Seneca Valley virus (SVV) has recently caused many vesicular diseases in pigs in different regions and countries. As a newly causative agent of porcine vesicular disease, SVV has evolved and spread quickly. It causes clinical signs similar to those of foot-and-mouth disease and results in significant economic losses. An increasing number of SVV outbreaks were reported in 2016 and 2017 in Brazil, United States, and China. However, few diagnostic methods have been established and no commercial vaccine has been available until now. Therefore, more attention needs to be paid to SVV, and urgent surveillance should be performed to prevent the spread of this virus. Although recent research has shed some light on SVV, there are still many aspects of the virus and the disease that are not yet fully understood, and many questions need to be resolved. This review presents current knowledge concerning SVV infection, epidemiology, pathogenicity, immune response, and diagnostic methods. This information will aid the design and adoption of effective prevention and control strategies to counter this viral pathogen. Senecavirus, family Picornaviridae , also known as Senecavirus A, is a non-enveloped, positive-sense, single-stranded RNA virus that belongs to the genus aviridae . SVV wasaviridae . SVV doeaviridae ; howeveraviridae . Therefoaviridae . The firaviridae . A murinaviridae . A firstaviridae . A Phaseaviridae .Six picorna-like viruses were isolated from pigs in the United States between 1988 and 2005, which showed a variety of clinical symptoms. They were closely related to each other and to SVV by sequencing analysis. Furthermore, each of these viruses is serologically related to the others, as well as to SVV-001 . This imSenecavirus. Swine are thought to be a natural host of SVV and previous studies have indicated that SVV is not prevalent in humans. Serological surveys have revealed the presence of specific SVV antibodies in pigs, cattle, and mice, but not in humans  (n = 8) challenged by an SVV strain (5 × 108.07 TCID50) isolated in the United States in 2015 presented lameness and lethargy by day 4 post-infection (pi), and these manifestations persisted for 2–10 days. The vesicular lesions were found either on feet or snout and in some cases these lesions were found both on feet and snout (6/8 pigs). Viral RNAemia was detected in serum during a period of approximately 7 days (3–10 days pi). The presence of SVV in tissues was assessed following convalescence from disease. SVV was detected in the lungs, lymph nodes, liver, spleen, small and large intestines, and tonsils of infected finishing pigs between 3 and 5 days post-infection, and it showed that SVV has a tropism for lymphoid tissues, with a higher level in the tonsils. Therefore, the tonsils are likely to be considered as the primary replication site for SVV that can be selected for virus isolation. The virus shedding lasts up to 28 days and can be detected in oral and nasal secretions and also in feces. The highest viral peak occurs between 1 and 5 days post-infection in oral secretions, suggesting the acute nature of the vesicular disease caused by SVV  which lasted for 1–3 days. The viral RNA can be detected in the blood after 1–7 dpc and the RNAemia disappeared after 9 dpc  . The affgure 1A) . Accordid by SVV . Anotherer 9 dpc . These sin situ hybridization. Seneca Valley virus infection has also been associated with pig neonatal mortality [recently named epidemic transient neonatal losses (ETNLs)], especially in pig herds with SVV-affected sows . A longiin vitro and in vivo. SVV capsid interacts directly and specifically with ANTXR1. This interaction is required for SVV binding to the host cells. ANTXR1 is frequently expressed on the surface of tumor cells compared with normal human cells , VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3Cpro and 3Dpol, and the four structural proteins make up the round viral particles with a diameter of ∼30 nm   and Aphthovirus (including FMDV) .The first SVV genome was determined in 2007 (SVV-001 strain) which has typical genomic feature of other picornaviruses including the standard L-4-3-4 layout . The virgure 1B) . The 5′ gure 1B) . Phyloge+ T cells that are initially detected by day 7 post-infection and increased in frequency until day 14 post-infection. An increase of CD8+ and double-positive CD4+CD8+ T cells associated with interferon-γ production are observed after day 10 post-infection  response in sows and finishing pigs regardless of the clinical manifestations of the disease . Mice alnfection .The innate immune system plays a significant role in recognition of viral components and triggering the antiviral response . Many viFigure 1B)  . Observagure 1B) .in situ hybridization are also established methods to identify SVV antigen and RNA in tissue samples  staining and  samples . SVV RNA samples , and pul samples . A speci samples . This me samples . An indi samples . For SVV samples . These RAs no SVV commercial vaccines have been developed currently and immunization with FMDV vaccine shows no cross protection against SVV infection , controlIn recent years, SVV has demonstrated its capacity to evolve and cause morbidity and mortality in pigs, accompanying the expansion into new countries. Surveillance, epidemiological investigations and genetic characterization of SVV associated with vesicular diseases and neonatal mortality in pigs are important for controlling SVV and supporting the development of specific and effective diagnostic tests. The lack of available commercial vaccines, and because SVV is not included in the OIE Terrestrial Animal Health Code, means that the increasing incidence of SVV infection in pigs in different countries will continue to cause unpredictable and substantial outbreaks. Therefore, in response to this threat, animal administration authorities and farm veterinarians must show continued awareness of this disease and remain vigilant in their surveillance, treatment, and provision of effective control measures. Further evaluation and study of the pathogenesis of SVV in swine, transmission among swine, and development of effective commercial vaccines will help limit the spread of SVV.XZ, HZ, and XL conceived the study and wrote the manuscript. ZZ, FY, WC, HT, and KZ collected data, drew the figures, and compiled the tables.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.


Background: Large spatial heterogeneity was observed in the dengue fever outbreak in Guangzhou in 2014, however, the underlying reasons remain unknown. We examined whether socio-ecological factors affected the spatial distribution and their interactive effects. Methods: Moran’s I was applied to first examine the spatial cluster of dengue fever in Guangzhou. Nine socio-ecological factors were chosen to represent the urbanization level, economy, accessibility, environment, and the weather of the 167 townships/streets in Guangzhou, and then the geographical detector was applied to analyze the individual and interactive effects of these factors on the dengue outbreak. Results: Four clusters of dengue fever were identified in Guangzhou in 2014, including one hot spot in the central area of Guangzhou and three cold spots in the suburban districts. For individual effects, the temperature (q = 0.33) was the dominant factor of dengue fever, followed by precipitation (q = 0.24), road density (q = 0.24), and water body area (q = 0.23). For the interactive effects, the combination of high precipitation, high temperature, and high road density might result in increased dengue fever incidence. Moreover, urban villages might be the dengue fever hot spots. Conclusions: Our study suggests that some socio-ecological factors might either separately or jointly influence the spatial distribution of dengue fever in Guangzhou. Aedes albopictus and Aedes aegypti [Dengue fever (DF) is an infectious disease transmitted by  aegypti . The Wor aegypti ,3. Along aegypti ,5,6,7.Previous studies have investigated the impacts of socio-ecological factors on dengue fever outbreaks and transmission. Hunter et al. examined the relationship between the construction of water impoundments and dengue fever in tropical areas, and found that the increasing water areas exacerbated the transmission of dengue fever, because they served as breeding places for mosquitos . Other sThough those previous studies showed that dengue fever was influenced by socio-ecological factors, several limitations are worth considering in future studies. First, majority of previous studies employed temporal methods, such as time-series study design to explore risk factors of dengue outbreaks ,15, and To fill these knowledge gaps, we attempted to examine the individual and interactive impacts of socio-ecological factors on dengue fever at a fine scale using the geographical detector in this study, which is based on spatial variance analysis of the spatial consistency of health risk or disease distribution with suspected socio-ecological factors . The finGuangzhou, the biggest city and the economic, political, and cultural center of Guangdong Province, is located between the 22°26′ to 23°56′ N and 112°57′ to 114°3′ E . A totalDaily data of dengue fever in Guangzhou in 2014 were obtained from the National Notifiable Infectious Disease Reporting Information System, which has access to the Guangdong Provincial Center for Disease Control and Prevention . Only clA patient was considered to be a clinically diagnosed case if he or she had an acute onset of rashes, headache, subjective fever, itching, anorexia, and leukopenia, and lived in the dengue fever outbreak area . All dat2) of each township/street in Guangzhou was downloaded from the Socioeconomic Data and Applications Center hosted by the Columbia University (http://beta.sedac.ciesin.columbia.edu/) [Three factors were chosen to describe different aspects of urbanization at townships/street level, including population density, urbanization level, and the ratio of urban village. Because the population density data in Guangzhou were not available in 2014, we used the 2015 data as a replacement. The population density data (persons/kmia.edu/) ,29. Urbaia.edu/) . An urbaia.edu/) . Guangzhhttp://www.geofabrik.de/index.html). We