0.001). The most frequent HPV type was HPV6/11 (46.9%), followed by HPV16 (35.1%), HPV18 (23.2%) and HPV35 (21.1%). HIV status  and never condom use ) were independently associated with prevalence of ‘anal-HPV16 infection’ when adjusting for confounding for age, other sexual and behavioural factors, for example, smoking and alcohol consumption.Complete data were available for 298 MSM and transgender women . The overall HPV-DNA prevalence was 65.1% and was higher in participants living with HIV as compared with HIV-negative (87% vs 48%; χHigh prevalence of HPV indicates a substantial future risk of anal cancer in Pakistani MSM and transgender women, and particularly in those living with HIV. Current findings support anal Pap-smear HPV screening for this particular group and vaccination efforts for future generations. This study is the first description of the epidemiology of anal human papillomavirus (HPV) infection among sexual and gender minorities  living with HIV in Pakistan.Given the strong stigma, homophobia and discrimination against sexual minorities in Pakistan, data collection included a careful process of gaining trust among study participants.The results may be of a public health importance in Pakistan, indicating the need for gender-neutral HPV vaccination before sexual debut, targeting both women and men.The cross-sectional study design precludes any conclusion on temporal relationships between potential risk factors and the outcome.Any under-reporting or over-reporting regarding sexual behaviours or lifestyle factors leading to information bias cannot be excluded.Human papillomaviruses (HPV) is the most common sexually transmitted infection (STI) worldwide,16–18In Pakistan, the demography of MSM is broadly classified as: MSM who are in relationships but outwardly closeted; as male sex workers (MSW) who are males and undertakes receptive anal or oral sexual activity with a man in return of money or other financial benefits; and as hijras (transvestites/transgender women) who are biologically male with male genitals but function in society as if female and take the receptive role in anal sex.21Pakistan is an authoritarian Islamic republic with conservative social values, particularly on issues of sex. MSM, and in particular MSW, face high levels of discrimination and contempt from society and are subject to harassment, sexual violence and detention by the state authorities. Their health-seeking behaviours are often poor due to stigmatisation in healthcare settings and for the fear of being identified. As a result, they are reluctant to seek healthcare not only in public setups but also in private facilities. One previous study on sex work and MSW in Pakistan showed that the majority of MSWs are poor, have little knowledge about safe sex, and cannot negotiate the use of condoms and safe sex with their paying clients, predisposing them to acquiring STIs.20Pakistan’s national government has lately recognised MSM as a vulnerable population, and prevention and control programmes targeting MSMs in its big cities are being organised .22So far there is no research on the burden of HPV infection and HPV-related anal squamous intraepithelial lesions in this key population in Pakistan. Currently, HPV vaccines, in which the bivalent vaccine protects against HPV16/18 and quadrivalent vaccine protects against HPV16/18 and the LR types HPV6/11,We conducted a cross sectional study in Karachi Pakistan in 2016–2017 with the objective to determine the prevalence and risk factors for anal HPV infection including sexual risk behaviours and the association with HPV infection in this dynamic population to inform future policy and prevention strategies.Enrolment occurred in two phases for this cross-sectional study. The initial recruitment (from March 2016 to May 2016) started at the ART clinic with transgender women, a centre run by the National AIDS Control Programme of Pakistan in Civil Hospital Karachi (CHK)—the largest (1900 bed) tertiary care public hospital of Karachi Pakistan (recruited n=33). Low pace, logistic issues and privacy concerns led to the relocation and setting-up of recruitment centre at a sexual health clinic run by a community-based organisation, Perwaaz Trust Karachi Pakistan, where the study population was reached using snowball technique and peer referrals. During the second phase between April 2017 and November 2017 a total of 265 study participants with different sexual orientations, that is, MSM  and transgender women living with and without HIV were recruited. Participants were eligible for the study who self-reported to have had anal sex in the last preceding at least 6 months and were age 18 and above.No patients or member of the public were involved in the design, conduct, reporting or dissemination plans of our study. Data were kept confidential and anonymous and were used for research purposes only.et al.28Verbal and written informed consent were obtained from all eligible and willing participants for all study procedures. For participants who could not read or write their name, a thumb impression was obtained. Participants were informed that they were free to interrupt the interview or withdraw from the study at any time without any negative consequences in terms of clinical services provided to them. A 35–40 min long structured questionnaire was administered at a private space to interview the study participants in order to collect data concerning sociodemographic information, sexual and reproductive history and medical history relevant to HIV, any anal disease and use of ART. A blood sample was obtained for confirmation of their HIV status (through Architect HIV Ag-Ab Combo kit by ABBOTT), viral load  and CD4 +T cell count . Two anal swabs for HPV testing were collected by a trained general physician. A water-moistened swab was inserted 5–6 cm proximal to the anal verge and the samples were obtained from anorectal transition zone around the dentate line in the anal canal. The swab was then agitated vigorously in a tube containing 3 mL of methanol-based fixative—a Sample Transport Medium  and transported to the lab. In Molecular lab the solution was stored at −70°C before further processing and was later used for the extraction and detection of HPV DNA by the PCR. The DNA from rectal swab was extracted by using Qiagen DNA mini kit and DNA was eluted in 80 uL of elution buffer and stored at −80°C for further use. HPV DNA was detected by using MY09/MY11 primers from L1 region resulted in amplification of 450 bp of L1 gene. PCR for Housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was done for every extracted DNA and samples tested negative for GAPDH gene were excluded from study. Nested multiplex PCR assay was usedFor the estimation of HPV prevalence, a sample size of 267 participants were needed with an anticipated prevalence of HPV of 50%, 6% precision and 5% level of significance. For the analysis of factors associated with HPV infection, we aimed for a sample of at least 256 participants to achieve 80% power with a range of exposures between 9% and 39% and an anticipated prevalence ratio (PR) of 2.5 or more with 5% level of significance. An additional sample of 10% was added to cater for inadequate biological samples.The database was analysed using Statistical Package for Social Sciences .2 or Fisher’s exact test (where needed) were used for p values. The overall HPV DNA prevalence (as determined by PCR) is reported as the proportion of HPV positive participants among all included individuals. Conversely, type-specific HPV positivity is presented as the proportion of HPV-subtype positive individuals divided by all HPV positive individuals.2. For multiple comparisons for each sexual orientation stratified by HIV status for ‘any anal HPV infection’ and ‘any anal HR-HPV infection’ Bonferroni correction method was used, the p value threshold was (α/n)=0.05/6=0.0083.The mean value was calculated with standard deviation (SD) for symmetrically distributed variables and independent sample t-test was used for statistical significance, while the median with IQRs was used for asymmetrical quantitative variables. For categorical variables, proportions were employed; for comparison of proportions between groups, χSince the design of the study was cross-sectional and the outcome of the interest was common ; for the association between independent risk factors and the outcome variables ‘any anal HPV infection’ that is, all those HPV types that we identified in our study sample and ‘anal HPV16 infection’, we employed a Cox proportion hazard model algorithm to estimate crude and adjusted PRs and their 95% CIs in univariate and multivariate models’. Moreover, we employed robust SEs to get the closest CIs. A significance threshold for the p<0.05 was used for the final model.The final analysis included 298 MSM and transgender women (235 MSM and 63 transgender women) to assess the prevalence of HPV DNA in the anal canal. Out of these 298, 131 (44%) had an HIV-infection and 167 (56%) were HIV-negative. Among MSM living with HIV, 33 (25%) were transgender women. Almost 90% of the participants living with HIV were on ART and two thirds of them had an undetectable HIV viral load.The overall mean age (±SD) of the 298 recruited participants was 28.8 (±8.06). The level of education was low: 28% reported no formal education and 12% less than primary level. Most respondents were currently not married. Among study participants almost 78% were smokers with mean age of smoking initiation 16.5 (±4.9) and duration of 12 (±8.5) years. Almost 80% were sex workers, with 12.4 (±8.5) years mean duration of sex work. The mean age at sexual debut was 16.4 (±4.7) years. Only 24.2% reported consistent condom use during sex .MSM with any anal HPV infection (n=194) were younger than MSM without detectable HPV infection (28.9 (±7.3) vs 30.2 (±9.1) years; independent sample t-test p=0.027). Smoking was more prevalent among HPV-positive MSM (82.5% vs 69.2%) than among those uninfected, with a significant difference in mean age (±SD) of 15.6 (±4.5) years at smoking initiation. The mean age (±SD) at sexual debut among study participants