Bacillus species is a well-documented causative pathogen of nosocomial bloodstream infection. The present study aimed to identify climatological variables that are associated with Bacillus-positive blood culture in Sapporo, Japan. All cases with Bacillus-positive blood cultures from January 2011 to December 2016 were retrospectively analysed. Climatological data from 2011 to 2016, including daily mean temperature and absolute humidity, were retrieved from the Japan Meteorological Agency. Employing a hazard-based statistical model to describe the non-homogeneous counting process in which temperature and absolute humidity act as explanatory variables, we computed all possible models with variable lengths of time lag. Akaike Information Criterion was computed to identify the best fitted model. High wavelet power at 12 months was identified for the period from 2013 onwards, which coincided with the time period in which sampling multiple sets of blood culture has been recommended. The temperature-only model with a lag of six days yielded a high sensitivity value (72.1%) and appeared to be the optimal model to predict Bacillus-positive blood culture with the highest area under the receiver operating characteristic curve value. Temperature was identified as a climatological driver of Bacillus-positive blood culture. Our statistical modelling exercise offers an important message for infection control practices to improve awareness among healthcare workers of the identified association and mechanically controlled in-room temperature.The  Bacillus, which are ubiquitous in the environment, are Gram-positive, rod-shaped spore-forming pathogens. Bacillus spp. optimally grow at temperatures ranging from 25 to 37 °C on standard culture medium [Bacillus spp., including highly abundant Bacillus cereus, are well-documented pathogens of nosocomial bloodstream infection (BSI) in immunocompromised patients and patients with malignancies [Bacillus spp. to catheters is considered to frequently induce catheter-related BSI [Bacillus spp. are also considered as a contaminant when isolated from clinical specimens, including blood culture. Hospital outbreaks of Bacillus spp. in a variety of settings such as the intensive care unit (ICU) [Bacteria of the genus e medium . Bacilluit (ICU) , neonatait (ICU)  have beeit (ICU) .B. cereus bacteraemia outbreaks due to contaminated linen and reusable towels have been reported. Many of these outbreaks occurred in summer [Bacillus spp. from blood culture is known to peak in summer in Japan [Bacillus spp. isolated from blood culture has also been implicated [Bacillus spp. have not been explored in detail, especially with the aim of predicting the risk of positive blood cultures using environmental data.In Japan, n summer ,10. Furtin Japan ,12. Seasplicated . NeverthBacillus spp. may be closely associated with climatological determinants. Whereas a small number of published studies have emphasized that high temperature is likely associated with Bacillus spp. [Bacillus-positive blood culture in Sapporo, Japan, by employing a hazard-based statistical model.The abovementioned published evidence endorses the concept that the recovery of lus spp. ,11, idenBacillus-positive blood culture from January 2011 to December 2016 were retrospectively analysed using the laboratory database of microbiological testing at Hokkaido Cancer Center, a 520-bed tertiary cancer hospital in Sapporo, Japan. Blood culture samples were obtained from patients with suspected bacteraemia according to physicians’ decision. The date on which sampling occurred for each Bacillus-positive blood culture was examined, regardless of the actual bacteraemia or contamination of the sample. Days without positive patients were also recorded. All other datasets were anonymised and unused. Because the present study aimed to identify environmental drivers of Bacillus spp., which are rare events and hereafter modelled the occurrence as the stochastic counting process over time, we discarded the information on individual patients’ characteristics. All admitted patients in the corresponding hospital have neoplasms for treatment, and the underlying characteristics of admitted patients do not greatly vary with time.All cases with Bacillus-positive blood culture , and the measurement unit was per day, allowing an identification of the day with a high risk of infection.Blood culture specimens were inoculated into BacT/ALERT SA and SN bottles in a BacT/ALERT 3D system . Species identification was performed with BBL Crystal  Gram-positive identification panels. Our data set is the binary data describing the daily occurrence of http://www.jma.go.jp/jma/indexe.html). All datasets were collected at the observatory weather station in Sapporo. Sapporo is located approximately at latitude 43°4′ and longitude 141°21′ and has a humid continental climate. The average daily mean temperature is around 22 °C in summer (August) and around −3 °C in winter (February). Temperature, relative humidity, and wind speed were provided as the daily average in each geographic observation point. Absolute humidity was calculated using temperature and relative humidity. Collected environmental data are available as Climatological data from 2011 to 2016, including daily mean temperature, mean relative humidity, total hours of sunlight per day, and wind speed, were retrieved from the website of the Japan Meteorological Agency  [3), hours of sunlight, and wind speed (m/s) were used as climatological variables. This process enabled us to reject a model with variables that are not associated with Bacillus-positive blood culture in the subsequent analyses.Second, we conducted univariate analyses to examine the association between climatological variables and m towel) ,10 and dBacillus-positive blood culture using a hazard-based model. Specifically, we employed a non-homogeneous counting process model to describe the temporal event frequency of Bacillus-positive blood culture, modelling the daily risk. For this infection of environmental origin, we assume that the underlying characteristics of inpatients were unchanged over time, and model the daily risk using the time-dependent hazard that is governed by environmental variables. Let tλ be the hazard of a positive outcome at calendar time t. To parameterize the hazard function, we used variables that appeared to be predictive in univariate analyses. Lag days of climatological variables were set to account for the background causal mechanism of environmental variables influencing the risk of Bacillus spp. The daily hazard function reads as follows:a0 represents the baseline, ia is the coefficient of variable i, and ix(t) is the input variable i that varies as a function of day t. The model was dealt with as a multivariate model. When we used multiple input variables ix, we also accounted for an additional term  to address interaction between two variables. Let U and W be the groups of days with and without Bacillus-positive blood cultures, respectively. The likelihood function to estimate parameters of the hazard (1) isThird, we analysed the risk of observing To obtain maximum likelihood estimates, the negative logarithm of Equation (2) was minimized.To compare different models , Akaike Information Criterion (AIC) was used, reflecting a penalty in the number of parameters. The diagnostic performance of the prediction model, including sensitivity, specificity, and area under the receiver operating characteristic curve (AUC), was then quantified along with the positive predictive value (PPV) and negative predictive value (NPV). To identify the optimal threshold of the predicted risk, we used the Youden index . The 95% confidence intervals (CI) of the sensitivity and specificity were computed using normal approximation to binomial distribution, while calculations of 95% CIs of the PPV and NPV, as well as the AUC, were based on the Wald method as described elsewhere . As our https://cran.r-project.org/) [All statistical analyses were conducted using JMP Pro 13  and statistical package R (ct.org/) .The present study only analysed the counts of data collected in a hospital, and the data were fully anonymised in advance of our analysis. Moreover, the climatological datasets are publicly available. Thus, we did not require patient consent. The Medical Ethics Committees at the Graduate School of Medicine, Hokkaido University and the Hokkaido Cancer Center approved this study.Bacillus spp. Of these, 42 (80.7%) were B. cereus, 4 (7.7%) were B. subtilis, 2 (3.8%) were B. megaterium, and 4 (7.7%) were reported as Bacillus spp. . The mean and standard deviation of the age were 62.9 and 14.7 years, respectively. Twenty-three (44.2%) were male. Bacillus spp. in contrast to the daily temperature, as well as the absolute humidity. Overall, 26 (50.0%) Bacillus-positive blood cultures occurred from June to September, i.e., during the summer season. The two clinical wards that patients were most frequently admitted to were gastroenterology and urology . Individual patients’ background data are available as From 2011 to 2016, a total of 52 blood samples tested positive for Bacillus-positive blood cultures are identified by ensuring routine testing with a multiple set culture, and in fact, the very high frequency of single set culture was seen during the period from 2011–2012 (ranging from 75 to 90%). Recommendation for sampling multiple sets of blood for culture was issued in 2013, and the recent multiple sampling rate was as high as 92.0%.Bacillus spp.; however, the univariate association could still reflect them as seasonal confounders. Including 2011–2012 data which did not frequently implement multiple set sampling, temperature was still a weak predictor (p = 0.05), but humidity was not significantly associated with the occurrence of Bacillus-positive culture (p = 0.74).Using temperature and/or absolute humidity with variable time lags, we computed all possible models and compared AIC values. Bacillus spp. Of a total of 43 events of Bacillus-positive blood cultures, 30 events (69.8%) were successfully predicted by our environment-based prediction model. Using temperature only, Bacillus-positive samples in 2015–2016 and 2016, a total of three and eight events were successfully predicted. Thus, success rates were 13.6%  and 66.7% , respectively, in 2015–2016 and 2016.To avoid overly optimistic results, we predicted 2015–2016 and 2016 data using the training data up to the end of 2014 and 2015, respectively. Out of the total of 22 and 12 events with Bacillus spp. from blood culture using the hazard-based counting process model. The dataset was derived from a designated cancer hospital in Sapporo, Japan, in which sampling multiple sets of blood culture has been issued since 2013 and an annual cyclical pattern of the incidence of Bacillus-positive blood culture has been observed. To model Bacillus-positive blood culture, we explored the predictive performance of climatological variables, including temperature and absolute humidity. Based on model comparison using penalized likelihood, we have shown that the mean temperature with a time lag of six days would optimally predict the incidence of Bacillus spp. from blood culture, irrespective of true bacteraemia or contamination.The present study explored the predicted risk of detecting Bacillus-positive blood culture. A published study indicated the presence of seasonality in the frequency of Bacillus-positive blood culture and identified a possible association with high temperature [Bacillus spp. While the environmental driver was identified, the predictive performance was limited with the calculated AUC at around 0.6. Moreover, when we sequentially predicted the incidence in 2015–2016 and 2016 using the rest of time as the training data, the success rate appeared to be very small. Thus, the predictive performance of the resulting model was weak, and the biggest contribution was the identification of temperature itself.To our knowledge, the present study is the first to explicitly identify a climatological driver of perature . While tBacillus bacteraemia is catheter-related BSI. Thus, risk factors of Bacillus bacteraemia that have been investigated include the presence of a central venous catheter [Bacillus spp. in patient rooms would act as the most probable driver of Bacillus-positive blood culture. In fact, Bacillus outbreaks that were associated with the contamination of reusable towels and linens have been reported [Bacillus outbreaks were associated with hospital reconstruction or renovation activities [Bacillus spp. and environmental predictors was not caused by coincidence of construction.The most common clinical presentation of catheter , prior ucatheter , and usacatheter . Becausereported ,19. Whiltivities ,20,21,22Bacillus-positive blood cultures and temperature would be crucial for preventing bacteraemia, as well as blood culture contamination. As part of infection control activities, healthcare workers should be informed of our finding to be aware of the elevated risk of infection during and following warm days. Second, the association between temperature and Bacillus-positive blood culture would also imply the importance of controlling temperature at caregiving settings, e.g., the use of air conditioning systems during the summer season.Our findings involve important clinical implications. First, awareness of the association