classified roads into eight types based on their location, administration, and utility, i.e., footway, living street, path, pedestrian, residential, road, step, and unclassified. All eight types in 2014 were used to calculate the road density.Data on Gross Domestic Product (GDP) per capita in 2010 at the township/street level in Guangzhou were retrieved from the National Bureau of Statistics of China, which was computed by multiplying the population and GDP per capita at the township/street level. We used the data in 2010 because this is the only available data closest to 2014. Road density was selected to reflect the accessibility of Guangzhou, and the road net data were obtained from website of the GEOFBRIK (NDVI) was acquired by the Moderate Resolution Imaging Spectroradiometer (MODIS) satellite imagery to represent the greenness of vegetation and photosynthetic rate. NDVI is an indispensable factor to assess whether the target areas contain living vegetation. The monthly NDVI data (MOD13A3) was obtained, and the annual NDVI value in 2014 was calculated. As NDVI can only reflect the greenness of target areas but not the vegetation coverage ratio, we applied NDVI to calculate the vegetation fraction (VFC) using the following equation [NDVI denotes the pixel NDVI value, minNDVI denotes the bare soil NDVI value, and maxNDVI denotes the live vegetation NDVI value. The vegetation fraction (VFC) was calculated using the ENVI5.0 software . The resolution of VFC was 1 km, but the urbanization level and ratio of urban village were 30 m, and all the satellite-derived data were unified into 1 km × 1 km resolution, although this may lose some information of the precise profile of the urban village area. Water body areas were chosen to reflect the environmental aspects. Water body data in 2014 in Guangzhou were obtained from the Engineering Technology and Research Center of Guangdong Geography Census Monitoring and Comprehensive Analysis.Normalized difference vegetation index (equation :(1)VFC=Nhttp://www.gscloud.cn/) were applied to calibrate the temperature with a lapse rate of 6 °C/km. For precipitation, a Kriging interpolation method was used first to obtain the precipitation interpolation result. Then, the multivariate regression relationship of Yprecipitation = X  was established. Finally, the interpolated precipitation result was adjusted based on the multivariate regression relationship [According to previous investigations , temperationship .Moran’s I measures the spatial autocorrelation using the following equation :(2)Ii=xiN and L stratums, h = 1, 2, 3 … L. In this study, L is the number of townships/street in Guangzhou. q was defined as the spatial heterogeneity attribute whose statistical properties change in space. The range of q is from 0 to 1, where 1 implies the disease is completely controlled by the chosen environmental factor, while 0 means the disease is totally unrelated to the chosen environmental factor.Geographical detector was used to assess the impact of environmental factors on disease occurrence or transmission by evaluating the spatial heterogeneity between environmental factors and the diseases. The hypothesis was that the environmental factors would have the similar spatial distribution with the disease, if the chosen environmental factors contribute to the disease occurrence or transmission. This method has been applied in the environmental-related disease investigation, such as typhoid and paratyphoid fever ; hand, fX1 and X2 weaken or enhance one another when they work together. Layers X1 and X2 were overlaid and their attributes were combined (X1∩X2) as a new attribute, X3. The explanation levels of X1, X2, and X3 were calculated using the geographical detector, respectively. The interactive effects were judged using the following rules with explanation levels of X1, X2, and X3:q(X1), q(X2), q(X1∩X2 = X3) refers to explanation levels of X1, X2 and X3 conducted using the geographical detector. Min (q(X1), q(X2)), Max (q(X1), q(X2)) refers to the minimum and maximum values of X1 and X2 explanation levels.Interactive effect was applied to determine whether factors Moran’s I was applied to detect the spatial cluster of dengue fever, while the geographical detector was used to examine the individual and interactive impact of socio-ecological factors on dengue fever.In 2014, 37,322 indigenous dengue fever cases were reported in Guangzhou. The average incidence rate was 289.97 per 100,000 population. Women accounted for 50.73% with an average incidence rate of 307.39 per 100,000 population. Elderly individuals had the highest incidence rate . Among the occupations, household/unemployed represented the largest proportion (23.36%) of indigenous dengue fever cases, which is followed by the retired (13.99%). The general information of dengue fever in Guangzhou in 2014 is shown in the In Nine socio-ecological factors were mapped in 2 , 204.81 mm , 8.86 km/km2 , 27.43 °C , 56.22% , 16.47% , 11.50 km2 , and 2.92 km2 . Most areas with high value of socio-ecological factors were located in the center or south of Guangzhou, while precipitation was heavy in the northeast of areas, and the majority of water bodies were located in northwest and south areas. The mean values of these factors were 128,500 CNY , 17,562.44 person per kmR = 0.49, p < 0.01), road density , temperature , urbanization level , the ratio of urban villages , and precipitation . However, GDP per capita , VFC , and water body areas  were negatively correlated with the incidence. The results of geographical detector showed that seven factors, including precipitation, road density, temperature, urbanization level, VFC, urban village, and water body areas, are statistically significant (p < 0.05), but the GDP per capita and population density were not significant. Monthly average temperature (q = 0.33) was the dominant factor of dengue fever, followed by monthly average precipitation (q = 0.24), road density (q = 0.24), and water body area (q = 0.23).When only the interactive impacts of two socio-ecological factors were taken into consideration, 32 pairs of joint impacts were statistically significant, apart for four pairs . All the interactive impact results are shown in Nowadays, dengue fever has been regarded as the most prevalent and rapidly spreading mosquito-borne viral disease in humans. Exploring the risk factors and implementing control interventions are the major strategies to control and prevent global dengue epidemics. In the present study, we used a spatial analysis method to investigate the independent and interactive effects of several socio-ecological factors on the 2014 dengue epidemic at a township/street level in Guangzhou. We observed four clusters of dengue cases here; while the dengue infection risks demonstrated positive associations with road density, ambient temperature, urbanization level, urban village ratios, and precipitation, but negative associations with GDP per capita, VFC, and water body areas.The effects of population density, road density, urbanization, urban village, and GDP level on dengue epidemics have been assessed in several studies previously ,17,39,40Aedes albopictus tended to be the semi domestic mosquitoes and semi wild living mosquitoes, and it usually established breeding sites under shadowed water containers or near construction site pool [Urban villages were found to have a higher dengue incidence rate compared with other areas, which was similar to that of Saudi et al.’s study . Urban vite pool ,46. In aite pool ,48, and Aedes larvae or pupae per 100 houses [2, which was significantly lower than that in the Yuexiu district . Therefore, even though some people are infected by dengue virus, the virus may be difficult to be transmitted to other people [We further observed that environmental factors, including VFC and water body areas, were negatively related to dengue incidences, indicating that areas with more vegetation and water body had lower dengue incidence, which is consistent with several previous studies ,13,49. T0 houses ) was higr people ,52. Hower people ,53. The The positive associations between dengue incidence and temperature and precipitation in our study are similar to some previous studies ,56,57,58For interactive impacts, we mainly found that temperature and precipitation could significantly enhance other factors’ effects on dengue fever, which indicates that they may be the dominant determinants of dengue fever in South China, which is consistent with that of a previous study . The aveThis study has several strengths. First, we collected data at a fine spatial scale using the GIS and RS techniques and examined both independent and joint impacts of socio-ecological factors on dengue fever incidence, which extends our understanding of dengue transmission in South China. Second, to the best of our knowledge, this is the first study to investigate the effects of urban village, a special geographical phenomenon in the process of urbanization in China, on dengue transmission in China.However, several limitations should be mentioned while interpreting our findings. First, adult mosquito data in this study were inadequate to further verify the effects of socio-ecological factors on dengue fever transmission because the establishment of vector surveillance in Guangdong was finished in 2015. Moreover, the mosquito population data were spatially uneven in Guangzhou area. However, we used BI to indirectly explain their mediation effects during the effects of socio-ecological factors on dengue fever transmission. Second, mosquito survival and dengue fever transmission has been reported to decrease when daily temperature is either cooler than 18 °C or warmer than 32 °C . TherefoFour clusters of dengue cases were identified in Guangzhou in 2014. The dengue incidence might either be independently or be jointly impacted by multiple socio-ecological factors, including meteorological, environmental, and socioeconomic factors. These findings extend our understanding of the nature of dengue fever transmission, and also have important implications for the planning and implementation of control and prevention measures in South China.