with an HPV infection was 15.7 (±4.3) vs 17.6 (±5.2) (independent sample t-test-p=0.001). HPV-infected had more sexual partners, more inconsistent condom users and more often practised receptive anal sex .2p=0.01), younger at initiation of smoking (independent sample t-test p≤0.001) and had smoked for a longer duration of time ((χ2p≤0.001). They also reported younger age at sexual debut (independent sample t-test p≤0.001), had more sexual clients (χ2p≤0.001), were mainly the receptive anal partner (χ2p≤0.001) and used condoms less consistently than their HIV negative counterparts (χ2p≤0.001).MSM living with HIV were, however, older than MSM who were HIV negative (mean (±SD): 30.9 (±0.70) vs 27.2 (±0.59) independent sample t-test p≤0.001), more frequently smokers . The most common HPV types were HPV6/11 (46.9%), HPV16 (35.1%), HPV18 (23.2%), HPV35 (21.1%) and HPV58 (8.2%). Moreover, the distribution of HPV6/11 was more among HIV uninfected participants (61.3%) as compared with HIV infected participants (36.8%) ((χ2p=0.001). However, HPV16 was more common among HIV infected participants (44.7% verses 21.2%) and the difference was statistically significant (χ2p=0.001)  of the total study population was positive for HPV DNA (95% CI 59.5% to 70.3%). MSM living with HIV had a significantly higher prevalence of HPV (87% (95% CI 80.0% to 91.0%)) compared with those who did not have HIV (48% (95% CI 40.0% to 55.0%)) as well as any oncogenic anal HPV infection (83.2% (95% CI 75.8% to 88.6%) verses (35.3% (95% CI 28.4% to 42.8%). Likewise, 28.9% of participants living with HIV were infected with two or more than two types of HPV as compared with 18.8% participants without HIV (χp=0.001) .2p=0.001)  .2p=0.001).For HIV uninfected participants, the prevalence of ‘any anal HPV infection’ was 50.9% for homosexual men, 31.0% for bisexual men and 53.4% for transgender women and the prevalence for ‘any anal HR- HPV infection’ was 37.1%, 20.7% and 43.3%, respectively. (χ2p=0.0014 and 0.0019) when compared with bisexuals and transgender women living with and without HIV.Post hoc analysis with the Bonferroni correction method determined that the homosexuals were significantly different with being infected with ‘any anal HPV infection’ and ‘any anal HR-HPV infection’ (χBeing HIV positive (PR 2.81 (95% CI 2.16 to 3.82)) and inconsistent/never condom use (PR 2.61 (95% CI 1.50 to 4.50)/(PR 3.08 (95% CI 1.69 to 5.60) were independent risk factors associated with increased prevalence of HPV16 infection, adjusted for all other demographic, sexual and behavioural risk factors .et al29 38To the best of our knowledge, this is the first report on anal HPV infection among MSM living with and without HIV in Pakistan. The high prevalence of anal HPV among MSM living with HIV (87%) in Pakistan is similar to Thailand (85.0%)For the distribution of anal HPV types, our study results are similar to the studies done elsewhere.7The prevalence of HR-HPV type 35 (21.1%) in our study was similar to the levels reported by India (20%),Similar to previous research,Our study detected a high prevalence of multiple HPV types—that is, at least 2 types (either LR and HR or HR & HR)—of 47% in HPV positive MSM as a group, which is similar to other, studies.29 45The results of our study suggest that the most frequently distributed HPV types among Pakistani MSM could be covered by the currently available nonavalent (93.3%) and quadrivalent (84%) vaccines, which means that most of HPV-related diseases including cancers and anogenital warts may potentially be preventable if offered to MSM at initiation of sexual debut. However, the 20% carrying HPV35 would not be covered by the currently available vaccines.More than 50% of HPV-positive MSM were young, below 25 years of age, most were single and sexually very active, and most make a living and support their families through sex work.Among other sexual behaviours, inconsistent or never use of condom was the only variable that was significantly associated with any HPV, specifically HPV16 infection. For many in our study population, negotiating safe sex is not an option as, on an average, a MSW will service 3–5 clients per night with a one-time service charge as low as PKR100–PKR150 (US$0.7–US$1.03),40Our study had some limitations. First, the design of the study was cross-sectional, yet many HPV infections are non-persistent in nature.HIV care and treatment programmes in Pakistan represent a potential window of opportunity for providing preventive clinical interventions to avert an anal cancer epidemic nested within the HIV epidemic. Providing cost-effective, life-saving anal cancer prevention services  linked to HIV care could not only avert preventable HPV-related malignancies but could also lead to a more efficient utilisation of resources and improve healthcare access for those most at risk. Moreover, our study findings emphasise the need for government support of interventions that can improve access to and quality of services to MSM including MSW. Prevalent cultural and social stigma must also be addressed since this is essential for protecting the dignity and autonomy of this population.We believe that our study findings are of importance for clinical practice and public health policy. The relatively high prevalence of HR HPV indicates a future risk of anal cancer among MSM in general and among MSM living with HIV in particular. Given that HPV35 currently is not included in the 9-valent HPV vaccine, we strongly feel that this is one of our most important contributions and that there is a need for regular anal pap screening among MSM and transgender women to enable early detection and treatment to prevent anal cancer.


Background and Objectives: Seroepidemiological studies indicate that parvovirus B19 circulates in all areas of the world, although with some differences. The aim of this study is to analyze the seroprevalence of parvovirus B19 in the Croatian population. Materials and Methods: From 2010 to 2021, 1538 serum samples from different populations were tested for the presence of parvovirus B19 IgM/IgG antibodies. Serological tests were performed using a commercial enzyme-linked immunosorbent assay. Results: IgG antibodies were detected in 986/64.1% of participants with differences (p < 0.001) among the following population groups: 42.4% of children and adolescents, 67.1% of the adult general population, 66.7% of hemodialysis patients, and 65.6% of liver transplant recipients. Seroprevalence increased with age, from 30.0% in the 6 months–9 years age group to 69.0% in the 40–49 years age group, and remained stable thereafter (68.8–73.3%). There was no difference in the seropositivity among males (66.1%) and females (63.1%), as well as the place of residence . IgM antibodies (current/recent infection) were found in 61/4.0% of participants with the highest seropositivity in the youngest age group (11.1%). In pregnant women, seroprevalence was higher in women with an unfavorable obstetric history compared with a normal pregnancy , but these differences were not significant. Logistic regression showed that the adult population had almost three times higher risk of IgG seropositivity compared to children/adolescents . A one-year increase in age increased the risk of IgG seroprevalence . Conclusions: Older age was the main risk factor for IgG seropositivity. Hemodialysis and organ transplantation seem unrelated to the increased parvovirus B19 seroprevalence. The role of parvovirus B19 in the etiology of TORCH infections needs to be studied further. Erythroparvovirus genus of the family Parvoviridae. The capsid consists of 60 viral proteins (VP): VP2 constitutes about 95% of the viral capsid proteins, while VP1 constitutes about 5%. The primary receptor for parvovirus B19 is globoside or the P antigen, while various other co-receptors are presumed to be involved in the viral entry. The replication takes place in the nucleus of the infected cells. Parvovirus B19 mainly infects human erythroid progenitor cells; however, recent clinical studies indicate that the virus also infects nonerythroid lineage cells and can be associated with different disease outcomes [Parvovirus B19 is a common viral pathogen with a worldwide distribution. It is a small, non-enveloped virus with a linear, single-stranded DNA genome that belongs to the Seroprevalence studies indicate that the virus circulates in all areas of the world, although with some differences. Seasonal outbreaks occur every 3–5 years. In the general adult population, seroprevalence rates vary from 29% to 72% ,5,6. MosIn childhood, parvovirus B19 causes erythema infectiosum (“fifth disease”), a febrile disease with a rash, while, in adults, infection is more commonly presented as a non-specific febrile illness. Arthralgia is more common in adolescents and adults, affecting up to 60% of persons . In addiAcute parvovirus B19 infection in pregnant women is an important cause of fetal morbidity. The estimated risk of transplacental infection is 17–33% . There dIn Croatia, there are no data on the prevalence of parvovirus B19. The aim of the present study was to analyze the seroprevalence and possible risk factors for parvovirus B19 infection in different population groups.During an 11-year period (2010–2021), a total of 1538 consecutive serum samples from Croatian residents were tested for the presence of parvovirus B19 IgM and IgG antibodies at the Croatian Institute of Public Health, the largest public health institute in the country. In the tested group, there were 502 (32.7%) males and 1036 (67.3%) females aged from 1 month to 87 years. The study included different population groups: children and adolescents , the general adult population , patients on hemodialysis (N = 90), and liver transplant recipients (N = 90). Pregnant women (N = 304) were further separated in a subgroup with normal pregnancy (N = 211) and unfavorable obstetric history  (N = 93).Serologic tests were performed using a commercial enzyme-linked immunosorbent assay . The results were interpreted according to the manufacturer’s recommendations, as follows: IgM ratio < 0.8 negative; 8.8–1.1 borderline; >1.1 