between Bacillus-positive blood culture was consistent with published studies, and we believe that the plausibility is well supported. Second, our study relied on observation at a single healthcare centre; thus, representativeness may be limited and our findings may not be directly applicable to all other settings. Third, environmental variables were observed at an outer environment in Sapporo. However, ordinary clinical wards of our subject cancer hospital, built in the 1970s, have not been equipped with air conditioning facilities  and windows are continuously opened to lower in-room temperature during the summer season.The present study has some limitations. First, this study was conducted in a retrospective manner with a relatively small sample size. As such, the level of evidence is limited. Moreover, sampling error cannot be immediately overcome. Nevertheless, our finding of the seasonality and identification of temperature associated with the incidence of Bacillus-positive blood cultures exhibit an annual cyclical pattern with high incidence during the summer season, and the incidence was weakly but validly predicted by mean temperature in Sapporo. We believe that our statistical modelling exercise offers an important clue for infection control practice to improve awareness among healthcare workers of the identified association and mechanically controlled in-room temperature.In conclusion, we have shown that Bacillus-positive blood culture in Sapporo, Japan, by employing a hazard-based statistical model. Using temperature and/or absolute humidity with variable time lags, we computed all possible models. Based on model comparisons, the temperature-only model with a lag of six days yielded a high sensitivity value (72.1%) and appeared to be the optimal model to predict Bacillus spp. with the highest AUC value. Our statistical modelling exercise offers an important message for infection control practice to improve awareness among healthcare workers of the identified association and mechanically controlled in-room temperature.The present study aimed to epidemiologically identify climatological variables that are associated with


From more than 40 years African swine fever (ASF) is endemic in Sardinia. Historically, areas at higher risk are located throughout some inland parts of this island where domestic pigs are still illegally kept in semi-wild conditions, living in contact with the local wild boar population, thereby creating perfect conditions for disease endemicity. A new eradication plan (EP-ASF15-18) has been ongoing for the past 3 years, based on a comprehensive strategy adapted to the local situation and focused on strong actions on domestic pig farms, wild boars (WB), and the third Sardinian typical involved population [illegal free-ranging pigs (FRPs)]. A fundamental aspect of the plan is the classification of pig farms as “controlled” or “certified,” based on clinical, structural, and biosecurity characteristics. The eradication plan also provides for strong action against illegal farms and pig meat marketing channels. In addition, this plan establishes specific control measures for WB hunting and ASF checks. Each control strategy is specifically based on municipality risk level, to focus actions and resources on areas at higher risk of endemic or re-emerging ASF. Thus, precise risk classification is fundamental to this goal. The aim of the present work was to establish an ASF risk index, to provide a summary measure of the risk level in the Sardinian municipalities. This synthetic measure can express the different aspects of a multidimensional phenomenon with a single numerical value, facilitating territorial and temporal comparisons. To this end, retrospective data (years 2011–2018) were used. The ASF risk index is the result of the algorithmic combination of numerical elementary indicators: disease prevalence in the suid populations, WB compliance with EP-ASF15-18, domestic pig compliance with EP-ASF15-18, and presence of FRPs. A negative binomial regression model has been applied and predictors calculated to obtain a risk index for each municipality. The result of the risk analysis was discussed and considered according to expert opinion and consensus. The results of this study, expressed as risk score and classified into five risk levels, can be used to help define actions to be carried out in each Sardinian municipality, according to the risk assessment for the territory. Asfivirus), and characterized by up to 100% mortality (African swine fever (ASF) is one of the most serious infectious diseases affecting domestic and wild pigs, responsible for serious economic and production losses . ASF is ortality . The conortality . The quaortality , 5. At tortality . Since 2ortality , 8. Starortality . More reortality .Forty years have passed since ASF entered Sardinia, probably owing to the upon arrival of processed meat contaminated by African swine fever virus (ASFV) from the Iberian Peninsula . The conOrnithodorus and other natural reservoirs, such as the warthog (Phacochoerus africanus) , hunting management, and observance of rules governing animal identification and registration. Given that the spread of ASFV in DPs is facilitated by human activities and animal movement , as demonstrated in many studies  –22, whicricanus) , 24, are studies , 25–27,  studies . An excl studies , 29, and studies . Notwith studies . AlthougFrom the first ASF notification in Sardinia several eradication plans have been put in place at regional level, with special focus on DPs and WBs populations. From the first eradication program in 1982, many others have been carried out, with widely varying results. Some of these were able to came close to the ASF eradication, but none was able to solve the problem presented by FRPs, which in Sardinia have a key role in the spread and persistence of disease –19, 31. 2, located in the middle of the Mediterranean Sea (40°03′N 9°05′E). The island characterized by various ecosystems, mounts, woodlands, lowlands, largely uninhabited areas, rivers, long sandy beaches, and rocky coasts. Sardinia is administratively divided into 377 municipal territories, covered by eight different Local Socio-Sanitary Areas (ASSL).Sardinia island has an average area of 24,000 km2) and the presence of pristine areas, 48% of the island is used for pastoral and agricultural activities; of this proportion, 60% is used for breeding sector, 35% for planting, and the remainder for wood cultivation  were as follows: the number of SVDPs for ASF; the number of registered and active farms, including those active throughout the year (activity start date January 1 or later and end date not before December 31); the number of pigs, using data from March 31 as this is the date of the official census; data of animal movement . These data were collected from the official veterinarian databases: the Italian Veterinarian National Database (BDN), Veterinary Information Systems of the Italian Ministry of Health (VETINFO), and SIMAN. All data collected have been verified on the globally official site for animal health disease , taking into account the possible inconsistencies due to different update time between Italian national database and OIE international database. The number of official veterinarian checks on pig farms was determined, to calculate the percentage of compliance among DPs. From 2015, this measure is largely used in Sardinia to evaluate the performance of DP farms in terms of ASF management  and included data regarding to the three suid populations involved in ASF persistence: DPs, WBs, and FRPs. Each of the 377 Sardinian municipalities was considered as epidemiological unit, arranged by study year and linked to all 59 variables collected . For thenagement . This menagement . Using dnagement . Furthernagement , all WBsnagement , 38, 43 nagement , 45, we nagement . To descnagement  were colnagement ; these iad-hoc database has been created using Microsoft Office Access system and all information collected was double blinded and password-protected stored to ensure privacy. Extensive data checking was performed to evaluate the consistency and accuracy of the data collected and any disagreement was analyzed and corrected. Considering epidemiological, experimental, and statistical issues , several putative and potentially relevant predictors were detected. The baseline distribution of each explanatory variable was summarized and described, according to municipalities with zero or more/equal to at least one case of ASF  <10  <10 –57. InteVIF) <10 –61. The VIF) <10 . An asseVIF) <10 . The munp-value level of 0.05 or less has been considered significant. The statistical analyses were made with R Version 3.3.2  and Stata 13 Release 13 .n = 8) and municipality (n = 377). The ad-hoc database contained a total 3,016 records. Only 127 records represented municipalities with SVDP cases; all others were equal to a zero value . Data were collected by year . Twenty one of these were finally included, The results obtained by the analysis of the 21 variables are presented in p-values. The number of farms and pigs in each municipality revealed a role of significant risk factors favoring ASF outbreak, with ORadj 1.013 (95% CI: 1.007–1.025), p < 0.001 and ORadj of 2.581 (95% CI: 1.314–5.067), p = 0.006 with ≥ 120 pigs on the farm. However, increased farm and veterinary check compliance with the EP-ASF15-18 the previous year significantly decreased the probability of counting one case or more in the same municipality the following year . The final results of the NBRM showed that a total of eight different features related to the WB population that was live and/or tested positive in the previous hunting season significantly contributed to the risk of ASF cases (p < 0.05). In particular, the effect was equal to 1% greater risk  for each WB estimated to live in the same municipality, and 20%  and 15% greater risk  if hunted and tested WBs were ASFV positive or ASF-antibody positive, respectively. In addition, an increasing percentage of male WBs that were virus positive and seropositive increased the risk of new SVDPs in the same municipality the following year by 1%  and 1.7% , respectively. Likewise, the probability grew about 2%  if positivity (virus or seropositivity) was found in young animals (between age 0 and 6 months). As well as the effect found for DP compliance, compliance with hunting season management rules was a protective factor against the risk of SVDPs the following year. In particular, when WB compliance was greater than 20%, the risk was significantly lowered by 40% . As hypothesized, the presence of FRPs in the same municipality increased the risk of SVDPs fivefold , and about twice if a positive WB was found the previous year in the same municipality as FRPs . Older age of farmers seemed to be protective upon an increased number of outbreaks  whereas the opposite effect was seen for male vs. female sex of the farmer . Comparison between the first MDI level (lower deprivation) and others, suggested an increased probability of ASF outbreaks on farms, with statistically significant results between MDI level-1 and MDI level-3 (medium deprivation level)  or MDI level-5 (very deprived level) . Regarding to ISTAT socioeconomic indicators, a low probability of outbreaks on farms located within municipalities with high employment rates  has been highlighted by the NBRM. Higher counts of outcome variables were observed with higher rates of micro-criminality and tourism in the low season: ORadj = 1.432; 95% CI: 1.418–1.469; p < 0.0001 and ORadj = 1.196; 95% CI: 1.081–1.325; p = 0.001, respectively. Finally, regions with asphalted road area of more than 70,000 m2, high forest surface coverage, and a human population of <5000 people showed a significantly higher risk of SVDPs (p < 0.05). The results of the likelihood ratio test  supported the choice of the mixed-effects NBRM against the mixed-effects Poisson regression model. Based on internal validation criteria  and external validation criteria , it is possibile to affirm that the NBRM could properly predict the correct outcome with a strong goodness-of-fit.Overall, 50 variables collected for the period of interest, were considered for inclusion into the final mixed-effects NBRM . TotallySardinia is the European area that has been affected by ASF the longest, since its first notification in 1978. Furthermore, Sardinian territory is the only region “where the epidemiological situation has become stabilized and the disease has become endemic,” such that the region is the only one on the European continent included in Part IV (highest risk) of the European Commission Decision on ASF control measures (Decision 2014/709/EU). Despite the rapid spread of ASFV across Europe and parts of the Asian continent, excluding an isolated and quickly resolved case in northern Italy in 1983 owing to illegal introduction of pork from Sardinia, there is no evidence of disease spread from Sardinia to other countries . As demoAll datasets generated for this study are included in the manuscript/FL, SC, AC, and SR: conceptualization, investigation, writing—original draft, review, and editing. FL: data curation, formal analysis, and validation. FL and SR: methodology. SR and AC: project administration, resources, and supervision. SC and FL: visualization.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.