Pneumonia is a leading infectious cause of morbidity and mortality among adults. Pneumococcal pneumonia (PP) is the most common vaccine-preventable bacterial etiology of pneumonia. In this study, we estimated the incidence of community-acquired pneumonia (CAP) and pneumococcal diseases among Korean adults.Clinical and microbiological databases from three hospitals were retrospectively reviewed to determine the incidence and case fatality rates of CAP and pneumococcal diseases in Korean adults aged ≥19 years from 2011 to 2014. Incidence and case fatality rates of CAP, PP and invasive pneumococcal diseases (IPD) were evaluated based on the catchment population. Catchment population was calculated using national health insurance data, estimating the proportion of patients with pneumonia that were medically attended at each hospital.Among 5,783 patients with medically attended CAP, 833 (14.4%) had PP. For IPD, a total of 91 culture-confirmed cases were identified. The overall incidence of CAP was 307.7 cases per 100,000 persons per year with an in-hospital mortality rate of 6.2%. The estimated annual incidence of pneumococcal pneumonia was 42.2–49.4 cases per 100,000 persons per year, increasing with age to >280 per 100,000 persons per year in older patients over 70 years. The annual incidence of IPD had a range of 4.1–6.5 cases per 100,000 persons per year. The overall case fatality rate for invasive pneumococcal diseases was 30.8% with the highest rate of 66.7% in patients over 80 years.Over the study period, incidences of CAP, PP and IPD were consistently high, particularly in older people. These results provide baseline data to establish healthcare strategies and estimate their impact among Korean adults. Streptococcus pneumoniae (pneumococcus) is the most common bacterial pathogen causing community-acquired pneumonia (CAP). However, the reported burden of pneumonia and pneumococcal diseases varies temporally and geographically [Pneumonia is a leading cause of morbidity and mortality worldwide, with 450 million annual cases with 1.3 million annual deaths in children and 1.6 million in adults aged ≥60 years [Annual incidence of CAP is reported to be 520–610 cases per 100,000 adults, increasing in older people ,8. In paIn South Korea, the National Immunization Program (NIP) provides free vaccination with PPSV23 for old adults; NIP was implemented for everyone aged ≥65 years in May 2013. Since May 2014, pediatric PCV10/PCV13 vaccination is included in the NIP, so all children can receive pneumococcal vaccinations. However, the burden of PP and IPD among adults is not well understood, impeding selection of optimal strategies and cost-benefit analyses for pneumococcal vaccination in South Korea.This study was planned to better understand the burden of pneumonia and pneumococcal diseases among adults in South Korea. We estimated the population-based incidence and case fatality rates of CAP, PP and IPD using a hospital catchment population.This retrospective study was conducted at three university hospitals  in different areas of South Korea between January 1, 2011 and December 31, 2014. This study was designed to determine the annual incidence and case fatality rates of CAP, PP and IPD, stratified by age group. To estimate incidences, numerators were defined as number of eligible cases (inpatient and outpatient) with CAP, PP and IPD in the study hospitals. Hospital catchment population was the denominator, calculated using the National Health Insurance Sharing Service (NHISS) database.The study was performed with approval of the institutional review boards at Korea University Guro Hospital, Hallym University Kangnam Sacred Heart Hospital and Chungbuk National University Hospital. Written informed consent was waived because all data were collected retrospectively by reviewing medical records. In addition, all medical records were fully anonymized before we accessed them.For each study year, the pneumonia catchment population of the university hospitals was calculated using data from the hospital and the NHISS database . Calculai Σj ΣkAijkPneumonia catchment population of each hospital = Σi = age, j = gender, k = areaAijk = pneumonia catchment population of a particular gender/age group in a particular areaHijk = number of pneumonia patients in a particular area with a particular gender and age who visited the hospitalMijk = number of pneumonia patients in a particular area with a particular gender and age who visited any medical institutionsPijk = number in the population in a particular gender/age group in a particular areaWe defined the catchment area of a study hospital as the collection of administrative divisions that had a visit from one or more patients with pneumonia. In South Korea, administrative divisions are composed of 262 areas that include 75 cities, 86 counties and 101 districts across the country. In each regional area, the proportion of patients with pneumonia visiting each study hospital was estimated by age group and gender using the NHISS database. The number of patients with pneumonia registered in catchment areas of the three study hospitals was 33,353 in 2011, 42,519 in 2012, 38,957 in 2013 and 28,778 in 2014. The number of hospitals that provided medical services to the patients was 2,718 in 2011, 2,818 in 2012, 2,693 in 2013 and 2,850 in 2014. For each area, the proportion was multiplied by the number in the population by age group and gender. Using this process, age group-specific and gender-specific catchment populations were estimated for each area, and the sum of these estimates was used as the catchment population for each of the hospitals. During the study period from 2011 to 2014, the catchment population was estimated annually .To estimate disease incidence of CAP, PP and IPD, annual numbers of cases were estimated by reviewing clinical and microbiological databases during the study period from January 1, 2012 through December 31, 2014. For each hospital, two infectious disease specialists reviewed all cases of pneumonia (ICD-10 codes J10–18 and J69) and selected those meeting the CAP criteria [Streptococcus pneumoniae urinary assay were also diagnosed as having PP [S. pneumoniae from a normally sterile site such as blood, cerebrospinal fluid, or pleural or ascitic fluid [CAP was defined based on clinical and radiological criteria of: (a) acute pulmonary infiltrate evident on chest radiographs and consistent with pneumonia within 48 h after admission, (b) confirmatory findings on clinical examination, and (c) acquisition of the infection outside a hospital . For pataving PP . Adequatic fluid .P values < 0.05 were considered statistically significant.Incidences were calculated as proportions by dividing the number of patients (numerator) with the catchment population (denominator), and expressed as number of cases per 100,000 persons per year with 95% confidence intervals. Age-dependent and year-dependent trends for disease incidence and case-fatality rate were analyzed by linear-by-linear chi-square test. SPSS version 20.0  was used for statistical analysis. During periods from 2011 to 2014, the study hospital catchment areas covered 193 areas of administrative divisions across the country. The number of patients in the pneumonia catchment population in the study hospitals was 1,871,312 . The proAge-stratified incidence and case-fatality rates for CAP significantly increased with age (p < 0.001): the highest incidence was in the age group of ≥80 years with 4,865.7 cases  per 100,000 persons per year . The ageS. pneumoniae-caused CAP cases from reports on CAP etiology. Although a few Korean reports present the characteristics and etiology of CAP, no data provide information on the population-based incidences of CAP, PP or IPD [In this study, the overall estimated incidence of CAP was annually 307.7 cases per 100,000 persons. This number increased with age, up to 4,865.7 cases per 100,000 people aged 80 years and older. Overall estimated annual incidences were 44.3 cases per 100,000 for PP and 4.8 per 100,000 for IPD, increasing to 676.1 for PP and 57.1 for IPD per 100,000 persons in adults aged ≥80 years. From incidence and population data based on 2015 resident registration for South Korea , we calculated around 128,000 cases of CAP, 18,500 cases of PP and 2,000 cases of IPD in Korean adults annually. Although CAP is a relatively frequent infectious disease in developed and developing countries, only a few studies are available on the incidence of CAP . The incP or IPD –28. ThisThe most notable findings of this study were the epidemiological trend of the annual incidence of CAP, PP and IPD from 2011 to 2014. Although there was a relatively brief period for understanding the epidemiological trend, CAP incidence annually tended to increase from 2011 to 2014. This trend was also observed in a recent study for hospitalized CAP in South Korea . AccordiThe annual incidence rate of CAP in adults was estimated to vary between 162 and 1001 per 100,000 persons depending on the country: United States , Germany , Spain  and Japan  –23. CompWe documented the annual incidences of PP  and IPD  among adults and their increase with age. PP comprised 14.4% of all-cause CAPs. Although Song et al. previously reported an increased clinical and economic burden of IPD by age and risk groups in South Korea, they did not reported the IPD incidence because of study limitations . EstimatThe primary strength of this study was that we identified CAP, PP and IPD cases through a reliable review process by infectious disease experts. We estimated the incidence and case-fatality rate longitudinally over four-year study periods. Contrary to studies using the NHISS database alone, we avoided overestimation by excluding healthcare-associated pneumonia cases by reviewing medical records. In the recent study in South Korea, the annual CAP incidence was estimated to be 626 per 100,000 persons, which was higher than that of the present study . In addiIn conclusion, the incidences of CAP, PP and IPD were consistently high, particularly in older patients over the study period. The proportion of PP increased markedly with age. These results provide baseline data to establish healthcare strategies and estimate their impact among Korean adults.S1 Table(DOCX)Click here for additional data file.