positive; IgG RU/mL < 4 negative; 4–5.5 borderline and >5.5 positive.The frequencies are presented with 95% confidence intervals (CI). The differences between groups were tested using Fischer’s exact test. The strength of the association between dependent and independent variables was assessed using logistic regression. Due to mother-to-child passive immunity, the youngest age group (<6 months) was excluded from the logistic regression model. For statistical analysis, the software package STATA/IC ver 11.2  was used. The level of statistical significance was α = 0.05.The results of serology testing are presented in p < 0.001) among population groups: children and adolescents (42.4%); the general adult population (67.1%); hemodialysis patients (66.7%); and liver transplant recipients (65.6%). According to age, 65.2% of children less than 6 months old were parvovirus B19 IgG seropositive, due to transplacentally derived maternal antibodies. IgG seroprevalence increased steadily, from 30.0% in the 6 months–9 years age group to 69.0% in the 40–49 years age group, and remained stable thereafter (68.8–73.3%). There was no difference in the seropositivity among males and females . In addition, no difference was observed among residents in suburban/rural areas (63.9%) and residents in urban areas (64.1%).Parvovirus B19 IgG antibodies were detected in 986 (64.1%) participants. The seropositivity differed significantly (p < 0.001). The prevalence of acute infections did not differ between males (4.3%) and females (3.2%).Parvovirus B19 IgM antibodies (current/recent infection) were found in 61 (4.0%) participants . Acute ip = 0.192; IgM p = 0.098).In a group of pregnant women, both IgG (71.0% vs. 62.6%) and IgM (6.5% vs. 2.4%) seroprevalence was higher in women with an unfavorable obstetric history compared with normal pregnancy, but these differences were not statistically significant : the adult general population OR = 0.342, 95% CI = 0.192–0.607; hemodialysis patients OR = 0.104, 95% CI = 0.013–0.798. In addition, residents of urban areas showed a lower risk of IgM positivity compared to residents of suburban/rural areas  .In this study, which is the first seroepidemiological study on parvovirus B19 in Croatia, the overall seroprevalence was 64.1%. Similar seroprevalence rates were reported in the Netherlands (61%) , Israel Parvovirus B19 IgG seroprevalence increased significantly with age in the Croatian cohort population, with 52.2% seropositive persons at the age of 19 years. Gradually rising seroprevalence rates of up to 69.0% were observed in 40–49-year-olds; subsequently, seroprevalence rates remained stable (69.1–73.3%). A similar age-related seroprevalence was found in Poland. The first peak of seropositive persons was observed in pre-school children with the proportion of seropositive patients achieving 65.5% among 15–19-year-olds and 81.2% among 40–49-year-olds . In addiIn this study, no difference was found in the IgG seropositivity between males (66.1%) and females (63.1%). No gender differences in the parvovirus B19 seroprevalence rates were found in several other studies  ,4,20. HoSome studies showed difference in the parvovirus B19 seroprevalence, according to the area of residence. A higher level of seropositivity among residents in urban areas compared to rural areas was found in Iran (88% vs. 84.3%) . Our stuFew studies analyzed the seroprevalence of parvovirus B19 in hemodialysis patients, mainly from the Middle East. Chronic anemia is a common problem in hemodialysis patients that require blood transfusion. Our results found no difference in the seropositivity in hemodialysis patients (65.6%) compared to the general adult population (67.1%), suggesting no increased risk of parvovirus B19 infection regarding receiving blood transfusions. In Iran and Brazil, the prevalence of parvovirus B19 IgG antibodies among hemodialysis patients was 54% and 67.5%, respectively ,24, whilIn general, the recipients of solid organs or hematopoietic stem cells are at an increased risk of viral reactivation originating from previous latent or persisting viral infections or pathogens transmitted by the transplant. However, the parvovirus B19 seroprevalence of liver transplant recipients (66.7%) did not differ from other adult populations in our study, and none of the tested patients had an acute infection. Seroprevalence studies in the transplant population are scarce, but similar seroprevalence rates were also confirmed in Iranian kidney transplant recipients (69.2%) who suffered from anemia . A GermaWith a reduced incidence, acute infections can occur in adults. In this study, acute parvovirus B19 infections were the most frequent in the youngest age group (11.1%) and adolescents (8.9%). However, 5.5% and 4.9% of acute infections were also reported in the age group of 20–39 years, which could be explained by the close contact with small children because of family circumstances, which probably increase the risk of virus transmission.Data on the seroprevalence of parvovirus B19 infection in pregnant women are important for identifying seronegative women who are at risk of primary infection during pregnancy. Seroprevalence studies in European countries showed seroprevalence rates in pregnant women ranging from 55% to 74% ,30,31,32The limitation of this study, which needs to be stressed, is that consecutive serum samples were tested; therefore, people tested were not necessarily representative of that area population.The results of this first large seroepidemiological study demonstrates that 64.1% of the Croatian population shows the exposure to parvovirus B19, with (increasing) age as the main risk factor for IgG seropositivity. Gender specific differences in the seropositivity were not observed. Hemodialysis patients and transplant recipients were not found to be at an increased risk for the parvovirus B19 infection. The role of parvovirus B19 as a TORCH agent needs to be studied further.


The threshold-based (TB) trabeculated and papillary muscle mass (TPM) quantification method for cardiac MRI (CMR) calculates different values than conventional contouring techniques. We aimed to identify the sex- and age-related normal reference ranges for left ventricular (LV) myocardial mass values, volumetric and functional parameters and the correspondence of these parameters using the TB method.i).Healthy European adults  were examined with CMR and evaluated with a TB postprocessing method. They were stratified by sex and age . The calculated parameters were indexed to body surface area (i) and TPMi differences remained significant after stratification by age. Unlike other LV volumetric and functional parameters and mass-to-volume ratios, the TPMi, the LVCMi and the TPMi-to-LVCMi ratio did not differ among age groups in males or females. This finding was strengthened by the lack of correlation between TPMi and age.The normal reference ranges for the studied parameters were assessed in each age group. Significant biometric differences in LV parameters and mass-to-volume ratios were found between males and females, and the left ventricular compacted myocardial mass (LVCMi and TPMi values were established using a TB postprocessing method. TPMi, LVCMi and their ratio did not change over time. The TPMi-to-LVCMi and the mass-to-volume ratios might have clinical utility in the differential diagnosis of conditions with LV hypertrabeculation.Age- and sex-related normal reference ranges for LV volumetric and functional parameters and LVCM Several studies have been published about left ventricular (LV) functional parameters in both pathological and physiological conditions –3. The bA number of studies have already been performed in various populations using different methods and vendors since trabeculae quantification became available –8.However, the established values of LV trabeculation are not comparable due to the use of different techniques, the lack of intervendor agreements, and the heterogeneous study populations.The threshold-based (TB) trabeculated and papillary muscle (TPM) quantification method is based on the differing signal intensities of blood and myocardium and has been proven to be an excellent evaluation method with better interobserver agreement than conventional techniques –11. The To the best of our knowledge, no studies have been performed to determine the normal reference ranges for LV functional parameters and myocardial mass values using the TB method. In addition, there are no data about sex- and age-specific normal values of the LV trabecular mass.We aimed to describe the normal reference ranges for LV functional parameters, compacted myocardial mass (LVCM), TPM and the correspondences of these parameters based on a healthy European population divided by sex and age using the TB method.Two hundred healthy adult European volunteers were enrolled in this single-center study . Each participant completed a questionnaire about demographic characteristics, cardiovascular symptoms, medical history, medication and sport activity. The following exclusion criteria were applied: presence of any congenital cardiac abnormalities or acquired ischemic heart diseases, arrhythmias, valvular heart diseases, cardiomyopathies, other cardiac diseases or sudden cardiac death in the family history. Furthermore, participants with extracardiac disorders, including hypertension-related, pulmonary, nephrology, gastrointestinal, metabolic, autoimmune, hormonal, psychiatric, oncologic or neuromuscular diseases or other hereditary conditions, were excluded. None of the participants had received medical therapy. Athletes with competitive sport activity (>6 hours/week) were also ruled out .In addition to cardiac MRI (CMR) examinations, blood pressure measurements and 12-lead resting electrocardiography were performed for each participant.The study population was divided by age as follows: 20–29 years , 30–39 years , 40–49 years  and ≥ 50 years , and each subgroup contained an equal number of male and female participants. The baseline characteristics are seen in All procedures performed in this study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical approval was obtained from the Central Ethics Committee of Hungary, and all participants provided written informed consent.CMR examinations were performed on 1.5 T MRI scanners .Retrospectively gated, balanced steady-state free precession (bSSFP) cine images were acquired in conventional two-chamber, three-chamber and four-chamber long-axis views. Breathhold short-axis cine images from base to apex were obtained. Contrast agent was not administered.Medis Suite software was used for the postprocessing analysis .The LV parameters were calculated by a TB algorithm of the software (MassK module of the Medis Suite program). The method is based on the differential signal intensity of blood and myocardium. The program identifies each voxel within the epicardial contour as either blood or myocardium according to the chosen threshold, which was set to the default (50%). Endocardial contours include LV trabeculation and papillary muscle; therefore, the voxels detected as myocardium within the endocardial borders are identified as trabeculation . Unlike The end-diastolic and end-systolic phases were identified, and semiautomatic tracing of the epicardial and endocardial contours was corrected manually in all slices from base to apex. The contours were made by two observers  with excellent interobserver variability, and it was determined by the global intraclass correlation coefficient, which was 0.92 .Short-axis images were applied to calculate the following LV parameters using the TB method: end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), cardiac output (CO), EF, end-diastolic total myocardial mass and end-diastolic TPM. In our study, LVCM was calculated as the difference between LV end-diastolic total muscle mass and TPM. In the following, we used only LVCM and TPM for the characterization of the myocardial mass.i). The trabeculated and papillary muscle mass-to-myocardial mass (TPMi/LVCMi), the myocardial mass-to-end-diastolic volume (LVCMi/EDVi) and the trabeculated and papillary muscle mass-to-end-diastolic volume (TPMi/EDVi) ratios were created to assess the correspondence of the LV parameters.All of the measured parameters were indexed to body surface area (The Kolmogorov-Smirnov test was used to assess data distribution. Continuous variables were reported either as the mean ± standard deviation (SD) or the median [interquartile range] as appropriate. The 95% confidence intervals (CIs) were calculated to assess the normal range for LV parameters. Independent-sample t-tests were applied to compare parameters that fit a normal distribution; otherwise, the Mann-Whitney test was used. The comparison of the functional parameters of the different age groups was performed with one-way analysis of variance (ANOVA) with the Tukey–Kramer post hoc test. Linear correlations were assessed using the Pearson correlation coefficient. A p value < 0.05 was considered statistically significant. The intraclass correlation coefficients (ICCs) were calculated to assess interobserver variability. The statistical analysis was performed using MedCalc Statistical Software version 17.9.5. .The normal reference ranges of the LV functional parameters, the LVCM, the TPM and the derived parameters were assessed in the total study population and in each age group divided by sex .i, ESVi, LVCMi and TPMi were higher and the EF was lower in males than in females.Regarding the total population, the following functional parameters differed significantly between sexes: the EDVi and TPMi values differed significantly in all age groups; however, the difference in the TPMi/LVCMi ratio remained nonsignificant between sexes , and LVCMi correlated with TPMi in our study.Interestingly, there was an inverse correlation between TPMresearch . The invi, LVCMi and TPMi/LVCMi ratio were independent of sex and age, and this result is strengthened by the correlation between them.We defined age- and sex-related normal values for LV functional parameters and myocardial and trabecular mass using a TB trabeculated and papillary muscle mass quantification method in a healthy European cohort. The TPMIncreasing the number of the subgroups would provide even more accurate results. Overall, normal values would be helpful for the determination of trabecular mass and myocardial mass/volume ratios, which might have clinical utility in the differential diagnosis of physiological and pathological conditions with LV hypertrabeculation.In the TB method, the papillary muscles are added to the trabeculated myocardial mass because of the nature of this technique. Although this study was performed on a large cohort, after dividing by sex and age, the number of subgroups decreased, which may have affected the statistical findings.Different vendors and third party software companies may have differences in how they specifically implement the algorithm separating the trabecules and papillary muscles from the blood pool and the myocardium. A simple algorithm will not fit every scanner, and some fine-tuning is necessary for different MR vendors. So the different trabecular measuring techniques are not comparable because their intervendor agreements are not established yet.Current EF and volume quantification uses a stack of thick short-axis slices, and 8–10 mm is usual for Z-direction spatial resolution. Moreover, trabecules and papillary muscles will not cross the slice in an exactly perpendicular fashion, which creates partial volume effects. Depending on the actual path of the trabecules, this will influence the threshold-based quantification. With the advent of advanced isotropic 4D cine techniques, this drawback will be less important in the future. 6 Jul 2021PONE-D-21-13794Sex- and age- specific normal values of left ventricular functional and myocardial mass parameters using threshold-based trabeculae quantificationPLOS ONEDear Dr. Szűcs,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.ACADEMIC EDITOR: All issues raised by reviewers are required.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Aug 20 2021 11:59PM. 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Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained . If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.https://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research3. Please note that outmoded terms and potentially stigmatizing labels should be changed to more current, acceptable terminology. Examples: “Caucasian” should be changed to “white” or “of [Western] European descent” (as appropriate); “cancer victims” should be changed to “patients with cancer.” [Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Cardiac MRI is nowadays considered the most reproducible diagnostic tool for in vivo investigations of excessive trabeculation phenotypes. This paper raises a quite relevant issue, i.e., the current limits in the knowledge of sex-related (besides age-related) normal reference values to be used in the CMR evaluations of myocardial trabeculation. Assessing the normal variability range in different age and sex groups is an important issue in the imaging field for the correct assessment of cardiac diseases, and this topic has been addressed in recent studies .In previous echocardiographic and CMR studies, these volumetric parameters have been shown to be age-dependent, as correctly acknowledged by the Authors in Discussion section. Unfortunately, comparison with previous studies is not always feasible, because of different imaging technologies and/or of different investigation methods . Riffel et al. have also underlined the impact of different strategies on the assessment of the CMR reference values concluding in their CMR study on 362 healthy volunteer that “Quantitative assessment of LV volumes and mass with inclusion of papillary muscles and trabeculae to myocardial mass resulted in significantly different values” . Some of the Authors of the present (under consideration) paper have previously shown that “The TB algorithm could be a consistent method to assess LV and RV CMR values, and to measure trabeculae and papillary muscles quantitatively in various level of experience in CMR (Int J Cardiovasc Imaging 2018 34:1127).Few data are available as to sex-related differences, corrected by age. The Authors confirmed that males had significantly higher volumes and myocardial mass values and lower EF than females; they also showed that LVCMi and TPMi values (after stratification by age) differed significantly between sexes.Controversial results have been reported as to possible aging-related changes in LVCMi; anyhow the results of this study regarding LVCMi such as the lack of connection between TrMi and BMI, are confirmed by other studies  and/or by anatomic post-mortem investigations that have been conducted in the past (Ref. # 33), respectively.Although this paper adds a few data to our knowledge of normal populations values for CMR evaluations, its major limit is due to the small number of the subgroups, as stated by the Authors in the final (Limitations of the study) section. Then further studies are required to confirm their  data. Such comment might be included in the Conclusions section.Reviewer #2: This paper is interesting in trying to define "normality values" by sex and age. But it fails in the method, in my opinion. It uses the tool that already exists in the market and is known . The problem is the ventricular volumes that do not coincide with those observed so far .MRI has another tool to assess precision in the quantification of ventricular volumes. We can quantify the flow that leaves the left ventricle in each beat in the aortic root. If they are normal "healthy" patients, this flow must be the same as that obtained by volumes. This part is not included in this paper. If authors want to measure it like that and validate their method they must demonstrate that it is the correct one. I think that is the first thing before defining normality values.Reviewer #3: The Authors measured the LV mass and volumes in 200 healthy caucasian controls, using a threshold-based (TB) method; in particular, they focussed on the trabeculated and papillary muscle mass (TPM) and on the left ventricular compacted myocardial mass (LVCM), which have been overlooked in previous studies. The article is interesting, even though several points should be addressed.