Increasing proportions of people, including older adults, live alone. Studying living arrangements of the elderly is important because these affect and reflect general well-being of the elderly and inform communities’ response to elderly housing needs. We analyze data from the 2006 Canadian Census and the 2006 American Community Survey to examine living alone among non-married older adults aged 55 and older in Canada and the U.S. The paper has two parts. First, we compare native- and foreign-born elderly to see if immigrants are less likely to live alone. Second, we examine factors associated with living alone among older immigrants. While older immigrants in both countries are less likely to live alone, the large differences are substantially reduced once various explanatory variables are considered. Comparisons of four gender/country groups of older immigrants show the positive role of economic and acculturation factors on living alone among older immigrants. With few exceptions, predictors of living alone are similar for older immigrants in Canada and the U.S.: living alone is mainly explained by a combination of economic and acculturation factors, taking demographic variables into account. Findings underline the need for age-friendly housing with innovative design and technology that can accommodate older people who live alone, including older immigrants who may have different needs and cultural preferences.  Studying the living arrangements of older or elderly populations is important for several reasons. First, living arrangements affect and reflect family type and household structure among older people. These are in turn related to social support, inter-generational relations, health status, social isolation, satisfaction with life, and general wellbeing ,2,3,4,5.Second, another reason for studying elderly living arrangements pertain to the idea of “age-friendly communities” that incorporate suitable physical environments, including housing, transportation services, and home modification programs with innovative assistive technology and designs, with a supportive social environment that promotes positive social relations for older residents ,6. OlderFactors that influence living arrangements of the elderly include preferences and resources that people have, and health and other constraints as they age. Residential options for older people include living alone if not married or partnered; living with spouse or partner only if married or partnered; co-residence with family members or extended family living; co-residence with non-family members; and institutional living, including retirement homes and assisted living facilities. Researchers often refer to the first two types of living arrangements  as residential independence or independent living arrangements ,8,9. In There has been a rise in independent living arrangements, as defined above, among older people in many countries, particularly in the west and more developed countries ,11,12,13Over the past fifty years, there have been absolute and relative increases in the number of Canadian elderly people in independent living arrangements, mainly for married or partnered couples to live with spouse or partner only ,5,14. ThLiving arrangement patterns for the U.S. population aged 65 and older are fairly similar. Data for 2012 show that about 59 percent lived with a spouse or unmarried partner only and another 28.5 percent lived alone. Together, almost 88 percent of the population aged 65 and older were in independent living arrangements  , public and private sectors can use this information to promote age-friendly housing and communities . The concept of “environmental gerontology” highlights the need for a multi-disciplinary approach to designing neighborhoods and housing that facilitate “mobility, independence, and quality of life of older people living in the community”  (116).Finally, this study is the first comparative analysis of living alone among older immigrants in Canada and the U.S., offering a useful comparison of two large immigrant-receiving countries that have older immigrants from many different countries of origin. While Canada and the U.S. are distinctive in many ways, both countries have long histories of immigration with relatively high levels of current immigration from diverse source countries. In the following section, we discuss why a comparative study of Canada and the U.S. can be especially productive in advancing understanding of living alone among older immigrants.While there are distinctive challenges in cross-country research, including the need for comparable variables and sensitivity to historical and contextual differences, a comparative analysis can advance knowledge in many unique ways. A comparative analysis has the advantage of allowing researchers to conduct similar analyses using different data from the countries selected to identify similarities and differences in how various factors and characteristics affect the outcome being examined. If the influence of particular factors is similar, this increases confidence in the validity and reliability of the findings.Comparing Canada and the U.S. for this analysis is not simply based on the fact that they are North American neighbors with a long mostly amicable joint history, and that the two countries have been strong allies in modern times. Canada and the U.S. are far from “two peas in a pod” , but besFirst, immigration has always been a major factor in both countries, historically and today. Canada and the U.S. are among the leading destinations for global migration streams in recent decades . Both coHowever, we should note differences in immigration policies and systems. Canada has a selective points-based immigration system whereby potential immigrants are screened based on such human capital characteristics as age, education, English and/or French language proficiency , and adaptability whereas the U.S. immigration system is primarily based on family reunification. While the U.S. continues to admit more immigrants than other major immigrant destination countries , immigraSecond, population aging is another demographic trend shared by both countries. The Canadian population is aging, indicated by increased median age of the population from 26.2 in 1971 to 40.0 in 2011 . ElderlySimilar population aging trends are observed in the U.S. . The medAging-in-place of younger foreign-born cohorts and the immigration of older immigrants contribute to the growth of the elderly immigrant population in each country. While both the Canadian and U.S. populations are aging, and aging immigrants are part of this demographic trend, statistics cited above show that Canada’s population is older and elderly immigrants are a larger proportion of its elderly population. Third, closely related to the role of immigration is the expanded racial, ethnic, and cultural diversity of the Canadian and U.S. populations. Mainly because of immigration from Asia and other non-traditional  sources in recent decades, Canada’s population has evolved from one dominated by the two founding peoples (British and French) and the indigenous  population to the current situation where over two hundred ethnic origins were reported, and thirteen different ethnic origins had one million or more responses . Employment Equity Act of Canada defines visible minorities as ‘persons, other than Aboriginal persons, who are non-Caucasian in race or non-white in colour’. The visible minority population consists mainly of the following groups: South Asian, Chinese, Black, Filipino, Latin American, Arab, Southeast Asian, Korean, and Japanese [In 2011, close to 20 percent (19.1 percent) of Canada’s population identified as members of “visible minority” groups, that is, racial minority groups other than Aboriginal peoples. The Japanese .The U.S. population has also been transformed by increased racial and ethnic diversity, also closely related to immigration. The main group, non-Hispanic White, has been slowly declining as a percent of the total population, to 63.7 percent in 2010, down from 69.1 percent in 2000. This means that other racial groups and Hispanics have been increasing in numbers and proportions, and together comprise about 36 percent of the total U.S. population in 2010 . Asians were the fastest growing racial minority between 2000 and 2010, but account for just 5.6 percent of the population in 2010. The most notable change has been the growth of the Hispanic or Latino population to become the largest minority population in the U.S. since the 1990s. Hispanics are now 16.3 percent of the U.S. population compared with 13.6 percent Black and 5.6 percent Asian, the other main racial minority groups . While both Canada and the U.S. have become more racial and ethnically diverse, there are important differences, including the larger share of racial/ethnic minority populations in the U.S. (36 percent), compared with 19 percent of visible minority groups in Canada, and the large presence of Hispanics in the U.S. We analyze data from two data sets. Data for Canada are from the Public Use Microdata File (PUMF) on individuals in the 2006 Census of Canada (see  for detaU.S. data are from the 2006 American Community Survey (ACS) Public Use Microdata Sample (PUMS)  does not always imply declining economic or health status. Many statistics on the elderly refer to persons aged 65 and older, or persons aged 55 and older. In this study, we use age 55 in order to show more clearly potential differences that occur between age groups and to cover a wider age range at the “older” ages. Given the outcome variable—living alone—we exclude persons who are married or living common-law  or co-habiting. We include men and women, who are never married, separated, divorced, or widowed. We exclude older persons living in group quarters. Recent statistics show that for the population aged 65 and older, over 92 percent in Canada live in private households , and theWe identify Canadian-born or U.S.