Lung cancer (LC) screening will be more efficient if it is applied to a well-defined high-risk population. Characteristics including metabolic byproducts may be taken into account to access LC risk more precisely. Breath examination provides a non-invasive method to monitor metabolic byproducts. However, the association between volatile organic compounds (VOCs) in exhaled breath and LC risk or LC risk factors is not studied. Exhaled breath samples from 122 healthy persons, who were given routine annual exam from December 2015 to December 2016, were analyzed using thermal desorption coupled with gas chromatography mass spectrometry (TD-GC-MS). Smoking characteristics, air quality, and other risk factors for lung cancer were collected. Univariate and multivariate analyses were used to evaluate the relationship between VOCs and LC risk factors. 7, 7, 11, and 27 VOCs were correlated with smoking status, smoking intensity, years of smoking, and depth of inhalation, respectively. Exhaled VOCs are related to smoking and might have a potential to evaluate LC risk more precisely. Both an assessment of temporal stability and testing in a prospective study are needed to establish the performance of VOCs such as 2,5-dimethylfuranm and 4-methyloctane as lung cancer risk biomarkers. Lung cancer has become the most commonly diagnosed cancer as well as the leading cause of cancer death globally . EffectiIn this paper, we proposed a method to identify individuals at a high risk for lung cancer using VOCs pattern in exhaled gas, which was analyzed by thermal desorption coupled with gas chromatography mass spectrometry (TD-GC-MS). As of now, smoking is a strong lung cancer (LC) risk factor and plays an important role in the carcinogenesis of cells in lung and affects the immunological activity and metabolic procedure . How theVOCs in exhaled breath are one kind of metabolic byproduct that can be used to monitor health status and the progression of diseases ,9. StudiThe lung carcinogenesis can take years or even decades. Studies provided evidence that tobacco smoking is a major risk factor for lung cancer ,19. CigaWe used thermal desorption coupled with gas chromatography mass spectrometry (TD-GC-MS) to analyze exhaled breath samples of 122 healthy volunteers who were undergoing a routine annual medical checkup. The exposure to LC risk factors were recorded for all of the subjects. We identified an association study to analyze the relationship of VOCs and risk factors of lung cancer. Our study was undertaken to find VOCs that may serve as markers of risk and may help define a group at high risk of lung cancer more precisely.The characteristics of subjects are presented in The number of VOCs detected in each breath sample ranged from 108 to 452  a. The in377 VOCs, which were present in more than 30 subjects out of all 122 samples, were observed. The following statistics are based on these 377 VOCs. Results showed that 7, 7, 11, and 27 VOCs were correlated with smoking status, smoking intensity, years of smoking, and the depth of inhalation , respectp-values are shown in cis-1,3,5-hexatriene), which were highly significantly correlated with smoking status, were compounds in cigarette gas. CAS No. 112-31-2  was highly significantly correlated with smoking intensity. In addition, 4 and 3 VOCs were highly significantly correlated with years of smoking and the depth of inhalation, respectively. It is worth noting that DL-sec-butyl acetate (CAS No. 105-46-4), which is highly significantly correlated (p = 0.006) with years of smoking, was greatly influenced by the ambient air , which was negatively correlated with smoking intensity in our study, was the only ester reported to be linked to lung cancer [Seven and 11 VOCs were related to smoking intensity and years of smoking, respectively. The mean concentration of most VOCs increased as the smoking intensity and duration increased. This shows that the exhale metabolites can reflect the cumulative exposure of smoking. It is worth noting that butyl acetate (g cancer . A decrep < 0.01) with years of smoking. 2,4-Dimethyl-1-heptene [In our study, 2,4-dimethyl-1-heptene, DL-sec-butyl acetate, 2,3-dimethylheptane, and 4-methyloctane indicated a strong positive correlation  with the depth of smoke inhalation in our study, was reported as a breath biomarker of lung cancer [Decane, which had a correlation  after Bonferroni correction, the p-values are somewhat liberal to most other VOCs. Moreover, our study was only taken in Sir Run Run Shaw Hospital, Zhejiang province, China. This study population might not represent the entire population of China. Though the power of this study was limited, it does suggest that the metabolic VOCs detected in breath may reflect LC risks. Multi-center screening trials are needed to verify the findings. Furthermore, studies have shown not only tobacco smoke but other factors such as asbestos fibers are significant inhaled carcinogens that contribute significantly to lung adenocarcinoma development [VOC assessment provides a non-invasive approach to identify LC risk factors by evaluation of components of exhaled breath. Breath detection of LC biomarkers, such as butyl acetate, decane, heptane, 2,5-dimethylfuranm and 4-methyloctane, not only can be used to diagnose lung cancer but can also be used to estimate the risk of lung cancer. However, our study has limitations. Although 2,5-dimethylfuranm has a significant elopment . In our We continuously recruited 122 healthy people from December 2015 to December 2016 at Sir Run Run Shaw Hospital, Hangzhou, China. The participants performed exhaled breath test and filled out a questionnaire. People who were undergoing an annual routine examination were asked to participate in the study. The exclusion criteria were as the following: (1) people with an age of detection less than 18 years old; (2) pregnant women; (3) people with other tumor history or suffering from severe infection; (4) people who did not understand or cooperate with any part of the gas collection process. This study was approved by the ethics committee of Sir Run Run Shaw hospital , and written informed consent was obtained from every participant.To ensure the accuracy of test results, the participants were required to comply with the following requirements: (1) fasting 12 h before collecting samples; the participants were instructed to avoid high-fat foods at dinner of the evening before the collection; (2) the participants were asked to abstain from smoking for 12 h before collecting samples; (3) antioxidants should not have been used for 24 h prior to the sample collection; (4) gargling was required 15 min before the collection.Each participant completed a questionnaire. The survey items mainly focused on risk factors of lung cancer. The major risk factor was smoking behavior, so information on smoking status, years of smoking, smoking intensity, the depth of smoke inhalation, and second-hand smoking for participants who never smoked was obtained via the questionnaire.The VOCs were captured and concentrated from 1000 mL of exhaled breath into a Tenax TA stainless steel tube . The capture was realized by a portable breath collection device, which was made in our own lab . The subm/z) range. The scan time was 2.5–38 min. The temperatures of the electron impact ion source and interface were set at 200 °C and 250 °C, respectively.The composition analysis procedure was completed via a thermal desorption (TD) instrument  and gas chromatography-mass spectrometry (GC-MS) . TD was used to desorb the VOCs adsorbed in the Tenax TA stainless steel tube. GS-MS analyses were performed following the desorption procedure. The VOCs, then, were sent into an Rtx-5 column  to be separated. The carrier gas was helium. The column temperature program was 40 °C initially, held for 1 min, and increased to 250 °C at 5°/min, and held for 1 min at 250 °C. The MS was in scan mode in the 45–500 mass to charge  was dissolved in methanol . The mixed solution was converted into mixed gas by a gas generator . The concentrations of 2,5-dimethylfuranm in the mixed gas were 0.1, 0.2, 0.5, 1.0, and 2.0 ppb, which covered the range in exhaled gas. The mixed gas was kept in a 2 L Tedlar bag. The process of analysis was as same as that for exhaled gas. Results indicated the detection system had a good linear response , and quality of fit, were automatically downloaded into a text file.Ambient air in the room, where the breath test was performed, was also collected as a background for the test. The VOCs in ambient air were also managed according to the procedure described above.A VOC is not only identified by spectral library match, but also by its retention time . AccordiAmbient air data as a baseline would be subtracted from the sample data. VOCs presented in breath and in air whose CAS number was the same and the relative time difference of RT was less than 5% were regarded as the same compound. Equation (3) describes the process. Equation (4) shows that the peak area of a VOC in ambient air is subtracted from its corresponding peak area in breath. VOCs data and data from questionnaire of each volunteer were recorded in a dataset for further analysis. The CAS No., retention time, and peak area of each VOC were included in the dataset. Meanwhile, age, gender, and smoking behavior were also recorded in the dataset. Statistical analysis was done by Statistical Package for the Social Sciences (SPSS) , GraphPad  and MATLAB  for Windows.p-value of less than 0.05. A multivariate linear regression analysis was performed to adjust age and gender. Conservative Bonferroni correction was applied to test the significance in multiple analyses.Pearson correlation coefficient was used to screen VOCs that were correlated with risk factors of LC and to evaluate their linear correlations. The t-test was used to compare the correlative VOCs. The level of significance was set to a In conclusion, we found 7, 7, 11, and 27 VOCs were correlated with smoking status, smoking intensity, years of smoking, and depth of smoke inhalation, respectively. Exhaled VOCs are related to smoking, and might have a potential to evaluate LC risk more precisely. Both an assessment of temporal stability and testing in a prospective study are needed to establish the VOCs’ performance such as 2,5-dimethylfuranm and 4-methyloctane as lung cancer risk biomarkers.