-As far as patient stratification is concerned, the older group (>50 year old people) might not be representative of elderly people (>60 y.o.), who might display a different phenotype and different reference values than middle-aged patients (50-60 y.o.).-The threshold-based (TB) method relies on signal thresholding between the myocardium and the bloodpool, so that different thresholds produce different results by including/excluding not only any small trabecula but also any blood/myocardiuam partial volume areas. This is apparent even in Figure 1, where some small trabeculae (not highlighted in green) can be hardly distinguished from the bloodpool. Besides intra- and inter-operator variability, the variability of TB measurements should be tested on cineSSFP images acquired with/without Gd, as well as with different spatial and temporal resolutions.-Total LV myocardial mass should be measured in systole and diastole, as a double-check, also because this TB method should detect all trabeculae  and yield very similar total mass values between sytole/diastole.-Were all the volunteers free from medical therapy? Please specify in the Methods.-Table 2 and 3 are more or less the same, with only different subgroup analysis (per sex vs. per age group). I would suggest to merge the two tables. Moreover, I would suggest to represent a column/row about total LV mass (which should be the sum of TPM+LVCM).**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  12 Aug 2021Responses to the ReviewersReviewer #1 Cardiac MRI is nowadays considered the most reproducible diagnostic tool for in vivo investigations of excessive trabeculation phenotypes. This paper raises a quite relevant issue, i.e., the current limits in the knowledge of sex-related (besides age-related) normal reference values to be used in the CMR evaluations of myocardial trabeculation. Assessing the normal variability range in different age and sex groups is an important issue in the imaging field for the correct assessment of cardiac diseases, and this topic has been addressed in recent studies .In previous echocardiographic and CMR studies, these volumetric parameters have been shown to be age-dependent, as correctly acknowledged by the Authors in Discussion section. Unfortunately, comparison with previous studies is not always feasible, because of different imaging technologies and/or of different investigation methods . Riffel et al. have also underlined the impact of different strategies on the assessment of the CMR reference values concluding in their CMR study on 362 healthy volunteer that “Quantitative assessment of LV volumes and mass with inclusion of papillary muscles and trabeculae to myocardial mass resulted in significantly different values” . Some of the Authors of the present (under consideration) paper have previously shown that “The TB algorithm could be a consistent method to assess LV and RV CMR values, and to measure trabeculae and papillary muscles quantitatively in various level of experience in CMR (Int J Cardiovasc Imaging 2018 34:1127).Few data are available as to sex-related differences, corrected by age. The Authors confirmed that males had significantly higher volumes and myocardial mass values and lower EF than females; they also showed that LVCMi and TPMi values (after stratification by age) differed significantly between sexes.Controversial results have been reported as to possible aging-related changes in LVCMi; anyhow the results of this study regarding LVCMi such as the lack of connection between TrMi and BMI, are confirmed by other studies  and/or by anatomic post-mortem investigations that have been conducted in the past (Ref. # 33), respectively.Although this paper adds a few data to our knowledge of normal populations values for CMR evaluations, its major limit is due to the small number of the subgroups, as stated by the Authors in the final (Limitations of the study) section. Then further studies are required to confirm their  data. Such comment might be included in the Conclusions section.Author’s responseWe would like to thank you for the detailed opinion and useful advice. After studying the recommended references, we revised and completed our manuscript, which increased its quality.We agree with the Reviewer that the small number of subgroups is a limitation that should also be mentioned in the Conclusion section. We added an extra sentence to the final section; please find the corrected version of the Conclusion in the modified manuscript file .Reviewer #2 This paper is interesting in trying to define "normality values" by sex and age. But it fails in the method, in my opinion. It uses the tool that already exists in the market and is known . The problem is the ventricular volumes that do not coincide with those observed so far .MRI has another tool to assess precision in the quantification of ventricular volumes. We can quantify the flow that leaves the left ventricle in each beat in the aortic root. If they are normal "healthy" patients, this flow must be the same as that obtained by volumes. This part is not included in this paper. If authors want to measure it like that and validate their method they must demonstrate that it is the correct one. I think that is the first thing before defining normality values.Author’s responseWe would like to thank the Reviewer for the comment on the methodological section. The Reviewer is right that the threshold-based (TB) method has been used for years and is commercially available. However, the literature is incomplete in terms of normal values concerning sex and age, particularly when using the TB technique. The advantage of this method is that this technique is highly reproducible and is independent of the experience of the observer [1-3]. Furthermore, it is more accurate than conventional contouring techniques, especially for the determination of trabecular mass. Based on the altering signal intensities, the program automatically identifies each voxel as blood or myocardium, as the trabeculae are counted to the compact mass and extracted from the intracavital volume, resulting in lower volumetric parameters than the conventional contouring technique. This is in line with the Reviewer’s observation.Riffel et al. observed a strong correlation between conventional and TB methods in measured myocardial mass parameters with the exclusion and inclusion of trabecular and papillary muscles [4]. Another investigation used the TB method of Medis Medical Imaging Systems and compared this semiautomatic segmentation algorithm with the standard method of manual contour tracing for subjects with normal heart function and on a dynamic anthropomorphic heart phantom. The volumetric values calculated in that study were similar to ours, and the correlation between the TB method and manual contouring technique was high in terms of EDV and ESV parameters [2].We agree with the Reviewer that validation of the software is absolutely necessary for defining normal values. Validation of the TB method was conducted by Varga-Szemes et al., who performed aortic flow analysis to provide independent reference standards for SV measurements, and they described that the software has excellent agreement in SV and CO with the flow reference [1]. Since it has been proven to be a validated method, we did not perform this measurement in our study.We also made a representative sample from our studied population with 10 people from each subgroup. Please find the presented volumetric, functional and myocardial mass parameters  in Table 1 calculated with conventional technique, with values corresponding to the normal values [5]. The left ventricular mass (LV_Massi) values were calculated as the myocardium between the endo- and epicardial contours and indexed to the body surface area.IDENTIFIER EDVi (ml/m2) ESVi (ml/m2) SVi (ml/m2) COi (l/m2*min) EF (%) LV_Massi (g/m2)A_1 68.2 28.9 39.2 2.9 57.6 34.2A_2 93.8 40.6 53.2 3.7 56.7 48.0A_3 69.8 24.3 45.4 2.9 65.1 38.8A_4 78.4 29.7 48.8 3.4 62.2 37.5A_5 93.2 37.6 55.6 5.5 59.7 35.7A_26 96.0 43.0 53.1 3.5 55.3 49.4A_27 70.2 27.7 42.5 3.3 60.5 35.9A_28 86.9 35.9 51.0 3.1 58.7 53.1A_29 101.2 47.7 53.4 4.8 52.8 50.4A_30 107.9 42.6 65.3 5.3 60.5 54.9Females 80.7 32.2 48.4 3.7 60.3 38.8Males 92.4 39.4 53.1 4.0 57.6 48.7Average of Group A 86.6 35.8 50.8 3.8 58.9 43.8B_1 87.0 30.9 56.2 3.2 64.5 43.1B_2 81.0 32.9 48.1 3.7 59.4 37.2B_3 77.2 32.5 44.7 3.1 57.9 38.5B_4 88.0 31.7 56.4 3.5 64.0 47.3B_5 73.2 28.2 45.0 2.8 61.5 38.0B_26 85.3 34.4 50.9 3.9 59.7 40.8B_27 109.5 49.4 60.1 4.9 54.9 64.2B_28 79.3 35.4 43.9 2.4 55.3 42.6B_29 82.5 36.2 46.4 3.1 56.2 53.7B_30 94.1 40.7 53.4 3.4 56.8 50.5Females 81.3 31.2 50.1 3.2 61.5 40.8Males 90.1 39.2 50.9 3.5 56.6 50.4Average of Group B 85.7 35.2 50.5 3.4 59.1 45.6C_1 83.4 37.8 45.6 3.7 54.7 43.5C_3 80.4 29.7 50.7 4.2 63.0 41.4C_4 73.7 29.2 44.4 2.6 60.3 32.4C_5 82.3 30.2 52.1 3.9 63.3 39.4C_6 92.8 37.5 55.2 3.4 59.5 50.3C_26 91.1 34.0 57.1 3.2 62.7 44.9C_27 82.9 37.1 45.8 3.0 55.3 48.5C_28 104.3 43.5 60.8 4.7 58.3 67.9C_29 88.1 29.0 59.2 4.1 67.1 64.4C_30 81.2 33.4 47.7 2.2 58.8 43.6Females 82.5 32.9 49.6 3.6 60.2 41.4Males 89.5 35.4 54.1 3.4 60.4 53.9Average of Group C 86.0 34.1 51.9 3.5 60.3 47.6D_1 76.5 25.9 50.6 3.6 66.1 38.1D_2 76.9 31.6 45.4 2.8 59.0 45.7D_3 60.6 24.9 35.7 3.3 58.9 37.1D_4 76.0 23.8 52.2 4.2 68.6 33.3D_5 76.9 23.5 53.3 3.8 69.4 43.1D_26 73.5 23.2 50.2 3.0 68.4 42.9D_27 101.7 39.8 61.9 3.6 60.8 45.3D_28 66.9 23.8 43.1 2.8 64.4 49.6D_29 64.1 23.4 40.7 2.3 63.4 42.3D_30 106.6 39.8 66.8 5.0 62.7 64.7Females 73.4 25.9 47.4 3.6 64.4 39.5Males 82.5 30.0 52.5 3.3 63.9 49.0Average of Group D 78.0 28.0 50.0 3.5 64.2 44.2Table 1 – Representative sample from the studied population divided by age group and sex; five males and five females from each subgroupEDVi: end-diastolic volume index, ESVi: end-systolic volume index, SVi: stroke volume index, COi: cardiac output index, EF: ejection fraction, LV_Massi: left ventricular end-diastolic myocardial mass index, LV: left ventricular Group A: 20–29 years, Group B: 30–39 years, Group C: 40–49 years, Group D: ≥ 50 yearsReviewer #3 The Authors measured the LV mass and volumes in 200 healthy caucasian controls, using a threshold-based (TB) method; in particular, they focussed on the trabeculated and papillary muscle mass (TPM) and on the left ventricular compacted myocardial mass (LVCM), which have been overlooked in previous studies. The article is interesting, even though several points should be addressed.