-born elderly and immigrants from responses to the questions on citizenship at birth and place of birth. Persons who are Canadian or U.S. citizens at birth are considered native-born while persons who are not Canadian or U.S. citizens at birth are considered immigrants. This avoids including persons born abroad to Canadian or U.S. citizens as immigrants (based on foreign place of birth) as these persons are not considered immigrants in Canada or the U.S., respectively.Variables included in the analysis are as directly comparable as possible across the two data sets. We note where it is not possible to develop directly comparable categories for some variables.The outcome variable, living alone, is coded as a binary variable  based on responses to questions on household type, family structure, and individual family status.We include explanatory variables that previous research had shown to influence living arrangements of older adults. Expected results are based on previous research and published statistics. Age is coded in seven age groups, 55–59, 60–64, 65–69, 70–74, 75–79, 80–84, and 85 years or older for descriptive analysis. We use 5-year age groups because 2006 Canadian census public-use microdata are limited to five-year age groups. Age is recoded into a continuous variable in the multivariate analyses. We use the mid-point of each 5-year age category to assign age values for the Canadian sample .Statistics on living alone from Statistics Canada  and the Gender is a binary variable . Living alone is expected to be more likely among women because of women’s longer longevity. However, this may not be the case once other factors are considered.Marital status includes four non-married categories: divorced, separated, widowed, and never-married. Living alone may be more likely among widowed adults.The influence of economic variables is expected to be positive, as living alone requires adequate income to pay for a housing unit that is inhabited by only one person. Education is coded in five categories: less than high school, high school graduate, some post-high school education but less than a Bachelor’s degree, Bachelor’s degree, and post-Bachelor’s degree. Education is expected to have a positive influence on living alone.Individual income is coded in six categories: less than $10,000; $10,000–19,999; $20,000–39,999; $40,000–59,999; $60,000–99,999; $100,000 and over for descriptive analysis. Income and other monetary variables are measured using Canadian dollars in the Canadian sample and U.S. dollars in the U.S. sample. In 2006, the Bank of Canada exchange rate was around US $1 equals CAD 1.14.In the multivariate analyses, individual income is a continuous variable. Living alone is expected to increase with income.Government retirement income is a binary variable (0 = received less than $100 in government retirement income during the past year or 1 = received $100 or more in government retirement income during the past year). In Canada, government retirement income refers to benefits from the Canada or Quebec Pension Plan. In the U.S., government retirement income refers to payments and benefits from the Social Security Administration. Having government retirement income increases the likelihood of living alone.Guaranteed retirement income is a binary variable . Guaranteed retirement income refers to regular income received from being a member of an employer’s pension plan, payments from individual annuities, private pensions paid to widows or widowers, pensions of retired civil servants, and other annuities paid to individuals by a private insurance company. Having guaranteed retirement income is expected to increase the chances of living alone.Homeownership is a binary variable (0 = does not own home or 1 = own home). Homeownership’s influence is expected to be positive on living alone because owning a home implies having sufficient economic resources to own a home. In addition, homeownership facilitates living alone, removing the need to look for alternative housing in the event of marital dissolution or widowhood.Culture and acculturation are closely related but distinct concepts. Ethnic origin, language background, religion, and other characteristics are usually used to indicate cultural background. Given differences across ethnic groups on other characteristics such as marriage, fertility, and family patterns, ethnicity has frequently been used as a sociodemographic variable to indirectly measure these differences. We describe ethnic origin as a demographic variable in the descriptive analysis. For immigrants, acculturation is usually indicated by duration of residence in the host country and proficiency in host country language ,43,44. Ethnic origin is coded using fifteen groups. These are “American” (in the U.S. sample) or “Canadian” (in the Canadian sample); British; French; Other European; Arab or Middle Eastern; South Asian/Asian Indian; Chinese; Filipino; Korean; Vietnamese; Other Asian; Latin American/Latino/Hispanic; African, Black, or Caribbean; Other single ethnic origins ; and Multiple ethnic origins. Persons reporting ethnic origins that are culturally closer to mainstream American or Canadian culture  are more likely to live alone.For immigrants, knowledge of official languages (in Canada) or proficiency in English (in the U.S.) is coded using four categories. Besides being an indirect indicator of acculturation, language knowledge or proficiency implies an ability to communicate and navigate social and other situations and understanding of broader societal norms.The four categories range from excellent to poor competence in Canada’s two official languages (English and French) or in English (for the U.S. sample), although the specific definitions differ for Canada and the U.S. For the Canadian sample, the four categories are (1) English or French mother tongue, and English or French home language; (2) other mother tongue, and English or French home language; (3) other mother tongue, and other home language, knows English or French; and (4) other mother tongue, other home language, and does not know English or French. For the U.S. sample, the four categories are: (1) only speaks English; (2) speaks English very well; (3) speaks English well or not well; and (4) does not speak English. Cultural closeness to the host country and/or acculturation decreases from category 1 to 4 for both the Canadian and U.S. samples, and living alone is expected to decrease from the first to the fourth category of the language variable. Duration of residence for immigrants measures how many years immigrants have resided in Canada or the U.S. It is coded in five categories for descriptive analysis, from 0–9, 10–19, 20–29, 30–39, and 40 years or more. In the multivariate analyses, duration of residence is a continuous variable. Duration of residence is expected to have a positive influence on living alone as increased duration implies greater acculturation.Place of residence indicates metropolitan and non-metropolitan residence, and residence in several specific Canadian and U.S. cities. Metropolitan categories for this variable include three Canadian and five U.S. cities with the largest number of older immigrants. We include only three Canadian cities because immigrants in Canada are highly concentrated in them: 63.4 percent of Canada’s immigrants reside in these three cities .The codes for place of residence are (1) Montreal (Canada) or Chicago (U.S.); (2) Toronto (Canada) or Los Angeles (U.S.); (3) Vancouver (Canada) or Miami (U.S.); (4) New York City; (5) San Francisco; (6) other metropolitan areas; and (7) non-metropolitan areas. Living alone is expected to be higher in non-metropolitan areas because of lower cost of housing which facilitates independent living arrangements, including living alone.We begin with descriptive analyses to describe and compare the study samples. For multivariate analyses, we use Stata 12 statistical software  to estimFor the second research question on predictors of living alone among older immigrants, we limit analysis to older immigrants only, and estimate a logistic regression model of living alone (Model III) for four groups: females in Canada, males in Canada, females in U.S., and males in U.S., to identify and compare predictors of living alone among older immigrants. Model III includes duration of residence, in addition to other explanatory variables.For interpreting the logistic regression results, we calculate predicted probabilities for each explanatory variable using the margins command in Stata 12 . The preSelected characteristics of the Canadian and U.S. study samples are shown in Gender: There are more females in both samples, about 69 percent. There are also more women in the immigrant samples, at around 73–74 percent in both the Canadian and U.S. samples. Age: The distribution across age categories is as expected, with higher percents in the younger age categories. The U.S. immigrant sample has higher percents in the younger age categories. Marital Status: Marital status refers to non-married categories only. Being widowed is the most common marital status for both Canadian and U.S. samples, with a higher percent widowed among immigrants in Canada. The percent divorced is higher among the native-born in both samples. Ethnic Origin: For describing the sample, ethnic origin is considered a demographic characteristic. Most native-born Canadian and U.S. elderly report European or multiple origins but immigrants are distributed over a wider range of ethnic origins compared with the native-born. There are two striking differences between the Canadian and U.S. immigrant samples. First, older immigrants in Canada have higher percentages of people reporting European origins (72.8 percent—this percent includes the 25 percent reporting multiple origins), compared with 33.8 percent (including the 5.9 percent reporting multiple origins) in the U.S. sample. Studies of ethnic origin in Canada show that persons reporting multiple origins are mainly reporting “Canadian” in combination with other European origins, and persons reporting “Canadian” used to report European origins, particularly French or British ,48,49. SEducation: On average, older immigrants in Canada have more years of schooling compared to Canadian-born elderly (a mean of 12.1 years versus 11.5 years) whereas in the U.S., older immigrants have fewer years of schooling, with a mean of 10.3 years versus 12.3 years for the U.S.-born. The distribution across levels of educational attainment of Canadian-born and immigrant elderly is generally quite similar but higher percents of immigrants in the U.S. sample are in the lower educational categories. Income: In both samples, older immigrants have lower mean incomes, and the percent of immigrants in the two lowest income categories exceeds that of the native-born in both countries. Homeownership: Homeownership is higher for the U.S. sample (70 percent are homeowners compared with 62 percent for the Canadian sample). However, immigrants in Canada are more likely to own their homes , while immigrants in the U.S. sample are less likely to be homeowners .Metropolitan Residence: Notably higher percentages of elderly immigrants in both samples reside in metropolitan areas. The metropolitan concentration of older immigrants in the U.S. is higher, at 94 percent, compared with 86 percent in Canada. U.S.-born elderly are also more likely to reside in metropolitan areas (73 percent) compared with Canadian-born elderly (at 59 percent).There are two immigrant-specific characteristics in Duration of Residence: On average, older immigrants in Canada have resided in Canada for 37.7 years compared with 34.8 years for older immigrants in the U.S. Higher percentages of elderly immigrants in the Canadian sample have resided in Canada for 40 or more years (43.8 percent) compared with 36.4 percent of immigrants in the U.S. sample. More immigrants in the U.S. sample are recent arrivals: 10.7 percent have been in the U.S. for less than 10 years, compared with 5.5 percent of immigrants in the Canadian sample. Language Proficiency: As noted earlier in the section describing variables and in Logistic regression results from Models I and II focus on the role of nativity on living alone. We begin with results from Model I, which was estimated separately for Canada and the U.S. Complete logistic regression results for Model I are in While the role of nativity is statistically significant , the observed large gaps in living alone between native- and foreign-born elderly are substantially reduced once other factors in the equation are taken into account. Specifically, the observed difference of 15.9 percent between Canadian-born and immigrant elderly living alone is reduced to 3.4 percent, and the observed difference of 21.5 percent between U.S.