This was a prospective multicenter observational study, aiming to explore the effects of acupuncture on smoking cessation in Hong Kong. From March of 2010 to August of 2015, a total of 5202 smokers were recruited based on inclusion criteria and treated with acupuncture for 8 weeks. As a result, 2940 subjects finished the study with a drop-out rate of 43.48%. The self-reported 7-day point abstinence rate was 34.00% in Week 8 and 18.40% in Week 52. The exhaled carbon monoxide level and the number of cigarettes smoked per day were reduced significantly after treatment. The time to relapse was calculated to be 38.71 days. In addition, “cigarettes smoked per day,” “Fagerstrom Test for Nicotine Dependence,” “total sessions of acupuncture,” “whether finished 8 acupuncture treatments in the first month,” and “total sessions of acupuncture” were believed to be essential factors for abstinence success. It was concluded that acupuncture was a safe method for smoking cessation and was effective in helping smokers to quit; therefore, acupuncture could be considered as one of the methods to help smokers quit. Further studies regarding the effect differences between acupuncture and medications were needed to clarify the overall benefits of acupuncture. Smoking  Regulations, was established. After then, Hong Kong government started to make a comprehensive tobacco control from aspects of legislative control, tobacco tax, and public education. The latest survey by Census and Statistics Department of Hong Kong in 2013 [Smoking is believed to be a serious health threat in the world, which is estimated to involve approximately 6 million deaths each year . As one  in 2013  showed t in 2013 , includiAs one of the essential components of the smoking cessation services, acupuncture is widely accepted in Hong Kong, but its effectiveness is still controversial. The research by Vincet and Richardson  showed “Therefore, the effect of acupuncture for smoking cessation in Hong Kong was in need of comprehensive evaluation. In order to achieve this propose, with the support from Tobacco Control Office of Health Department of HKSAR as well as State Administration of Traditional Chinese Medicine (TCM) of China, this study was conducted by Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, and Hong Kong Pok Oi Hospital. It was hoped to provide references for further application of acupuncture for smoking cessation in Hong Kong.This was a prospective observational study, which is aimed at preliminarily evaluating the 8-week and 52-week effect of acupuncture for smoking cessation in Hong Kong. This study composed of the preparation period, 8-week acupuncture treatment, and 44-week follow-up visit. The study duration was from March of 2010 to August of 2015. The study sites included 5 clinics of Pok Oi Hospital in Tin Shui Wai, Tsing Yi, Taikoo, Huafu, and Cheung Chau as well as Kwun Tong Community Health Center, Ping Shek TCM Community Clinic, and Causeway Bay Acupuncture Center. In addition, 20 TCM mobile clinics were applied to provide smoking cessation for 48 major areas in Hong Kong.The subjects who met the inclusion criteria were allocated to the study sites to receive acupuncture for smoking cessation. However, control group was not established in this study, because the protocols for sham acupuncture have not been well validated and need to be future studied. The clinical evaluation was performed 1 week, 2 weeks, 8 weeks, 26 weeks, and 52 weeks after treatment. All the subjects were recruited via local newspaper, hotline of smoking cessation, community events, hospital websites, and recommendation from other medical institutions. Subjects were included into this study if they voluntarily participated in this study; they were daily smokers, aged 18 to 70 years, and were willing to quit smoking; the subjects were excluded if they had mental diseases, serious cardiovascular diseases, apoplexy, or nervous system diseases, had disturbances of blood coagulation, or were pregnant.ChiECRCT-2013014).The subjects were firstly preliminarily evaluated by researches, and data including personal information, general health condition, and smoking background were recorded. After setting a quit date, subjects were allocated to the study sites. All the subjects signed informed consent before receiving any acupuncture treatment. This study was approved by China Ethics Committee of Registering Clinical Trials , Lieque (LU 7), Hegu (LI 4), Zusanli (ST 36), Sanyinjiao (SP 6), and Taichong (LR 3). Yintang (EX-HN3) was added if there were withdrawal symptoms of cough, running nose, dry eyes, and so forth, while Neiguann (PC 6) was added if there were withdrawal symptoms of dysphoria, melancholy, insomnia, and so forth.Body Position. Subjects were in supine position to expose the body acupoints.Acupoint Location. Refer to the Acupuncture Point Locations in the Western Pacific Region by World Health Organization .Manipulation of Acupuncture. Routine fertilization was given on the selected acupoints with 75% medicinal alcohol before acupuncture. Needles were horizontally inserted at Baihui (GV 20) while vertically inserted at other acupoints with a depth of 25 to 50 mm. The mild reinforcing and reducing technique was applied at all acupoints. The arrival of qi (needle sensation) was required and needles were required to stay at the acupoints for 30 min. Meanwhile, electroacupuncture device (HuaTuo SDZ-III type) was used and connected at Lieque (LU 7) and Zusanli (ST 36) with continuous wave (15 Hz) for 30 min. The needles were withdrawn quickly to avoid bleeding or hematoma.Treatment Frequency. It was twice per week for 8 weeks.Selection of Acupoints. Shenmen (TF4), Neifenmi (CO18), Pizhixia (AT4), Jiaogan (AH6), Fei (CO14), and Wei (CO4) were the acupoints. Kou (CO1) was added if there were withdrawal symptoms of nausea, while Zhiqiguan (CO16) was added for cough phlegm.Body Position. Subjects were in sitting position to fully expose the auricular acupoints.Acupoint Location. Refer to China National Stand Nomenclature and Location of Auricular Points (GB/T 13734-2008) [34-2008) .Manipulation. After routine disinfection, the patch of vaccaria seeds with 2 mm in diameter were used and stuck to different auricular acupoints. Each auricular acupoint was pressed for 1 min. In addition, subjects were instructed to press each auricular acupuncture point for 20 s every 1 to 2 hours or whenever they had a craving for smoking.Treatment Frequency. Treatment frequency was twice per week for 8 weeks.The primary outcome was self-reported 8-week 7-day point abstinence rate , which wThe secondary outcomes were self-reported 7-day point abstinence rate in the 1st week, 2nd week, 26th week, and 52nd week, number of cigarettes smoked per day, and exhaled breath carbon monoxide level . All the results were acquired by inquiring and testing subjects at each visiting point.Besides, the safety of acupuncture was assessed. The blood clotting was tested before acupuncture, while blood pressure, pulse, breath, and body temperature were measured before and after treatment as well as in follow-up visit; adverse events were recorded during the study, including the symptoms, occurrence date, and intervention and its relationship with treatment.According to our plot study , the post-test was used for comparison which met Gaussian distribution and homogeneity of variance, while nonparametric test was used for comparison which did not met homogeneity of variance. Chi-square test was used for numeration data. The Kaplan-Meier curve was used for analyses of time to relapse. The logistic regression analysis was used to calculate the relationship between possible influence factors and abstinence results, represented with OR value and 95% credibility interval. P < 0.05 was taken as statistical significance. The authors had no access to information that could identify individual participants during or after data collection.Intent to Treat (ITT) was applied in this study. SPSS 19.0 statistical software was used for statistical analysis. The measurement data were represented with means ± standard deviation ; 2605 subjects finished 8 acupuncture treatments in the first month, while 2721 subjects finished at least 8 treatments in the study.According to the self-reported 7-day point abstinence rate, 1106 subjects succeeded in smoking cessation in the 1st week, 1237 subjects succeeded in the 2nd week, 1769 subjects succeeded in the 8th week, 1142 subjects succeeded in the 26th week, and 955 subjects succeeded in the 52nd week . This inP < 0.05); the number of cigarettes smoked per day was also significantly decreased from (17.67 ± 7.96) before treatment to (4.34 ± 5.43) after 8-week treatment (P < 0.05) but was significantly increased in follow-up (P < 0.05).As was shown in The results of Kaplan-Meier analysis showed the time to relapse was 38.71 days (95% CI 37.12–40.32) for acupuncture .The relationship between baseline characteristics and study outcomes was calculated by logistic regression analysis. As was shown in This study was aimed at exploring the effects of acupuncture for smoking cessation in HKSAR. It was indicated that 8-week acupuncture was safe for smoking cessation; the abstinence rate was 34.0% in Week 8 and 18.4% in Week 52, respectively. In addition, the exhaled breath CO level and number of cigarettes smoked per day were significantly reduced in subjects who failed in smoking cessation; the time to relapse was 38.71 days, which was longer than 35 days in E-cigarette and 14 days in nicotine patch [This result was consistent with conclusions of 27.15% by Wu et al. , 4.1% byThe strengths of our study included rigorous study design and the use of a conservative outcome measure and to minimize the risk of bias.Firstly, as was demonstrated, a high drop-out rate in smoking cessation study was inevitable, even if the adherence to treatment of different research designs was not satisfactory 19% to 62%) % to 62% , one-yeaSecondly, although several researches –23 recom qi; acupuncture at Zusanli (ST 36) can regulate qi movement of spleen and stomach to relieve the discomfort caused by smoking cessation; acupuncture at Baihui (GV 20) can calm the mind to relieve the anxiety. The use of auricular acupoints can not only enhance the effects of body acupoints but also induce subjects to have a negative taste and smell to tobacco. Consequently, the desire for cigarettes was reduced [Thirdly, our acupuncture plan was based on literature research  and pilo reduced . Due to Fourthly, besides the unified treatment plan, more individual acupuncture for smoking cessation was provided. For example, in order to relieve withdrawal syndrome during the smoking cessation, subjects were instructed to press each auricular acupoint at home or when they felt a strong craving for smoking, especially after dinner or intellectual work. Furthermore, acupuncturists, after took course provided by researchers, were asked to add or reduce acupoints according to different withdrawal symptoms.Fifthly, a total of 20 TCM mobile clinics for smoking cessation were introduced for subjects who had busy work schedule or lived in the outskirts. Unfortunately, a high drop-out was still inevitable, indicating that the effects of acupuncture on smoking cessation may be related to personal environment and living factors . It was Our study had several limitations. Firstly, it was difficult to design an appropriate control group for acupuncture. Sham acupuncture is often used as control, but the protocols for sham acupuncture have not been well validated and need to be future studied . SecondlWe conclude that acupuncture is a safe method for smoking cessation and is effective in helping smokers to quit; therefore, acupuncture could be considered as one of the methods to help smokers quit in Hong Kong. Further studies regarding the effectiveness differences between acupuncture and nicotine replace treatment, Varenicline, and Bupropion are needed to establish the overall benefits of acupuncture.