-As far as patient stratification is concerned, the older group (>50 year old people) might not be representative of elderly people (>60 y.o.), who might display a different phenotype and different reference values than middle-aged patients (50-60 y.o.).-The threshold-based (TB) method relies on signal thresholding between the myocardium and the bloodpool, so that different thresholds produce different results by including/excluding not only any small trabecula but also any blood/myocardiuam partial volume areas. This is apparent even in Figure 1, where some small trabeculae (not highlighted in green) can be hardly distinguished from the bloodpool. Besides intra- and inter-operator variability, the variability of TB measurements should be tested on cineSSFP images acquired with/without Gd, as well as with different spatial and temporal resolutions.-Total LV myocardial mass should be measured in systole and diastole, as a double-check, also because this TB method should detect all trabeculae  and yield very similar total mass values between sytole/diastole.-Were all the volunteers free from medical therapy? Please specify in the Methods.-Table 2 and 3 are more or less the same, with only different subgroup analysis (per sex vs. per age group). I would suggest to merge the two tables. Moreover, I would suggest to represent a column/row about total LV mass (which should be the sum of TPM+LVCM).Author’s responseQuestion #1: We agree with the Reviewer that we should have enrolled more elderly people in our study (>60 y.o.). However, in this single-center prospective study, it was quite challenging to include healthy participants older than 60 years, as most of the people in this age group are under medical therapy and have severe comorbidities. As the Reviewer mentioned, elderly people might display different phenotypes than middle-aged people (50–60 y.o.). Although there are investigations reporting normal values for an older population, a different approach was used in these larger center studies .Question #2: The Reviewer’s observation is correct. Visualization of the myocardium  can slightly differ from the calculated myocardium due to the nature of the threshold-based technique: small trabeculae may be calculated as myocardium, even though they are not marked with green color on the image. The threshold was set to default (50%) and was not modified manually in either case; thus, there was no interoperator variability regarding the threshold settings. The Medis Medical System recommends using the software with the default settings, which makes it the most applicable. Other studies have examined the changing threshold to 70% and found that this value might too high [2].Testing the variability of TB measurements on cineSSFP images acquired with and without Gd is an appropriate question; however, the usage of the contrast agent modified the calculation of the volumes and the masses during the postprocessing evaluation as the border between blood and myocardium became less defined [7]. Because of this, we did not use contrast agent in our study. Please see this text on page 6 line 112–113.We did not find any data on whether altering spatial and temporal resolution changes the measured TB parameters; however, it would be interesting to compare different settings, which would constitute a completely new investigation. To set the appropriate normal values, we prefer the original setting used in everyday practice in our research.Question #3: The Reviewer is correct: a double-checking method for systolic and diastolic myocardial mass measurements would be useful mainly in the conventional techniques. However, the TB method calculates the total myocardial mass within the epicardial contour and measures the trabeculae within the endocardial contour automatically with minimal manual correction. Thus, in our study, we did not focus on double-checking because the TB method is an already validated technique with excellent reproducibility .Question #4: Thank you for this comment. We agree that this was not clearly emphasized in the Methods section. All of the participants were free from medical therapy, and information was added to the manuscript. Please see page 4, line 87.Question #5: The Reviewer requested that we merge Table 2 and Table 3. We hope that the tables are still clear and logical in this form and fit with the Reviewer’s conception. We have also represented the total LV mass values in the table. Please see the new version of it as the new Table 2 on page 9.1. Varga-Szemes, A., et al., Clinical feasibility of a myocardial signal intensity threshold-based semi-automated cardiac magnetic resonance segmentation method. Eur Radiol, 2016. 26(5): p. 1503-11.2. Jaspers, K., et al., Improving the reproducibility of MR-derived left ventricular volume and function measurements with a semi-automatic threshold-based segmentation algorithm. Int J Cardiovasc Imaging, 2013. 29(3): p. 617-23.3. Csecs, I., et al., Left and right ventricular parameters corrected with threshold-based quantification method in a normal cohort analyzed by three independent observers with various training-degree. Int J Cardiovasc Imaging, 2018. 34(7): p. 1127-1133.4. Riffel, J.H., et al., Cardiovascular magnetic resonance of cardiac morphology and function: impact of different strategies of contour drawing and indexing. Clin Res Cardiol, 2019. 108(4): p. 411-429.5. Petersen, S.E., et al., Reference ranges for cardiac structure and function using cardiovascular magnetic resonance (CMR) in Caucasians from the UK Biobank population cohort. J Cardiovasc Magn Reson, 2017. 19(1): p. 18.6. Natori, S., et al., Cardiovascular function in multi-ethnic study of atherosclerosis: normal values by age, sex, and ethnicity. AJR Am J Roentgenol, 2006. 186(6 Suppl 2): p. S357-65.7. Szucs, A., et al., The effect of contrast agents on left ventricular parameters calculated by a threshold-based software module: does it truly matter? Int J Cardiovasc Imaging, 2019. 35(9): p. 1683-1689.AttachmentResponse to the Reviewers.docxSubmitted filename: Click here for additional data file. 27 Sep 2021Sex- and age- specific normal values of left ventricular functional and myocardial mass parameters using threshold-based trabeculae quantificationPONE-D-21-13794R1Dear Dr. Szűcs,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The Authors have revised the paper according to the reviewers' comments and the manuscript has been improved by the changes that have been made.Reviewer #2: As I said in the first review, this paper is interesting in its attempt to define "normality values" by sex and age. But it fails in the method, in my opinion. It uses the tool that already exists on the market and is well known . The problem is the ventricular volumes that do not match those observed so far .MRI has another tool to assess the accuracy in quantifying ventricular volumes. We can quantify the flow out of the left ventricle at each beat at the aortic root. In normal "healthy" patients, this flow should be the same as that obtained by volumes. This part is not included in this paper and the authors go on to say that it does not validate the method. If the authors want to measure it this way and validate their method, they must prove that it is correct. I think that is the first thing to do before defining normal values.Reviewer #3: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. 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The burden of chronic liver disease (CLD) in India is high, particularly among middle-aged men, with nearly 220,000 deaths due to cirrhosis in 2017. CLD increases the risk of infection, severe disease , and mortality. Hence, various countries recommend HAV vaccination for CLD patients. While historic Indian studies showed high seroprevalences of protective HAV antibodies among Indian adults with CLD, the most recent ones found that nearly 7% of CLD patients were susceptible to HAV infection. Studies in healthy individuals have shown that HAV infection in childhood is decreasing in India, resulting in an increasing population of adults susceptible to HAV infection. As patients with CLD are at increased risk of severe HAV infection, now may be the time to recommend HAV vaccination among people with CLD in India. CLD (disease that has lasted for ≥6 months) is a progressive deterioration in liver function, which can lead to fibrosis and cirrhosis.1 Ultimately, patients may require a liver transplant, which is the second commonest major organ transplantation.7Early treatment of patients with CLD is important. The goals of treatment are to stop disease progression  and to manage complications .8–12 and those who become infected have a nearly 4-fold higher risk of death compared with uninfected people with cirrhosis.2 Given the effect of cirrhosis on the immune response, such patients may also be at increased risk of HAV infection. Although we could not find any confirmation of this, patients with CLD certainly appear to be at increased risk of developing more severe HAV disease if they have superimposed HAV disease.13–15 For example, in an outbreak of >300,000 HAV cases in China, mortality was 5.6-fold higher among those with HAV infection superimposed on underlying HBV infection than in those with HAV but without HBV.15 Acute HAV infection in patients with CLD can also result in acute-on-chronic liver failure (ACLF), which is associated with high rates of mortality.16Patients with cirrhosis have a compromised immune system and are known to be at increased risk of bacterial infection,17 also known as fulminant hepatic failure (FHF). In an Italian study, 595 adults (29.1 ± 9.8 years) with chronic HBV or HCV (without