-born and immigrant elderly is reduced to 1.6 percent. This shows that differences in living alone between older native-born and immigrants are modest, once all other factors in Model I are considered.Results for Model II, estimated for four groups, Canada/female, Canada/male, U.S./female, and U.S./male, are shown in Model III is estimated for older immigrants only and addresses the second research question: what factors are associated with living alone among older immigrants? Complete results from estimating Model III for Canada/female, Canada/male, U.S./female, and U.S./male are shown in Age: Age is an important factor in living arrangements among older adults because of age-related health declines which can be expected to influence the ability to live alone ,11,18. TMarital Status: The results for marital status are shown in Panel A, Among female immigrants in the U.S., those who are separated are least likely to live alone while divorced female immigrants are most likely to live alone. Among males, widowed males have the lowest proportion living alone, while divorced and never-married males have the highest proportions living alone. In both samples, divorced older immigrants have higher proportions living alone. Individual income: Individual income results are shown in As expected, the proportions living alone increase with income, with the sharpest increase observed for females in the U.S. The influence of other economic factors are shown in Panel B, Government and private retirement income: Older immigrants who have government or private retirement income are more likely to live alone. These results hold across all groups, but the impacts are larger for males in both Canada and the U.S.Home ownership: Older immigrants who are homeowners are more likely to live alone, a pattern observed for all four gender/country groups. Older male immigrants in Canada who are homeowners have the highest proportion living alone.Educational Attainment: The proportions living alone increase for all four groups as educational attainment increases. The proportions living alone are higher among males in both countries at each level of educational attainment.The results for ethnic origin and language proficiency are shown in Panel C, Ethnic Origin: The influence of ethnic origin is not statistically significant for almost all ethnic origins. We interpret the influence of ethnic origin as a largely cultural and acculturation variable, but acknowledge that ethnic origin relates to other characteristics that can also influence living alone, such as ethnic group differences in marriage and fertility patterns, which relate to family size and availability of family for elderly co-residence. The lack of statistically significant results for all but a few ethnic origins suggests that this may not be an adequate proxy for characteristics that affect living alone among older immigrants.However, ethnic group differences in living alone do generally support cultural expectations: older immigrants of European backgrounds are culturally closer to “mainstream” Canadian and U.S. culture, and higher proportions of these groups live alone. In contrast, lower proportions of older immigrants reporting Asian, Latin American, and other non-European origins live alone. Older female immigrants of all ethnic origins in both Canada and the U.S. are less likely to live alone than male co-ethnics . Language Proficiency: This variable indicates high to low linguistic and related acculturation (from Category 1 to Category 4) and its influence is as expected. The proportions living alone decrease from Category 1 to Category 4 for both samples and for both genders. For example, 60 percent of older female immigrants in Canada in Category 1 live alone, compared with 43 percent of female immigrants coded Category 4. The difference by linguistic acculturation is larger among males: 72 percent of male immigrants in Canada classified in Category 1 live alone versus 53 percent of males classified in Category 4. Duration of Residence: Acculturation is also indicated by duration of residence, increasing as years of residence increase. As expected, living alone among older immigrants increases with duration of residence, shown in The increase is sharper among female immigrants in both Canada and the U.S., and immigrants in Canada  have higher proportions living alone compared to their U.S. counterparts at all values of duration of residence. Place of Residence: Older immigrants who live in non-metropolitan areas are more likely to live alone, a pattern that is similar for all four sub-groups. Lower proportions of older immigrants in Canada who reside in one of Canada’s three largest immigrant destination cities  live alone, compared to immigrants who live in other metropolitan and non-metropolitan areas. For older immigrants in the U.S., a similar pattern holds, except for those who reside in Miami where the proportion living alone (69 percent) is quite close to the percent living alone in non-metropolitan areas. We return to our two research questions in this section. The first question focuses on the role of nativity and asks whether non-married older immigrants are less likely than Canadian- or U.S.-born non-married elderly to live alone. Descriptive results show that older immigrants in Canada and the U.S. are much less likely to live alone than native-born elderly. The difference is larger in the U.S. This finding is consistent with other research showing lower rates of independent living arrangements, including living alone, among immigrants, including older immigrants ,24,43. However, once appropriate factors are taken into account, nativity differences, while still statistically significant, are substantially reduced. Differences by gender and nativity are also reduced or become modest once various factors are considered. These findings suggest that aggregate differences in living arrangements between older immigrants and native-born elderly are largely due to differences in demographic, economic, and acculturation characteristics between the older native-born and immigrant populations. Findings show that notable proportions of older adults, including immigrants, in both countries live alone, reinforcing the need to consider these groups when discussing age-friendly communities.Our second research question is directed at older immigrants and examines predictors of living alone among non-married older immigrants in Canada and the U.S. The main findings show higher levels of living alone for older male immigrants in both Canada and the U.S., across different characteristics, including age, marital status, income, education, and duration of residence. With some exceptions, the proportion living alone is higher among immigrants in Canada across different characteristics. Factors influencing living alone are generally similar for older immigrants in Canada and the U.S., suggesting that living alone among older immigrants is mainly explained by a combination of economic and acculturation factors, after taking demographic variables into account. More acculturated older immigrants, and immigrants with more economic resources, are more likely to live alone, findings that are consistent with previous studies on extended living arrangements among older immigrants: the predictors of living alone are opposite to those for extended living arrangements where less acculturated older immigrants with fewer economic resources are more likely to co-reside ,50.Aggregate statistics on older people living alone contribute to widespread beliefs and images that older women are more likely to live alone. It is of course correct that higher proportions of older women live alone, as shown in statistics from many countries ,18,20, aAnother widespread image of elderly people who live alone is that of elderly widows living alone. Again, this is not entirely wrong, given women’s longer longevity and the common age gap between spouses. However, once appropriate factors are taken into account, older immigrants who are widowed are not the most likely group to live alone, compared to other marital status groups. It is possible that widowed older immigrants have adult children with whom they can co-reside following widowhood, an option that may be unavailable to divorced, separated, and never-married older immigrants. Divorced and separated older immigrants are more likely to live alone than the widowed, and in the U.S., older male immigrants who are never-married are as likely as divorced males to live alone. Marital disruptions due to divorce is therefore a better predictor of living alone among older immigrants than widowhood. Marital disruptions can also be associated with other forms of disruptions such as moving away, which also disrupts previous family and social networks, thereby increasing the chances of living alone. Unfortunately, we are unable to examine the role of geographical mobility as well as other factors such as gendered differences in cultural norms about living alone and subjective preference for living alone with the data examined in this paper. This paper contributes to the literature on living alone and housing in two ways. First, we show that notable percentages of older adults, including older immigrants, in two large countries with aging populations, live alone. This trend is expected to continue and reinforces the need for more private and public policies to design and build age-friendly communities that allow older adults to continue to live independently and participate fully in their community. Second, the findings show that once appropriate factors are taken into account, there are only modest differences between native-born and immigrant elderly’s likelihood of living alone. This suggests that elderly immigrants should be included in housing initiatives that include more units geared towards elderly living alone, instead of mistakenly assuming, based on aggregate statistics, that elderly immigrants are somehow different, and are less likely to live alone and do not need to be included in these efforts.Many countries have already implemented initiatives on age-friendly communities in response to population aging and the rise in independent living arrangements among the elderly, including living alone ,18,20. SAdditionally, older immigrants may have different cultural preferences in housing design and use of technology. For example, Chinese immigrants may place great importance on the role of feng shui in housing alignment and design, immigrants may be less familiar with advanced technology, and some immigrants’ accented speech may pose challenges for voice-recognition software. These potential differences would have to be considered in elderly housing designs and use of technology in planning age-friendly housing. While this study has produced some new and useful findings, we note several limitations. First, this is a cross-sectional analysis, and findings refer only to the period when the data were collected in 2006. We do not know if the living arrangement recorded at time of data collection is temporary or permanent, and the findings cannot speak to trends in factors related to living alone. Second, the sampling frame for both data sets are private households and individuals and families in private households. This misses the population in group or institutional living quarters, an important limitation for studying the elderly. As health declines accelerate with increased age, the oldest old are less likely to live alone in private households and more likely to be in group housing such as retirement or assisted