Ixodes persulcatus is a predominant hard tick species that transmits a wide range of human and animal pathogens. Since bacterial flora of the tick dwelling in the wild always vary according to their hosts and the environment, it is highly desirable that species-associated microbiomes are fully determined by using next-generation sequencing and based on comparative metagenomics. Here, we examine such metagenomic changes of I. persulcatus starting with samples collected from the wild ticks and followed by the reared animals under pathogen-free laboratory conditions over multiple generations. Based on high-coverage genomic sequences from three experimental groups–wild, reared for a single generation or R1, and reared for eight generations or R8 –we identify the core bacterial flora of I. persulcatus, which contains 70 species that belong to 69 genera of 8 phyla; such a core is from the R8 group, which is reduced from 4625 species belonging to 1153 genera of 29 phyla in the wild group. Our study provides a novel example of tick core bacterial flora acquired based on wild-to-reared comparison, which paves a way for future research on tick metagenomics and tick-borne disease pandemics. Ixodes persulcatus, is a predominant hard tick species found in Europe, central and northern Asia, China, and Japan. Tick is the second most widely recognized transmission vectors of human diseases worldwide, second only to mosquitoes [Borrelia burgdorferi and Anaplasma phagocytophilum cause Lyme disease [I. persulcatus is a vector for the tick-borne encephalitis virus [Babesia [The tick, squitoes ,2. Ticks disease ,4 and gr disease , respect[Babesia , 7 and R[Babesia , 9.As progresses made through various microbial sequencing programs, such as the Human Microbiome Project , we haveI. persulcatus, we start from wild I. persulcatus, rear the tick in specific pathogen-free (SPF) mice in a sterile environment, and examine its flora over multiple generations. We then use a next-generation-sequencing method, acquire high-coverage metagenomic data, and analyze them by comparing the metagenome change among the samples. Here, we report our results for defining the core bacteril flora of I. persulcatus.To explore such core symbiotic bacterial flora of I. persulcatus strain Sfh has been reared in our laboratory; it is originally from female ticks collected with cloth-dragging methods from a forest region of Suifenhe City , Heilongjiang Province of northeastern China. The natural enzootic cycle is completed by growing larvae and nymphs on shaved skin of SPF Balb/C mice. We used female adults for all experiments. Larvae were obtained by placing 1.0 g hatched eggs onto feeding patches glued to the shaved patches of SPF Balb/C mice. After feeding, all larvae were removed before they molted to the nymphal stage. Nymphs were molted to the adult stage (first generation), and some adult ticks were maintained at room temperature for 1–3 weeks prior to hatching for propagation to the eighth generation. Other adult ticks were frozen at −80°C for DNA extraction.The Before DNA extraction, 30 to 40 adult female ticks were sterilized in 70% ethanol for 20 min and washed three times in distilled water. Total DNA was extracted using the Qiagen DNA extraction kit  according to the manufacturer’s instructions. Paired-end libraries were constructed according to the manufacturer’s instructions (Illumina) and sequenced by using the Illumina Hiseq 2500 platform.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed on both ends using the FASTX-Toolkit, leaving high-quality nucleotides (http://hannonlab.cshl.edu/fastx_toolkit). The high-quality reads were aligned to NCBI NR databases using DIAMOND [Paired-end library sequencing reads were quality controlled with FastQC , heatmap, and phylogeny (http://itol.embl.de/help.cgi) were used to show shared phyla and genera [Venn diagram , Heilongjiang Province of northeastern China. Our study didn’t involve any endangered or protected species.th generation. We use 40 and 30 adult females for DNA extraction for the wild and reared groups, respectively, since the entire animals are ground together. The sequences are from paired-end libraries and one lane for wild group, another lane for R1 and R8 group. Sequence coverage and read length are both within their standards; we note that the matched (protein sequences collected for Blastp) reads are an order of magnitude lower in R8 than the rest, both wild and R1 (We divide samples into three groups: wild, direct collection from the wild; R1, reared in the laboratory as the first generation; and R8, reared as the 8d and R1 .Our raw data from the three groups range from 23 Gb to 47 Gb in total nucleotides and from 111 million to 231 million in sequencing reads. Annotation based on Blastp narrows useful reads into 59 million for wild, 58 million for R1, 7.1 million for R8. Rare fraction curve analysis indicates  data satWe annotate 80.39%, 89.96%, and 26.31% of all assigned bacterial reads of the groups, wild, R1, and R8, respectively, at the kingdom level . The annAt the phylum level, the annotated read distribution is 29 for wild, 31 for R1, and 8 for R8, where all 8 phyla in R8 are shared by the rest, representing candidates for the core bacterial flora. Since most phyla are accounted for only a small fraction of the total reads, so we define the major bacterial phyla as those greater than 1% Figs . The majAt the genus level, there are 1153, 1193, and 69 genera for wild, R1, and R8 , respectContinuous rearing in a sterile environment has lead to only 69 genera in the R8 ticks; these bacteria belong to 8 different phyla that are shared among all three groups. Therefore, we tentatively attribute the 69 genera to be the core bacterial flora of the tick. After normalization, the sequence reads of all three groups Figs  and 6 arRickettsia genus, R1 has 22, and R8 has 7; the 7 species of R8 are all shared by the rest 2 groups.At the species level, the 69 genera of the core tick bacterial flora have 1219 species for wild, 1307 species for R1, and 74 species for R8, of which 70 species are shared by both wild and R1. For example, wild has 38 species of I. persulcatus has been studied by using metagenomics [As a predominant hard tick species in Europe, central and northern Asia, China, and Japan, genomics , 28. Up genomics , which hgenomics , 19. Howth generation the core bacterial flora becomes stabilized. We also need to know how stable such floras are for ticks from a single origin and how variable it can be for ticks from different origins, even at the individual level in an ultimate sense. There are, of course, more questions can be asked for much more details as to how the floras change gradually from the wild conditions to the rearing in each generation, especially under different controlled rearing, such as pathogen-free vs natural hosts. Ample categorization procedures are of importance for more systematic studies.In our study, concerning methodology, we took one of the two popular approaches, genome-wide sequencing approach rather than transcriptomes sequencing, and hope to provide an initial definition of the tick core bacterial flora. Previous reports on tick metagenomics have not used genome-wide sequencing approach  and theyI. persulcatus. Third, we have identified a tentative species-associated core bacterial flora that is composed of 69 genera and 70 species. We notice that the number of species is almost equal to that of the genera as the tick core bacterial flora, and such a phenomenon is rather common due to relative less certainty at the species level. Fourth, the largest number of species common in all 3 experimental groups are from the genus Rickettsia, which are endosymbiont of I. scapularis. This species plays an important role in sex determination in ticks [Nevertheless, such a study is already very informative for further definition of the core floras of the tick under various conditions. First, we notice that at the kingdom level R8 has much less annotated reads as compared to the rest two samples. Judging by the increase of eukaryotic reads, it is reasonable to attribute the reduced annotation to unknown eukaryotes. Another loss in R8 is Archaea, similar to the case of eukaryotes. Second, the dramatic reduction of bacterial phyla and genera after rearing suggests the fact that most of the tick dwelling microbes from the wild are both temporary and environment-associated. It is obvious that an effort to define a species-associated core bacterial flora is both necessary and important for in ticks .High-throughput sequencing, coupled with bioinformatics analysis, provides a powerful tool for defining microbiome or flora of ticks, and such floras and their variations provide diagnostic markers or clues for disease transmission and infection mechanisms. Our results represent initial steps that uncover the species-associated flora and its variability under the influence of environmental elements, including hosts, geography, climates, and etc. Our results justify further metagenomic studies on the tick, including further anatomic stratifications.


Pseudonocardia and Rubrobacter and unclassified fungi in the order of Capnodiales were dominant. The relative abundance of Pseudonocardia in the painted layer samples was higher than that in the dust sample, while Cyanobacteria dominated in the dust sample. Many of them have been discovered at other cultural heritage sites and associated with the biodeterioration of cultural relics. The presence and activity of these pioneering microorganisms may lead to an unexpected deterioration of the painted sculptures that are preserved in this heritage site. Thus, proper management strategies and potential risk monitoring should be used in the Maijishan Grottoes to improve the conservation of these precious painted sculptures.In this study, a culture-independent Illumina MiSeq sequencing strategy was applied to investigate the microbial communities colonizing the ancient painted sculptures of the Maijishan Grottoes, a famous World Cultural Heritage site listed by UNESCO in China. Four mixed samples were collected from Cave 4–4 of the Maijishan Grottoes, the so-called Upper Seven Buddha Pavilion, which was built during the Northern Zhou Dynasty (557-581AD). The 16/18S rRNA gene-based sequences revealed a rich bacterial diversity and a relatively low fungal abundance, including the bacterial groups Actinobacteria, Acidobacteria, Bacteroidetes, Cyanobacteria, Chloroflexi, Firmicutes, Proteobacteria and Verrucomicrobia and the fungal groups Ascomycota, Basidiomycota and Chytridiomycota. Among them, the bacteria genera of  The biodeterioration of cultural heritage is a ubiquitous and inevitable phenomenon that has attracted conservators, archaeologists and scientists who are involved in the conservation of cultural heritage and relics. Microorganisms are the most diverse group of known life in the world; they occupy almost all the niches of the biosphere due to their abundant diversity, tenacious vitality and exuberant metabolism. Previously, multiple reports have documented that almost all types of historic artifacts, including the mural paintings in temples, caves and catacombs –6, and eThe selection of an appropriate strategy for understanding microfloral characteristics is indispensable for clarifying the ‘disease’ mechanisms induced by these microbes. Historically, microbial communities were mainly investigated by a culture-based method. However, only less than 1% of the microorganisms present in environmental samples can