HAV antibodies) were enrolled during 1990–1997.18 Of these, 27 (4.5%) acquired HAV superinfection (10/163 of those with chronic HBV and 17/432 of those with chronic HCV). FHF developed in 0/10 chronic HBV patients and 7/17 chronic HCV patients, 6/7 of whom died. None of 191 controls (without CLD) who presented with acute HAV developed FHF.18 While this study implies that patients with chronic HBV are not at risk of FHF after HAV superinfection, results from a small Canadian study show that those with chronic HBV can have FHF after HAV superinfection. In the Canadian study, 4/60 cases of FHF during 1991–1997 were due to HAV.19 Three of these patients had CLD , and all 3 died (13–35 days after admission); the patient without CLD survived.19Patients with CLD are also at increased risk of developing fulminant hepatitis,20 Similarly, in the United Kingdom (UK), patients with various chronic liver conditions are recommended to receive HAV vaccination.21The United States (US) Advisory Committee on Immunization Practices recommends a 2-dose series of HAV or a 3-dose series of HAV+HBV vaccinations for all patients with CLD, including those with HBV, HCV, cirrhosis, NAFLD, alcoholic liver disease, autoimmune hepatitis, or alanine aminotransferase or aspartate aminotransferase level >2 the upper limit of normal.22 – live attenuated and inactivated – of which only the latter is appropriate for immunocompromised patients such as those with CLD. The World Health Organization (WHO) has endorsed that inactivated HAV vaccines are well tolerated by patients with mild-to-moderate CLD.17 It is recommended that HAV vaccination should be given as early as possible after CLD diagnosis for maximum efficacy and safety.14Two types of HAV vaccine are available23 One quarter of these patients had a new diagnosis of liver disease, of whom 19.8% had CLD.23 Among these 13,014 patients with newly diagnosed CLD (of whom 4413 [33.9%] had decompensated cirrhosis), mean age was 42.8 ± 14.4 years and the majority (73.0%) were male.23 The main etiologies were related to hepatitis viruses (54.9%), alcohol (17.3%), and NAFLD (12.8%).23 However, etiology varied widely by region, with HCV being the most common in the North (44.9%), HBV in the East (47.9%) and South (40.5%), alcohol in the North-East (31.9%), and NAFLD in the Central region (43.6%) and the West (39.6%).23 CLD etiologies reported in other studies have varied widely,24–34 likely due to variations by region, population, and over time. The latter has been shown in a study in a tertiary care referral hospital in Eastern India, where etiologies of CLD changed substantially from 2003 to 2011, with alcohol increasing from 22.5% to 42.0% (p = .01), cryptogenic (i.e. unknown cause) decreasing from 44.9% to 25.0% (p = .001), but no significant changes in HBV (mean 22.3%) or HCV (mean 10.9%).35In a multicenter prospective study conducted in different parts of India, 1.3% of nearly 21 million patients who attended 11 hospitals during February 2010 to January 2013 had liver disease.36 while one published in 2019 reported that 528 (53.5%) of male blood donors  in an urban community in North India had NAFLD on ultrasonography.37More recent studies have indicated that NAFLD could be becoming a major cause of CLD in India, with huge numbers of people potentially affected. For example, a study published in 2016 found that 30.7% of adults aged ≥35 years in a rural community in North India had NAFLD on ultrasonography,38 and 0.44–0.88%,39 respectively in India. Based on a population of approximately 1.38 billion,40 this would equate to approximately 20 million people in India having chronic HBV infection and around 6–12 million having chronic HCV infection, meaning that large numbers of people are potentially at risk for FHF, which has a very high mortality rate among patients with CLD.19 However, these numbers are dwarfed by the potential number of people with NAFLD, which, based on the two above-mentioned studies37 and the adult Indian population,40 could equate to hundreds of millions of adults with NAFLD in India. While we were unable to find data on the prevalence of alcoholic liver disease in India, given that 18% of liver-related deaths in India were due to alcohol,41 there are likely also many millions of people with alcoholic liver disease in India.Recent meta-analyses have estimated seroprevalences of HBV surface antigen (HBsAg) and anti-HCV to be 1.46%42 In 2017, 16.5% of global cirrhosis deaths  were in India.42In India, deaths due to cirrhosis nearly doubled – from 110,091 to 217,896 – between 1990 and 2017 .43 In that study, patients with alcoholic versus nonalcoholic cirrhosis were more often male (97% vs. 64%) and had more advanced disease (based on various parameters).43 The rates of death or orthotopic liver transplantation within 1 year are even higher among those with a first episode of decompensation , occurring in 22.2%, 28.0%, and 20.0% of these patients, respectively.33 In an Indian retrospective study of 392 patients  who had died of liver-related causes (except liver metastasis from non-hepatic cancers), the most common causes of liver-related death were alcohol (30.1%), nonalcoholic steatohepatitis/cryptogenic (23.2%), hepatotropic viruses (18.6%), and bacterial/other infections (11.5%).34 Most patients (85.5%) had CLD, and among those with CLD, most (70.7%) had presented with cirrhosis complications , while 29.3% presented with ACLF.34 Based on data from the WHO, approximately 54% of liver-related deaths in India are due to HBV, 18% due to alcohol, 10% due to HAV or HEV, and 10% due to HCV.41 However, acute hepatitis-related deaths are largely due to HBV (54%) or HEV (37%), with 6% due to HAV and 2% due to HCV.41Mortality rates vary among patients with CLD. For example, those with alcoholic cirrhosis had higher 1-month mortality than those with nonalcoholic cirrhosis (9.8% vs. 3.2%) in a single-center study from North East India.16) occurred in 121/3220 (3.8%) patients with cirrhosis and acute HAV or HEV admitted to a hospital in Lucknow during 2000–2006.32 The mean age of those with ACLF was 36.3 ± 18.0 years and 70.2% were male.32 Clinical features included jaundice (100%), ascites (78.5%), coagulopathy (77.7%), encephalopathy (55.4%), hyponatremia (41.3%), renal failure (35.5%), and sepsis (33.9%).32 ACLF was due to HEV in 66.1%, HAV in 27.3%, or both in 6.6%; the underlying CLD was mainly cryptogenic (36.4%), HBV (30.6%), or alcohol (10.7%).32 Three-month mortality among these patients with ACLF was high (44.6%).32 In another retrospective study, which included 1049 consecutive patients with ACLF  conducted in 10 tertiary centers from across India during 2011–2014, the most common precipitants of ACLF were alcohol consumption (35.7%), viral superinfection/flare  (21.4%), and sepsis (16.6%).29 The underlying CLD was mainly alcohol (56.7%), cryptogenic (19.4%), or HBV/HCV (15.9%). During a median (range) hospital stay of 8 (4–14) days, 42.6% of patients died.29 In a single-center study in Eastern India (2012–2014), the most common precipitants of acute decompensation among 123 patients with ACLF  were recent alcohol intake (42.3%) and bacterial infection (36.6%).44 Three-month mortality was very high (71.3%), more so in alcoholics than nonalcoholics .44 Lastly, among 64 patients  with ACLF in a hospital in Hyderabad, 2015–2016, the main precipitants were infection (43.8%) and alcoholism (37.5%). Twenty-eight day mortality was high (43.8%).45ACLF  had evidence of past infection with HAV (as shown by HAV-immunoglobulin G or HAV-IgG49 or anti-HAV antibodies), as did most healthy controls (71.2–100%) , possibly because they only included patients from the upper middle or upper socioeconomic classes. All of their seronegative CLD patients were vaccinated against HAV.50 The authors of most of the other studies suggested that CLD patients did not routinely require HAV vaccination ,46–48 while opinions on testing for HAV antibodies before potential vaccination were mixed (see 48Nine out of ten old studies from India (carried out up to 2007).2–100%) . The stuixed see .25,31,4848 which included patients during the mid 2000s, nearly 7% of CLD patients did not have anti-HAV/anti-IgG antibodies and were therefore susceptible to HAV infection. As suggested by Radha Krishna et al. in 200932 – based on their study that included 21 adults with ACLF due to HAV – this advice may now be outdated. In a more recent study (2011–2014), 21.4% of ACLF cases were due to HAV, HBV, or HEV superinfection, but unfortunately, the authors did not report results separately for HAV.29However, most of these studies are old, only including patients until 2007 at the latest and, as will be discussed further below, the epidemiology of HAV is changing in India, with declining HAV infection during childhood and subsequent increasing susceptibility in adulthood. It should also be noted that, in the two latest studies in 51 and providing long-term immunity against HAV.52 However, if HAV is encountered for the first time in adulthood, most people will have symptoms , and it is associated with a mortality rate of around 1%.51 HAV is also significantly more likely to result in more severe disease with increasing age.53 Historically, many people in India were exposed to HAV during childhood, resulting in life-long protection.52 However, with improved sanitation and hygiene, children are becoming less likely to be exposed to HAV, resulting in increasing number of adolescents and adults who are at risk of infection, and a paradoxical increase in morbidity and mortality due to HAV.55 Thus, and taking into account the high heterogeneity across the Indian continent, India is now thought to be shifting from high to intermediate HAV endemicity.54 This situation is particularly challenging, as in high HAV endemicity areas, most children are exposed, resulting in mild disease and lifelong immunity, while in low endemicity areas, the chance of exposure in adulthood is low.54 However, with intermediate HAV endemicity, the chance of childhood exposure is reduced, leaving more adults at risk of more severe disease.54If HAV is encountered during