living housing. This limitation may be implicated in the finding of age’s negative influence on living alone among U.S. female older immigrants. Third, the outcome, living alone, poses some conceptual challenges. Living alone is one type of living arrangement, and living arrangements are inherently dynamic and may be recursive. This means that an individual can transit through different types of living arrangements over her/his life . In this example, living alone occurs at different stages over the life course, and has different determinants and implications. The study of living arrangements has therefore to be particularly sensitive to age, gender, and life course influences, including marital status. Fourth, there are measurement challenges for studying living alone as a form of living arrangement. The data examined in this study do not tell us whether the person living alone is in a relationship with another person (the “living apart together” couples noted earlier). It is likely that the predictors and implications of living alone for such individuals would differ in important ways from others who live alone and are not in a relationship.Finally, while the census and ACS data used are appropriate for identifying and comparing sociodemographic, economic, and acculturation factors on living alone among older immigrants, there is no information on other factors that influence older immigrants’ living arrangements, including the key role of health status (the ACS includes a question on disability but there is no comparable information in the Canadian census). Other unmeasured factors include availability of family or friends to share housing, and community characteristics that either facilitate or discourage living alone .We began our analysis by making no assumptions about whether living alone is the “best” living arrangement for non-married older adults. The increased social trend to elderly residential independence suggests that most elderly prefer independent living arrangements , but we However, as we reflect on our findings, a picture emerged suggesting that living alone is associated with characteristics that can only be described as advantageous. Results from examining older immigrants show that those who live alone have higher income and education and are more acculturated. These characteristics may be related to other dimensions of wellbeing, such as more extensive social ties and support because of being more acculturated, and better health, given the well-known socioeconomic status-health gradient ,52. Stil


Henipavirus genus in ten hosts. NiV genome encodes nine open reading frames; and overall, no significant bias in codon usage was observed. Aromaticity of proteins had no impact on codon usage. An analysis of preferred codons used by NiV and the tRNA pool in human cells indicated that NiV prefers codons from a suboptimal anticodon tRNA pool. We observed that codon usage by NiV is mainly constrained by compositional and selection pressures, not by mutational forces. Parameters that define NiV and host relatedness in terms of codon usage were analyzed, with a codon adaptation index (CAI), relative codon deoptimization index (RCDI), and similarity index calculations; which indicated that, of all hosts analyzed, NiV was best adapted to African green monkeys. A comparative analysis based on the relative codon deoptimization index (RCDI) for host adaptation of NiV, Hendra virus (HeV), Cedar virus (CedV), and Hendra like Mojiang virus (MojV) revealed that except for dogs and ferrets, all evaluated hosts were more susceptible to HeV than NiV.A recent outbreak of Nipah virus (NiV) in India has caused 17 deaths in people living in districts of Kerala state. Its zoonotic nature, as well as high rate of human-to-human transmission, has led researchers worldwide to work toward understanding the different aspects of the NiV. We performed a codon usage analysis, based on publicly available nucleotide sequences of NiV and its host adaptation, along with other members of the  Henipavirus genus, Paramyxoviridae family, that infects both wild animals and humans  is an RNA virus in the d humans . In 1998d humans . Human-td humans . Thereafd humans , Cambodid humans , Malaysid humans , Vietnamd humans , the Phid humans , Indonesd humans , Thailand humans , Ghana , P (phosphoprotein), M (matrix), F (fusion), G (glycoprotein), and L (large polymerase), and possesses nine coding sequences (CDSs). Of these genes, two encode integral membrane proteins; one encodes a glycoprotein (G) that is essential for attachment, and another encodes a fusion (F) protein needed for cell entry. Both the G (602 aa residues) and F (546 aa residues) proteins of NiV are essential to mediate the cellular entry into the hosts . The M pAt present, there is no prophylaxis for NiV; however, vaccination and passive transfer of antibodies as interventions against NiV have been evaluated . SubunitThe purpose of the present study was to elucidate codon usage pattern of NiV, that can be applied to generate an attenuated NiV vaccine strain. An international coalition of governments and pharmaceutical companies was formed in 2017 to develop safe, effective as well as affordable vaccines to combat pandemic diseases like NiV . The preHenipavirus  ]  has also1 in FASTA format. For G, F, M, N, L, P, and C, complete sequences were obtained; whereas, for V and W, partial sequences were used in this study. Sequences downstream of the RNA editing site were excluded for V and W genes. A total of 149 CDSs corresponding to 101500 codons were analyzed in the present study. Similarly, 181 CDSs of HeV, 21 CDSs of CedV and 14 CDSs of MojV corresponding to 118113, 16413, and 10408 codons, respectively, were retrieved.Sequences of all the nine CDSs  for NiV were retrieved from the National Center for Biotechnology Information (NCBI)The nucleotide composition of CDSs, specifically the nucleotide at the third codon position  and the overall AU% , AU3% (nucleotides A and U present at third position of codon), GC% , GC12 (the average of nucleotide G and C present at first and second positions of codon), and GC3  were analyzed.Variation in the frequency of dinucleotide pairs may affect the codon usage. Dinucleotide frequency is often used to determine whether some dinucleotide pairs are favored by an organism or not. A maximum of 16 dinucleotide combinations are feasible. The patterns of dinucleotide frequency indicate both selectional and mutational pressures; which was calculated using the following formula:fx and fy are the frequency of individual nucleotides , and fxy is the frequency of dinucleotides (xy) in the same sequence.where The ratio of the observed to expected dinucleotide frequency is known as odds ratio; if this ratio is more than 1.25, the dinucleotide is considered overrepresented, whereas values below 0.78 indicate a underrepresentation .P. vampyrus were obtained from the codon usage database2, and NiV RSCU data was analyzed using the CAIcal server3  of a P. vampyrus “Shadow” isolate from the Lubee Bat Conservancy  were analyzed using the ‘Countcodon’ program of Yasukazu Nakamura4.As described by  server3 . To deteThe GRAVY value is the sum of hydropathy values of all amino acids in a sequence divided by the number of residues , which rThe AROMO value is the frequency of aromatic amino acids, i.e., Phe, Tyr, and Trp, in a given amino acid sequence.The similarity index indicates the effect of host codon usage on pathogen codon usage. Codon usage by different genes of NiV and by hosts was analyzed. This strategy was developed by D is a numerical value, ranging between 0.0 and 1.0 representing the effect of the overall codon usage of the host on that of NiV. The higher the value, the greater the impact of a host is on NiV codon usage.where ‘ai’ is the RSCU value for a specific codon for the NiV coding sequence, ‘bi’ is the RSCU value for the similar codon of the host. In a coding sequence, most amino acids (except Met and Trp), are encoded by two or more codons, known as synonymous codons. To identify the bias in the use of synonymous codons, A neutrality plot was used to analyze the influence of mutation bias and translation selection on codon usage. A regression line was plotted between GC12 and GC3 contents. A slope of the regression line is indicative of the mutational force. A parity rule 2 (PR2) bias was calculated based on the AT bias [A3/(A3 + T3)] as the ordinate and GC bias [G3/(G3 + C3)] as the abscissa .Codon adaptation index (CAI) is a quantitative value that indicates how frequently a favored codon is used amongst highly expressed genes. It is a primary indicator of translation efficiency and is used to engineer nucleotide sequences to obtain maximal protein production for vaccine purposes . CAI valHenipavirus genus  were also calculated for the selected 10 hosts.5. The program was also used to obtain AROMO values. GRAVY values were calculated using http://www.gravy-calculator.de/. CAI and RCDI values were also calculated using http://genomes.urv.es/CAIcal/ (6).The RSCU values were determined using CODONW 1.4.2/CAIcal/ . The tRNIn the present study, we analyzed a total of 149 CDSs corresponding to 101,500 codons of all nine CDSs  of various NiV isolates. The general sequence information of samples including the year of isolation, host and type of sample  are depicted in the The NiV CDSs were found to be rich in A and U nucleotides, in comparison to G and C nucleotides. To determine the compositional constraints in the NiV genome, the nucleotide compositions of the CDSs were determined . Mean usIn the present study, ApA (10.4%) was observed as the most abundant dinucleotide reflecting a high abundance of A in the NiV genome. Whereas the least abundant dinucleotide pair was CpG (1.8%)  with theIn order to determine the patterns of synonymous codon usage, RSCU values were calculated. RSCU values for the individual gene, as well as for the complete genome, were analyzed separately. All overrepresented codons are highlighted in their respective genes . The aveP. vampyrus showed similar codon preferences; whereas for Tyr and Asn, NiV and H. sapiens showed similar codon preferences. Different preferences in codon usage by NiV and its hosts indicate that the virus does not compete for the host tRNA pool. The heat map of RSCU values for all the nine CDSs of NiV and hosts indicated that A/U-ending codons were preferred over G/C-ending codons . The results of the neutrality plot revealed statistically significant correlations between GC12 and GC3, however, the slope value was less, indicating the role of mutational pressure, though not as the dominant pressure , the number of residues A = T and residues C = G in a DNA strand . The cenIn this case, A3/(A3 + T3) and G3/(G3 + C3) were plotted as the ordinate and abscissa, respectively . The meaDifferent isoacceptor tRNAs of various tRNA species bind to different codons that signify a particular amino acid residue. Codon adaptation index values are used to determine the level of expression of pathogen proteins in the host and adaptation of a virus to a host. Sequences with higher CAI values are considered more adapted to particular hosts than those with low values. The CAI values were 0.75 ± 0.019, 0.74 ± 0.017, 0.72 ± 0.017, 0.67 ± 0.021, 0.66 ± 0.02, 0.65 ± 0.027, 0.65 ± 0.021, 0.61 ± 0.022, 0.59 ± 0.022, and 0.58 ± 0.026 for the African green monkey, bat, human, hamster, dog, horse, cat, pig, squirrel monkey, and