be cultured in laboratory conditions, and these cultures only represent the isolation of easily cultivable microorganisms . Even soIn this study, the Maijishan Grottoes, a famous Buddhist cave temple located on the ancient Silk Road in China, was selected. The Maijishan Grottoes are known for their beautiful scenery, numerous caves, precious artwork and long-spanning history. The site was added to the World Heritage List in 2014 as one of the key historical sites of the “Silk Roads, the Route Network of the Chang'an-Tianshan Corridor.” The priceless painted sculptures that are preserved in the caves of the Maijishan Grottoes are severely threatened by multiple factors, including the poor stability of the mountain rocks, frequent earthquakes, water seepage, microclimate change and human disturbance, in addition to microorganisms. More severely, the cultural relics preserved in caves had been exposed to high humidity (RH ≥70%) in the atmosphere for long periods, namely, more than six months of the year . Generally, the physical conditions are considered extremely unfavorable to the conservation of various types of cultural relics –19. ConsAspergillus, Cladosporium and Flavobacterium [3O4). Recently, the microfloral characteristics that were present on the wall paintings with signs of damage, along with their temporal and spatial distribution patterns in different caves, were also investigated. Furthermore, the key environmental parameters that influence community distribution patterns and their relationship with the ambient conditions were further investigated [Previously, research on the microbial invasions of wall paintings was only carried out at the Mogao Grottoes, a cultural heritage site along the Silk Road. As early as the 1990s, the staff of the Dunhuang Academy first isolated cultivable microorganisms from 51 samples of discolored paintings that were collected from six ancient caves constructed in different dynasties. Several functional genera, including acterium , were idacterium . These pstigated ,25. The stigated ,27. OverA comprehensive understanding of the microflora colonized on the painted sculptures in the Maijishan Grottoes is a crucial step for the effective protection and management of this site’s ancient relics in the future. In recent years, a huge number of tourists have been attracted by the Maijishan Grottoes. Statistically, approximately 57,000 visitors entered the grottoes during the May Day Holiday (1 May to 3 May) in 2016, but the maximum tourist capacity at the site is under 6,400 per day. Undoubtedly, too many tourists flooded into a relatively limited space in a short time, resulting in a micro-climate alteration, which, in many cases, is harmful to the conservation of cultural heritage sites. For example, the Paleolithic rock paintings of the Lascaux Cave suffered successive biological invasions since its discovery due to artificial disturbances, most of which were tourists ,28. ThusOverall, to improve the conservation of the cultural heritage preserved in the Maijishan Grottoes, it was necessary to investigate the microbial communities on the painted sculptures. We adopted an innovative molecular strategy, namely, Illumina MiSeq sequencing, to analyze the samples of the discolored painted sculptures that were collected from Cave 4–4 of the Maijishan Grottoes. Our research objectives were to: (1) describe the complete bacterial and fungal communities on the painted sculptures in the Maijishan Grottoes; (2) define the core phylogenetic groups among the whole microbial community; and (3) assess the potential threat of invasive microbial groups at this study site. This is the first report investigating the characteristics of the microbial community on the painted sculptures of the Maijishan Grottoes. It may not only contribute to clarifying mechanisms of biodeterioration induced by the inhabiting microbes but also provide opportunities for microbiologists to formulate preventive and remedial strategies to control the undesirable growth of microorganisms on this priceless and fragile artwork.The Maijishan Grottoes are situated in Maijishan, 30 km southeast of Tianshui city in the Gansu Province, China . This ciThe Maijishan Grottoes are surrounded by the Qinling Mountains. The Grottos have a warm, semi-humid continental climate with an average annual temperature of 10.4°C, an average annual relative humidity of 69.2% and an annual rainfall of 680 mm. A unique geographical advantage gives the location a pleasant climate, without brutal heat in the summer or bitter cold in the winter, and a clear rhythm of the four seasons, with a beautiful landscape and various well-known natural sites.Three painted sculptures were selected for sampling in Cave 4–4 of the Maijishan Grottoes , the so-called “Upper Seven Buddha Pavilion,” which was built during Northern Zhou (557-581AD). For each statue, three independent sampling locations on the surface of painted layer were chosen, and the samples were carefully collected with sterile swabs and scalpels. Then, the samples were pooled together in three parallel repetitions and mixed thoroughly to form a single sample . Only a All the samples were collected under strict aseptic conditions. The sample materials were collected in a sterile centrifuge tube and stored at -20°C until further analysis. The sampling process was authorized and supervised by the administrative staff of the Art Institute of the Maijishan Grottoes.® ND-2000 .The four sample materials were used for genomic DNA extraction with a commercially available extraction Kit  according to the manufacturer’s protocols. For each sample, the integrity of the DNA was determined by electrophoresis on 1.0% agarose gels, and the concentration and purity of the DNA were measured spectrophotometrically with a NanoDrop5'-ACTCCTACGGGAGGCAGCAG-3', and 806R, 5'-GGACTACHVGGGTW TCTAAT-3' [5'-TTAGCATGGAATAATRRAATAGGA-3', and 1196R, 5'-TCT GGACCTGGTGAGTTTCC-3' [The universal primers 338F/806R for bacteria or 817F/1196R for fungi was used for the amplification and MiSeq sequencing of the PCR products. The 16S rRNA genes were amplified using the primer set: 338F, CTAAT-3' , which tTTTCC-3' , which t2O to the final volume. The PCR program included an initial denaturation at 95°C for 3 min; 35 cycles at 94°C for 30 s, annealing at 55°C for 30 s and an extension at 70°C for 45 s; and a final extension step at 72°C for 10 min. PCR was performed using an ABI GeneAmp® 9700 Cycler . Different “barcode” sequences were added at the 5' end of the forward primer to separate the corresponding reads from the data pool that was generated in a single sequencing run. The amplicons were extracted by electrophoresis in 2.0% agarose gels, purified using a Gel Extraction Kit  according to manufacturer’s instructions and quantified using a QuantiFluor™-ST Fluorimeter . The purified amplicons were pooled in an equimolar and paired-end sequence (2×300) on an Illumina MiSeq PE300 Sequencer  according to standard protocols.To minimize the impact of potential early round PCR errors, PCR was carried out in triplicate with a 20-μL reaction volume, containing 4 μL of 5-fold reaction buffer, 4 μL of dNTPs (2.5 mM), 0.8 μL of each primer (5 μM), 0.2 μL of BSA (20 μg/μL), 1 μL (approximately 10 ng) of template DNA and 0.4 μL of Pfu DNA Polymerase , with dd Hhttp://www.arb-silva.de) and the Unite database (http://unite.ut.ee/index.php) were used for bacteria and fungi, respectively. The β-diversity analysis, including Principal Component Analysis (PCA) and a hierarchical heatmap, was generated using the Vegan packages in R (3.2.0), while the Venn diagrams were implemented by the Venn diagram package. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (SRA accession: SRP095989).Raw FASTQ files were de-multiplexed and quality-filtered using Trimmomatic (Version 0.35) with the following criteria: (ⅰ) The 300-bp reads were truncated at any site that obtained an average quality score below 20 over a 50-bp sliding window, and the truncated reads shorter than 50 bp were discarded; (ⅱ) Extracted matching barcodes, two nucleotide mismatch in primer matching, and reads containing ambiguous characters were removed; (ⅲ) Only overlapping sequences longer than 10 bp were assembled according to their overlapping sequence. Reads that could not be assembled were discarded. Quality sequences were aligned in accordance with SILVA alignment  and clusFrom the four samples, 133,014 valid reads and 1,757 OTUs were obtained for bacteria. Each library contained 21,369 to 38,038 reads with different phylogenetic OTUs, ranging from 232 to 870. The average length of the quality sequences was 434 to 441 bp. In addition, 101,015 valid reads and 217 OTUs were found for fungi, and the average length of the quality sequences was approximately 403 bp. The total number of sequences, coverage, number of OTUs, and statistical estimates of the species richness for 15,595 sequences (bacteria) or 18,193 sequences (fungi), together with the subsets from each sample at a genetic distance of 3%, are presented in For bacteria, MJ4-4 had the highest richness , while MJ4-1 had the lowest . The Shannon index provides not only the simple species richness (the number of species present) but also the abundance of each species (the evenness of the species) as distributed among all the species in the community. MJ4-4 had the highest diversity (Shannon = 5.12), which was much larger than the diversity of MJ4-3 (Shannon = 3.42) among the four communities, and MJ4-1 had the lowest diversity (Shannon = 2.15). A total of 232 to 870 OTUs were achieved in the whole samples at a 3% distance. The rarefaction curve progression  was very close in all the samples. The Simpson index varied from 0.0208 to 0.2665, with the highest value for MJ4-4 and lowest value for MJ4-1 .For fungi, MJ4-4 also had the highest richness , followed by MJ4-3 , and MJ4-1 had the lowest richness . MJ4-3 had the highest diversity (Shannon = 2.41) whereas MJ4-2 had the lowest diversity (Shannon = 1.72). The number of OTUs ranged from 42 to 67 in these samples; MJ4-4 had the highest one and MJ4-1 had the lowest one .The bacterial OTUs can be assigned into 19 phyla, 202 families and 435 genera. Eight different phyla, namely, Actinobacteria, Acidobacteria, Bacteroidetes, Cyanobacteria, Chloroflexi, Firmicutes, Proteobacteria and Verrucomicrobia, out of the 19 total bacterial phylotypes were common to the four libraries , contribPseudonocardia and Saccharopolyspora accounted for only 5.41% of the total OTUs, but 54.06% of the total reads (18.80–78.90%). The subgroups of Micrococcaceae, Brevibacteriaceae, Geodermatophilaceae, Streptomycetaceae, Dermabacteraceae and Propionibacteriaceae were less than 1% of the total reads, accounting for 0.92%, 0.76%, 0.65%, 0.64%, 0.50% and 0.33%, respectively. A low relative abundance of Pseudonocardia (6.35%) was found in MJ4-4 compared to the other three libraries (41.54–65.57%) of this study. In contrast, MJ4-4 had a higher abundance of Cyanobacteria without rank (18.92%) than the other three samples (0.23–0.56%).At the genus level, the genera of 0–78.90%). RubrobaThe fungal communities were assigned to 6 phyla, 53 families and 46 genera. Ascomycota was the most dominant division, comprising 67.74% (147) of the OTUs and 94.73% of the total reads. Basidiomycota was the second largest division, with 28.57% (62) of the OTUs and 5.17% of the total reads; the other four fungal phyla, namely, Chytridiomycota, Ciliophora, Eukaryota_unclassified and Phragmoplastophyta, accounted for only 0.1%. The