early childhood, the resultant hepatitis is generally mild, causing no symptoms or nonspecific symptoms 56–59 For example, Arankalle et al.56 reported that anti-HAV positivity decreased significantly from 1982 to 1998 among children from urban high socioeconomic populations , but not in adults or urban lower middle socioeconomic populations. Das et al.57 reported that HAV-IgG seropositivity fell from 98.0% in 198260 to 54.1% in 1998 among those aged 15–24 years and from 98.6%60 to 58.7% among those aged 25–34 years (both p < .05). Hussain et al.26 reported that 71.2% of healthy subjects were positive for HAV-IgG in 1999–2003, much lower than the 94.8% reported in subjects in 1982 in an earlier study.60 Gadgil et al.58 found that HAV seropositivity among adult blood donors fell from 96.5% in 2002 to 92.1% in 2004–2005 (p < .01). Recently, Arankalle et al.59 reported that, while HAV seropositivity decreased from 1998 to 2017 among low/middle socioeconomic children and younger adults, it increased during the same time period among high socioeconomic children and adults  for 2017 and compared them with the same 1998 data as Arankalle et al.59Various serological studies have reported that the proportion of healthy Indian people with seroprotective anti-HAV antibodies (i.e. previous HAV infection) has fallen over time.d adults .59 This 59 also reported that 90–95% of 3-month-old infants in both 1995 and 2017 were seropositive for HAV, likely due to maternal antibodies. In 1995, seropositivity fell to 13.6% by age 9 months and then increased to 41.0% by age 15 months, which must have been due to natural infection as none were vaccinated. However, in 2017, seropositivity fell to only 2.2% among unvaccinated infants at age 15 months.59 These studies all support a decrease in natural HAV infection during childhood, resulting in an increase in the number of susceptible adults.Arankalle et al.50 Hussain et al.26 studied 1932 patients with acute viral hepatitis at a tertiary care center in Northern India, of whom 11.4% overall were HAV-IgM positive (indicating current infection). This increased from 3.4% in 1999 to 12.3% in 2003 among adults (Hussain line in p < .004); and from 10.6% to 22.0% in children (p < .003). At another tertiary care center in Northern India, Khanna et al.50 reported increasing proportions of acute hepatitis due to HAV from 1999 to 2004 among patients with acute hepatitis aged 13–20 years , 21–30 years , and >30 years .In line with declining HAV seroprotection, some studies have shown an increase in the proportion of acute viral hepatitis cases that are due to HAV over time.62 or have not studied their evolution over time63-69  of the Indian Academy of Pediatrics (IAP) recommends HAV vaccination for all infants, as a single dose of live attenuated vaccine at 12 months or 2 doses of inactivated vaccine at 12 and 18 months of age,51 which can be administered in a private setting paid for by the parents.22 The IAP particularly recommends HAV vaccination for various risk groups, including children with CLD and those who are carriers of HBV and HCV.51HAV vaccination is not included in the routine childhood immunization schedule in India.17 the Indian National Centre for Disease Control (NCDC) does not currently recommend HAV vaccination for adults with CLD in India, as “most adults have already been exposed to and are thus protected”.71 This recommendation is supported by 9/10 old studies from India (carried out up to 2007)46–48 46–48 . However72–74 The Association of Physicians of India (API) and the Indian Society of Nephrology (ISN) both indicate that patients with CLD who are not immune to HAV, those with other hepatitis virus infections, and patients awaiting or having received a liver transplant are at risk of HAV infection, but do not specifically recommend vaccination.73 The ISN, however, says that HAV vaccination “is indicated for all transplant candidates with CLD or those patients of end‑stage renal disease who have chronic hepatitis B or C” due to an increased risk of FHF.73 The Indian Medical Association (IMA) does not mention CLD or other hepatitis infection, but does recommend HAV for adults or children undergoing liver transplantation.74Indian associations and scientific society recommendations relating to HAV vaccination are detailed in 71 Instead, we propose that a recommendation for HAV vaccination of adults with CLD should be considered in India, as is already the case in the US,20 the UK,21 and Sri Lanka75 (a near neighbor of India), and also for children with CLD in India.51 While some Indian medical association/society guidelines recognize that seronegative patients with CLD are at increased risk of HAV infection, they do not clearly recommend HAV vaccination.73Based on the currently available evidence of shifting endemicity and increasing HAV susceptibility in adulthood in India, now may be the time to revisit the existing NCDC recommendation that HAV vaccination is not necessary for those with CLD in India.61 However, awaiting the results of such studies should not be a prerequisite for recommending HAV vaccination among patients with CLD in India.Up-to-date serological studies among Indian patients with CLD would be beneficial to confirm whether seroprotective HAV antibodies have decreased over time in these patients, in line with what has been shown in healthy people.25 However, given the limited facilities for serological testing, the associated cost, and the potential for missed opportunity for vaccination if patients do not return after serological testing, this should also not be a prerequisite.A more targeted approach, with serological testing prior to HAV vaccination, could be a more cost-effective option than universal HAV vaccination of patients with CLD.This was not a systematic review, so although we included all relevant manuscripts that we could find on PubMed and Embase, there may have been some manuscripts  that we did not manage to capture. Also, India is a large country with high socioeconomic status heterogeneity. As seropositivity rates vary considerably with socioeconomic status, age, HAV vaccination rates, region, setting, and local outbreaks, comparisons between studies from different time periods should be undertaken with caution. Futher, as already discussed, the studies that have assessed the HAV susceptibility of CLD patients are old and, while it is likely that susceptibility among these patients has increased as it has among general adults, this should be confirmed.42 Patients with CLD are at increased risk of severe HAV disease17 and ACLF, which has a very high mortality rate.45 Hence, such patients are recommended to receive various vaccinations, including HAV vaccination, in the US,20 the UK,21 and Sri Lanka.75 Old studies from India showed a high seroprevalence of protective HAV antibodies among Indian adults with CLD,46–48 although the most recent ones (≤2007) found that nearly 7% of CLD patients did not have protective HAV antibodies and were therefore susceptible to HAV infection. Studies in healthy individuals have shown that HAV infection in childhood is decreasing in India,76 resulting in an increasing population of adults without protective antibodies, and a higher risk of HAV infection in adulthood.50 This is likely also the case among patients with CLD.The burden of CLD in India is high, resulting in high morbidity and mortality.39 millions of people in India likely have chronic HBV or HCV infection. Even more adults could have NAFLD37 and, along with an increasing amount of alcoholic liver disease in India,35 this equates to a huge population of people with chronic hepatitis infection and/or CLD. Such people are at higher risk of severe disease17  and increased mortality.19Based on seroprovalence data,71–74 Patients with CLD who do not have HAV antibodies should receive HAV vaccination. This approach could reduce morbidity, mortality, and healthcare costs of HAV infection among patients with CLD.77 In situations where antibody testing is not available or practical, CLD patients should not be excluded from HAV vaccination.It may, therefore, now be time to reexamine the existing Indian recommendations.


Mycoplasma gallisepticum causes chronic respiratory diseases in poultry and economic losses to the chicken and turkey industry. We report the complete genome sequence of the field isolate strain KUVMG001 of Mycoplasma gallisepticum from South Korea. Mycoplasma gallisepticum causes chronic respiratory diseases in poultry and economic losses to the chicken and turkey industry. We report the complete genome sequence of the field isolate strain KUVMG001 of Mycoplasma gallisepticum from South Korea. Mycoplasma gallisepticum is an avian pathogen that causes chronic respiratory disease, decreased egg production, and decreased feed efficiency in poultry, which results in economic damage to the industry .DNA for Nanopore sequencing was extracted using the MagAttract HMW DNA kit (Qiagen) according to the manufacturer’s instructions. No shearing or size selection of the DNA was performed. The Nanopore sequencing was performed following the native barcoding genomic DNA protocol . The third barcode from the EXP-NBD104 kit and the SQK-LSK10 ligation kit were used. Sequencing was performed with a flow cell type R9.4.1 on the MinION Mk1B  for 48 h. Albacore v2.3.1 was used for base calling. Default parameters were used for all software unless otherwise specified. The output of Nanopore sequencing was 2.5 Gb . The subsample was assembled using Flye v2.7.1 (Nanopore sequencing reads were subsampled with 100× coverage using Rasusa v0.3.0  (read N5e v2.7.1 . CirculaThe complete genome sequence was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The size of the genome was 1,009,936 bp with a GC content of 31.6%, with 760 coding sequences, 41 RNAs, and 60 pseudogenes.CP070622, BioSample number SAMN16879834, and BioProject number PRJNA680431. SRR13148778 and SRR13148779 are the accession numbers for long-read and short-read data, respectively, in the NCBI SRA.This whole-genome sequence with the annotation data is available in the NCBI database under the accession number