ferret, respectively . The comr = 0.513; P < 0.0001). A correlation analysis between GC3%, Nc, CAI, GRAVY, and AROMO, is provided in There was a strong correlation between CAI and Nc  on expressivity of gene (indicated by CAI), wherein no correlation was obtained.Relative codon deoptimization index (RCDI) values indicate the cumulative effects of codon biases on the expression of a gene. It is measured by comparing the codon usage of a virus with that of its host. These values also provide insight into the possible co-evolution of virus and host genomes. A lower RCDI value indicates higher adaptation of a virus to its host. Our intragenic RCDI analysis revealed that for NiV, in all tested hosts, except for the squirrel monkey and ferret, the M gene showed the lowest RCDI, indicating a high degree of adaptation to these hosts . The L gp < 0.01; r = −0.227 to −0.411). A candidate virus genome rationally designed through modified codon usage possessing lower CAI and higher RCDI; that will result in production of viral proteins at a lower level, leading to decreased viral infectivity, i.e., a step closer to the development of an attenuated vaccine candidate. A comparative analysis of RCDI values of all the viruses in various hosts, indicates that except for Canis familiaris and Mesocricetus auratus, HeV is more adapted than NiV in all the hosts  impact on the NiV codon bias, followed by the ferret, whereas the African green monkey (0.054) had the lowest.Overall, from initial nucleotide compositional analysis, the NiV genome is AU rich; and A/U ending codons appear to be preferred over G/C ending codons.Codon usage bias, mutational bias, as well as the influence of selective pressure and compositional constraints, are reflected in the occurrence of dinucleotides. The relative abundance of dinucleotides has shown to affect codon usage in RNA viruses. CpG depOther dinucleotides that are commonly underrepresented in DNA and RNA virus genomes are TpA and UpA . That isy = –0.405x + 57.73, R2 = 0.110), further confirming the findings that mutational pressure is not the major factor affecting codon usage. Additionally, there was no correlation between Nc and GRAVY or Nc and AROMO, suggesting that codon usage bias is not affected by hydrophobicity or aromaticity. The correlation analysis between CAI, GRAVY, and AROMO was performed to determine the effect of GRAVY and AROMO  on the expressivity of the gene , which indicated that no correlation was observed. Parity analysis revealed that there is a bias for the third codon position which further reconfirms the role of selection pressure.In the case of NiV, the average GC and GC3 content were 42.245 and 40.173, respectively. If codon usage is affected only by GC3 content, this indicates mutational pressure; in such cases, the Nc values lie just over the expected Nc curve . In all Most preferred codons of NiV uses suboptimal isoacceptor tRNAs. This can be explained by the fact that, during the initial phase of infection, when the translation rate is low, usage of suboptimal codons does not represent a constraint, whereas, during later stages of infection, the overall host cellular machinery is hijacked and compromised, allowing preferential translation of virus proteins. In both situations, usage of suboptimal codons has shown no detrimental effects on the translation of viral proteins . In addir = 0.513; p < 0.00001) indicates that a higher expression (indicated by CAI) is associated with increased bias (Nc) and that selection pressure, rather than mutational forces, are the dominant pressure in the NiV genome.The CAI values, RCDI values, and similarity indexes were evaluated and examined among all hosts. The African green monkey was found to be the best suited for animal experiments, with the highest CAI and similarity index; and the lowest RCDI value. CAI values are associated with selection pressure, with highly expressed codons selected. On the other hand, Nc is associated with a bias that is due to selection or mutation pressure. The correlation between CAI and Nc can be based on the relative balance between the selection and mutation . A greatAedesalbopictus or Aedesaegypti that were higher than those of humans for Zika virus. The relatively low average values of D   suggests that NiV can replicate efficiently in hosts without much of an effect on host codon usage. In the case of the Marburg virus, the host Rousettus aegyptiacus (Egyptian fruit bat) also showed a higher similarity index than for humans , indicating the highest adaptation. This result is in accordance with the fact that a cell line derived from the kidney epithelial cells of African green monkey (Vero cell line) is commonly employed for isolation of NiV. A study encompassing 10 cell lines was conducted to compare the susceptibility of cell lines to NiV and it was revealed that the virus replicated best in Vero cells and BSR cells .A codon-optimized “humanized” green fluorescent protein has an RCDI of 1.31 . A low ROur codon usage study though, does not exactly explain the amplitude of pathogenicity/virulence in animal models; however, it is able to predict the virus replication inside the host (even when the presence of virus is asymptomatic as in case of bat). Thus, it can easily predict the carrier hosts as well, which without being affected by virus, may act as a source of infection in other co-circulating species. The study was carried out with the aim to predict which animals may be possible hosts for NiV, whether they could directly show symptoms or be asymptomatic hosts. In all tested hosts used in the study, NiV is known to cause infection and some of these live in close vicinity to human populations in the rural areas. Hence, the present study was carried out to compare the adaptation of NiV in different hosts. The study will also pave the way to evaluate and assess other animals for their potential to serve as hosts for the virus. The information will be helpful in elucidating the emerging health hazards to humankind as a result of living in close contact with these animals, which may serve as carriers of the virus, although asymptomatic, could serve as a source of infection.Our observation is corroborated with results obtained by comparing CAI values. NiV has recently emerged as a virus posing a major public health concern. There is an urgent need for the development of an effective vaccine and discovering viable therapeutics along with the identification of potential hosts. Information regarding potential hosts may be useful in identifying various preventive measures. Here, on the basis of codon usage, we attempted to identify hosts with the most potential to harbor the virus. The NiV sequences were found to be highly adapted to the bat and human sequences based on CAI values. Humans and bats were found to be highly supportive of virus replication, as evidenced by higher CAI values. Results of this study might also be useful in identifying the most suitable experimental animal models for vaccine and other pathogenicity-associated experiments.Another advantage of the study can be linked to the development of a codon biased rationally designed synthetic attenuated virus vaccine candidate against NiV. For any attenuated virus vaccine candidate, genetic stability and safety are major concerns. Introduction of codon-pair bias de-optimization in hemagglutinin (HA) and neuraminidase (NA) gene of Influenza A virus resulted in the generation of a virus phenotype, that produces inappreciable pathogenesis, but at the same time elicited a robust immune response after its intranasal administration . ColemanHenipavirus .NiV has recently emerged as a virus that poses a major public health concern. There is an urgent need for the development of an effective vaccine and to discover viable therapeutics along with the identification of potential hosts. Information regarding potential hosts may be useful in identifying various preventive measures. Here, on the basis of codon usage, we attempted to identify hosts with the most potential in harboring the virus. Based on the intragenic analyses, there are clear statistically significant differences in codon usage bias between the nine CDSs, and, along with compositional constraints, selection forces were found to play a major role in shaping NiV codon usage. The NiV sequences were found to be highly adapted to the bat and human sequences based on CAI values. Humans and bats were found to be highly supportive of virus replication, as evidenced by higher CAI values. The results of this study might also be useful in identifying the most suitable experimental animal models for vaccine and other pathogenicity-associated experiments. After evaluating CAI values, RCDIs, and similarity indexes, among all hosts examined, the African green monkey was found to be the best suited for animal experiments, with highest CAI and similarity index and lowest RCDI value. Among the 10 tested hosts, ferrets were found to be the least supportive of NiV replication. Considering other viruses like HeV, CedV, and MojV, RCDI analysis indicated that except for ferrets and dogs, HeV is more adapted than NiV. The comparative analysis of CAI value indicated that the African green monkey is the most suitable animal model for studying Various tools like DNAWorks, Jcat, Synthetic Gene Developer, GeneDesign, Gene Designer 2.0, OPTIMIZER, Visual Gene Developer, Eugene, mRNA Optimizer, Codon Optimization OnLine (COOL), and D-Tailor are freely available to optimize gene expression, without altering the amino acid sequence. These tools rely on various algorithmic measures like CAI, RSCU, Nc etc., and by using this information, both enhancement as well as reduction of protein expression is possible. On one hand, elucidation of the codon usage pattern is useful in optimizing protein expression. The information regarding enhanced protein expression will be useful in designing a subunit vaccine candidate, where a vaccine candidate is expressed in a prokaryotic/eukaryotic system. Apart from the subunit vaccine, codon optimization is also useful in constructing various other viral vectored vaccine candidates including vesicular stomatitis virus, rabies virus, canarypox virus, adeno-associated virus, measles virus, Newcastle disease virus and Venezuelan equine encephalitis virus harboring the G and N protein of NiV. On the other hand, contrary to protein expression enhancement, similar codon usage information can be used to reduce the NiV viral protein synthesis during replication of the pathogen. This rational systematic approach is called SAVE (synthetic attenuated vaccine engineering), by which choosing underrepresented codons and/or concomitant increase in rare dinucleotides like CpG and UpA, virus attenuation in a tunable manner is achieved. Codon usage information may also be obtained in a similar fashion for other viruses and investigations may be done for broad applicability.RK, SS, UK, AA, RT, KD, JD, AM, and RS substantially contributed to the conception, design, and analysis of primary data, interpretation of data, checking and approving final version of the manuscript, and agreed to be accountable for its contents. RK and AM conceived the concept. SS, UK, and AA retrieved and analyzed the data and generated tables and figures under supervision of RK, JD, and RS. RK, AM, and KD wrote the manuscript. RT assisted in writing. KD, AM, and RS finalized the manuscript.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.


In addition, we found that the PCOS women consumed less extra-virgin olive oil, legumes, fish/seafood, and nuts compared with control group. Despite no differences in energy intake between the two groups, the PCOS women consumed a lower quantity of complex carbohydrate, fiber, monounsaturated fatty acids (MUFA), and n-3 polyunsaturated fatty acid (PUFA), and higher quantity of simple carbohydrate, total fat, saturated fatty acid (SFA), PUFA and n-6 PUFA than the control group. The PCOS women have an adverse body composition when compared with controls, with the lowest values of phase angle (PhA) and fat-free mass (p < 0.001). Additionally, after adjusting for BMI and total energy intake, testosterone levels showed significant negative correlations with PREDIMED score (p < 0.001) and consumption of protein (p = 0.005), complex carbohydrate (p < 0.001), fiber (p < 0.001), MUFA (p < 0.001), n-3 PUFA (p = 0.001), and positive associations with CRP levels, simple carbohydrate, SFA, n-6 PUFA , and PUFA (p = 0.002). The cut-off for PREDIMED score ≤ 6  0.848, standard error 0.036, 95% confidence interval (CI) 0.768 to 0.909) could serve as a threshold for significantly increased risk of high value of testosterone levels. In conclusion, a novel direct association between the adherence to MD and the clinical severity of the disease was reported in women with PCOS. This association could support a therapeutic role of foods and nutrients of the Mediterranean dietary pattern in the PCOS pathogenesis likely involving their inflammatory status, IR, and hyperandrogenemia. In addition, we reported a different body composition that is characterized by lower PhA and fat-free mass than controls. These data suggested a role of PhA as a useful marker of the clinical severity of this syndrome and provided strong evidence regarding the strategic relevance of the nutritional assessment in the management of women with PCOS.Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder and is characterized by chronic anovulation, hyperandrogenism, and polycystic ovaries. Obesity, low-grade chronic inflammatory status, and insulin-resistance (IR) often coexist in PCOS. The Mediterranean diet (MD) is an anti-inflammatory dietary pattern, which is rich in complex carbohydrates and fiber, and high in monounsaturated fat. There is a close association among obesity, low-grade chronic inflammation, IR, and hormonal derangements in PCOS. The main aim of the present study was to evaluate the adherence to MD, the dietary intake, and the body composition and their association with PCOS clinical severity in a cohort of treatment-naïve women with PCOS when compared with a control group of healthy women matched for age and body mass index (BMI). In this case-controlled, cross-sectional study, we enrolled 112 patients with PCOS and 112 controls. PREvención con DIetaMEDiterránea (PREDIMED) and seven-day food records were used to evaluate the degree of adherence to the MD and dietary pattern, respectively. Body composition was evaluated by bioelectrical impedance analysis (BIA) phase-sensitive system. Testosterone levels and Ferriman-Gallwey score assessed the clinical severity of PCOS. C-reactive protein (CRP) levels were determined with a nephelometric assay with CardioPhase high sensitivity. PCOS women showed higher testosterone levels, Ferriman-Gallwey score, fasting insulin and glucose levels, and Homeostatic Model Assessment (HoMA)-IR when compared with the control group ( Polycystic ovary syndrome (PCOS) is a multifactorial endocrine metabolic disorder  that affBeyond the MD, to date, few data are available on the dietary intake in women with PCOS by using the seven-day food records, which is recognised as the “gold standard” for careful nutritional assessments ,19. DougIn clinical practice, body composition is commonly measured by bioelectrical impedance analysis (BIA), which is a non-invasive method for evaluation of body composition with a high agreement with Dual-energy X-ray absorptiometry . Phase aIn this context, the main aim of the present study was to evaluate the adherence to MD, the dietary intake assessed by seven-day food records, and the body composition assessed by BIA and their association with PCOS clinical severity in a cohort of treatment-naïve women with PCOS as compared with a control group of healthy women matched for age and BMI. When considering the coexistence of both inflammation and obesity, and while taking into account the well-known anti-inflammatory effects of MD, we suppose that PCOS women should be more prone to follow an unhealthy diet, poorly adherent to the Mediterranean style, which contributes to worsening their condition; furthermore, since various studies demonstrated the association between PhA and inflammation, we hypothesize to find this relationship also in PCOS patients.This cross-sectional, observational study was carried out in patients with PCOS attending the Unit of Endocrinology, Department of Clinical Medicine and Surgery, University Federico II of Naples , from January 2014 to January 2019. The study has been approved by the Local Ethical Committee (n. 05/14) and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments that involved humans. The aim of the study was clearly explained to all of the study participants and a written informed consent was obtained. The subjects were enrolled at the outpatient Endocrinology clinic. The study included 112 treatment-naïve women that were affected by PCOS attending the Outpatient Clinic of the Unit of Endocrinology in our Department. One hundred-twelve Caucasian healthy subjects , age, and BMI matched were chosen as the controls among hospital volunteers and employees from the same geographical area around Naples . To increase the homogeneity of the patient sample, we only included treatment-naïve women. Eligible patients were those with a diagnosis of PCOS classified by the European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine (ESHRE/ASRM) diagnosis . This in2), aged 18–40 years, a lack of underlying metabolic disease .The inclusion criteria for all groups were: premenopausal women who were normal-weight, overweight or obese ; pregnancy or lactation in the past 6 months;Hyperandrogenism and/or biochemical hyperandrogenemia, oligomenorrhea due to secondary etiologies as per the Endocrine Society Clinical Practice Guidelines and previous publications including endocrine disorders  ;Pre-existing systemic or psychiatric disease and use of medications that impact carbohydrate or lipid metabolism ;Specific nutritional regimens or hypocaloric diet in the last three months, including vegan or vegetarian diets; supplementation with antioxidants, vitamins or minerals;Occasional or current of use of drugs that could influence fluid balance, including non-steroidal anti-inflammatory drugs, diuretics, laxative use; and,Women patients with implanted pacemakers or defibrillators because of the theoretical possibility of interference with the device activity due to the field of current induced by the impedance measurements.The exclusion criteria for all groups were the following:The clinical and biochemical assessment were done between 8 am and 12 am, after an overnight fast.The sample size was calculated on the basis of a previous study that reported prevalence of PCOS as 12% in women of reproductive age . The calhttps://clincalc.com/stats/samplesize.aspx).The power was calculated by the differences of means ± standard deviation (SD) of PREvención con DIetaMEDiterránea (PREDIMED) score in the PCOS patients and control group . When considering the number of cases required in each group of 100, were set at 112 of group, a type I  error of 0.05 (85%), and a type II (beta) of 0.05, the calculated power size was 85%. The calculation of sample size and power were performed while using Sample Size Calculator Clinical Calc  were defined as physically active, as we have already fully reported in previous studies ,27,28. Measurements were performed between 8 am and 12 pm. All of the subjects were measured after an overnight fast. The anthropometric measurements were performed by the same operator , according to the International Society for the Advancement of Kinanthropometry (ISAK 2006). 2), kg/m2). Height was measured to the nearest 0.5 cm while using a wall-mounted stadiometer . Body weight was determined to the nearest 0.1 kg while using a calibrated balance beam scale . BMI was classified according to World Health Organization’s criteria with normal weight: 18.5–24.9 kg/m2; overweight, 25.0–29.9 kg/m2; grade I obesity, 30.0–34.9 kg/m2; grade II obesity, 35.0–39.9 kg/m2 59], and The PREDIMED score results from a brief and useful 14-item questionnaire, validated for the assessment of MD adherence ,62. The However, as diet is complex miscellanea of different foods and nutrients, the nutritional approach should be based on a meticulous dietary assessment, in terms of quantity and quality, like the one that was provided by the seven-day food records, the gold standard in self-administered food frequency questionnaires. The analysis of seven-day food records showed that PCOS women consumed a higher amount of high glycemic index carbohydrates, lower fiber, MUFA, and n-3 PUFA as compared to the control group. These results are in line with previous studies reporting that a high intake of simple carbohydrates exerts a pro-inflammatory effect, as demonstrated by increased CRP levels , and theIn parallel to carbohydrates, dietary fat, and in particular MUFA and n-3 PUFA, are also implicated in the PCOS pathogenesis. In our study, we found that the lowest intake of MUFA was among the three main predictors of the highest testosterone levels. The association between low MUFA consumption in PCOS women is in agreement with the clinical trial performed by Kalgaonkar et al. . MUFA arIt is clear that unhealthy diets, besides the effects on inflammation and IR, could negatively affect the body composition, which, in turn, is also responsible for worsening of the PCOS clinical severity. Besides BMI, limited data are available on the body composition of PCOS women. No difference in terms of body composition assessed by BIA has been detected in a pilot study that was performed on adolescents with and without diagnosis of PCOS . On the We are aware that there are some limitations in the current study. First, the cross-sectional nature of this study did not allow for any statements on a causal relationship between MD and clinical severity of PCOS, especially when considering the adherence and dietary patterns through a previous lifespan. Second, the sample size is relatively small and dietary intake is related to a larger number of other lifestyle factors. Nevertheless, the sample size was calculated using 95% power; in addition, the association between MD and testosterone levels was independent of the effects of other health-related behaviours . Third, the PREDIMED score, although being easy to perform by the participants, only allows relative but no absolute statements regarding the degree of adherence to the MD. The PREDIMED questionnaire was face-to-face administered and not self-reported in order to minimize any bias related to the filling of the questionnaire. In addition, to avoid the inter-operator variability, only one expert Nutritionist evaluated the anthropometric measures, administered the PREDIMED questionnaire and seven-day food records, and assesses, executes, and interprets BIA measurements. Anyway, the suggested cut-point for the PREDIMED score for identifying the highest of testosterone levels should be viewed with caution until results of studies in larger population samples have become available to perform an appropriate cross-validation.Nevertheless, the main strength of this study is the use of the seven-day food records. This tool, which is considered the “gold standard” in validation studies of different types of self-administered food frequency questionnaires, allows for more accurate measurement of the real dietary and macronutrient intakes as compared to those that were obtained by retrospective food frequency questionnaires . MoreoveIn summary, in the present study, we reported for the first time in women with PCOS: (i) a direct association between the adherence to MD and PCOS; (ii) a different body composition that is characterized by low PhA and fat-free mass. These data could support a therapeutic role of single foods and nutrients of the Mediterranean dietary pattern in the PCOS, by contributing to reduce the inflammatory status that paves the way for IR and hyperandrogenemia. In addition, the role for PhA as a useful marker of the clinical severity of PCOS is strongly suggested. Our data propose that the strategic relevance of the nutritional assessment and body composition evaluation of women with PCOS should be considered as the first and most important step in the management of this syndrome.