sequences affiliated with the Ciliophora were non-specific amplification products, and the application of universal eukaryotic primers resulted in the sequences of some Eukaryota that could not be classified.At the family level, abundant unclassified or no rank fungal sequences were found in each sample. The total relative abundances of the unclassified and no rank fungi in MJ4-1, MJ4-2, MJ4-3 and MJ4-4 were 83.93%, 97.23%, 92.15% and 93.71%, respectively . The memTo analyze the similarity of the microbial communities among the samples, a heatmap was conducted using hierarchical cluster analysis. For bacteria, the heatmap  was basePseudonocardia dominated the family of Pseudonocardiaceae , indicating a significant community succession on the painted sculptures in the Maijishan Grottoes over time. Pseudonocardia was discovered in many heritage sites, particularly in catacombs and subterranean caves. Subterranean caves are generally regarded as oligotrophic ecosystems with almost no natural light, little or no organic matter, high salinity and a relatively constant temperature and relative humidity, all of which represent extreme environmental conditions for most microorganisms [For bacteria, the results suggested that Actinobacteria and Firmicutes were the most predominant bacterial taxa on the painted sculptures in the Maijishan Grottoes. Actinobacteria are well known for their high secondary metabolism, such as the metabolism of pigments, organic acids, polysaccharides and potent antibiotics. Over the past decades, an abundance of Actinobacteria was recorded in many caves or subterranean environments ,33–35, wrganisms .3O4) and vermilion (HgS), which are both widely used red pigments at our study site; these pigments have a strong toxicity that inhibits the reproduction of the majority species in bacterial communities. Thus, most species of microbes cannot survive in this especially hostile microenvironment. However, Pseudonocardia is suitable for niches of poor nutrition because they can develop their filamentous growth and utilize a variety of nitrogen and carbon sources. Thus, Pseudonocardia colonized the wall paintings in caves at an early stage of microbial colonization, before the subsequent deterioration and damage to the substratum materials [Pseudonocardia can alter the local nutritional conditions and pH in a manner that benefits other life forms, mostly fungi and algae. The common, core metabolically active group was responsible for the degradation and discoloration of the wall paintings in many caves [Generally, the surface of the wall paintings was also an extreme environment for most of the surviving bacteria because of its poor nutrient availability and dry conditions. In the process of creating the painted sculptures in the Maijishan Grottoes, some of the pigments contained heavy metal compounds as main components, such as lead red  acid by other microorganisms, causing acid corrosion to wall paintings, marble and limestone [Arthrobacter, Bacillus and Kocuria, were identified in diverse environments, such as soil, aerosols and ancient buildings [Sphingomonas, Pseudomonas and Enterobacter were the representative genera in Proteobacteria. Clearly, the main reasons for this phenomenon were that these two phyla were inhibited on the painted sculptures due to the extreme environment and fierce competition from the other dominant genus, Pseudonocardia.Many heterotrophic bacterial genera, such as atacombs –50. Theyaux Cave . Some stimestone . Other buildings ,49,53 anuildings ,54,55. IFor fungi, the results showed that a large number of sequences belonged to the unclassified or no rank genera based on the current database of 18S rRNA gene sequences, and most of the sequences were affiliated with Capnodiales (unclassified and no rank) and Ascomycota_unclassified. The results revealed a weakness of the fungi primers 817F/1196R for MiSeq sequencing due to their narrow taxonomical variety coverage ,56,57. OComparing the microfloral structures on the painted layers and in the deposited dust, we found that the diversity and abundance of bacteria were far higher in the dust than on the painted layers. Apart from 78 shared OTUs detected in all samples, a total of 396 bacterial phylogenetic OTUs were observed in the dust sample only. In addition, there were slightly more fungal phylogenetic OTUs in the dust than the number shared among all four libraries. In other words, almost all of bacteria and fungi detected on the painted layers can be found in the deposited dust, which was derived from the surrounding environment. To a large extent, the microfloral characteristics of the deposited dust were highly consistent with the surrounding atmosphere of the painted sculptures, suggesting that the air is an important source of microbes for painted sculptures.2 concentration and an increase in the diversity and abundance of airborne microorganisms [2 concentration rose 51 ppm after one group of visitors entered the cave; these changes could not be restored to the initial background levels immediately, and, sometimes, they could not even be restored by the next day. Finally, the cave was closed for an indefinite period in 2002 due to the serious microbial outbreaks that were mostly caused by human disturbance [Pseudomonas, Brevibacterium, Sphingomonas and Staphylococcus, are pathogenic to humans and cause a potential danger to tourists and managers, but their existence and infection potential in humans are currently not understood [In recent years, the Maijishan Grottoes and its elegant artwork and beautiful scenery have attracted an increasing number of tourists, exerting tremendous pressure on the conservation and management of this site. Previous studies revealed that when masses of tourists suddenly swarm into a cave in a short time, they may destroy the original conservation environment of the cultural relics due to their disturbances, such as the resuspension and floating of dust, an increase in temperature, relative humidity and COrganisms ,25. Thesturbance ,59. Furtderstood ,27,60. TIn summary, the characterization of the microbial communities on the painted sculptures in the Maijishan Grottoes showed the presence of complex bacterial and fungal groups on these painted sculptures, many of which are common core species associated with the biodeterioration of painted sculptures. In many cases, tiny fractions of microorganisms may result in severe damage to wall paintings due to a lack of sufficient attention or inappropriate management. Although no obvious visible microbial damage exists on the painted sculptures in the Maijishan Grottoes, the priceless and fragile artwork is now under multiple threats by different microorganisms and human activities. Furthermore, the local environmental conditions are favorable for microbial growth and reproduction and are dangerous to the painted sculptures, particularly the high relative humidity and human disturbances, increasing the potential risk of microbial outbreaks . Thus, mS1 FigRarefaction curves show the number of reads with the number of phylotypes at 97% sequence similarity level for the different samples. A: Bacteria, B: Fungi.(DOCX)Click here for additional data file.S1 TableThe operational taxonomic units (OTUs) were defined with 97% similarity threshold. The coverage percentages, richness estimators (ACE and Chao), and diversity indices (Shannon and Simpson) were calculated.(DOCX)Click here for additional data file.S2 TableThe operational taxonomic units (OTUs) were defined with 97% similarity threshold. The coverage percentages, richness estimators (ACE and Chao), and diversity indices (Shannon and Simpson) were calculated.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableEighteen OTUs with abundances higher than 0.5% in the bacterial community were sorted from total of 1,757 OTUs, and defined as predominant OTUs. S: = species; G: = genus; F: = family; O: = order; C: = class; P: = phylum.(DOCX)Click here for additional data file.S6 TableFifteen OTUs with abundances higher than 0.5% in the fungal community were sorted from total of 217 OTUs, and defined as predominant OTUs. S: = species; G: = genus; F: = family; O: = order; C: = class; P: = phylum.(DOCX)Click here for additional data file.


The median fibre intake was 18.2 g (interquartile range [IQR] 13.2–25.0) in children and 20.7 g (IQR 14.3–28.7) in adults. We found that 42.3% (95% CI 40.5–44.1%) of children and 28.2% (95% CI 27.3–29.1%) of adults met the Adequate Intake (AI), and less than 20% of adults met the Suggested Dietary Target (SDT) to reduce the risk of chronic disease. Older children (aged 14–18 years), girls, young adults (19–30 years), males, and those of lower socio-economic status were less likely to meet the AI (p < 0.001). Those with a higher energy intake were more likely to meet the AI. Anthropometric measures were not associated with fibre intake or the likelihood of meeting the AI. Fibre is a nutrient of concern in Australian diets, with most children and adults falling short of recommendations. Adolescents, girls, young adults, men, and those of lower socio-economic status were less likely to meet the recommendations and may benefit most from public health interventions.Dietary fibre is important for regular laxation and reduces chronic disease risk. The National Health and Medical Research Council outlines daily fibre intake targets, yet the proportion of the population that meets these targets is unknown. Using the 2011–2012 National Nutrition and Physical Activity Survey, we profiled fibre intake among Australian children and adults. Data from one-day dietary recalls were analysed ( Dietary fibre is a central component of a healthy diet. Important for regular laxation , a largeDespite the benefits, the mean intakes of fibre in Australia are below the target intakes. The recommended levels of fibre are outlined by the National Health and Medical Research Council and include the Adequate Intake (AI), based on adequate laxation, and the Suggested Dietary Target (SDT) to reduce chronic disease risk. The AI ranges from 14 to 28 g for children and adolescents (children) aged 1–18 years-old, while, for adults, it is 25 g for women and 30 g for men . The SDTGlobally, numerous studies have utilised nationally representative data to profile the fibre intakes . The appThe aim of this study, therefore, was to determine the fibre intake, the proportion of persons meeting the AI and SDT, and the associations between fibre intake and demographic, socioeconomic, and anthropometric factors in a nationally representative sample of Australian children and adults from the 2011–2012 NNPAS. A more sophisticated understanding of fibre intake in Australia will help to establish the need to address fibre intake inadequacies in at-risk populations.n = 2812) or adults . Interview responses were provided by an adult proxy for children aged 2–8 years, with children aged 6–8 years allowed to assist. An Automated Multiple-Pass Method, developed by the Agricultural Research Service of the United States Department of Agriculture, was used to capture all foods and beverages consumed by the respondents within the 24 h prior to the interview day. The quantity of each food consumed was measured in grams and kilojoules (kJ), and dietary intake data were analysed using the survey specific 2011–2013 Australian Food Composition Database (AUSNUT) developed by Food Standards Australia New Zealand (FSANZ) [The 2011–2012 NNPAS is a nationally representative survey carried out by the Australian Bureau of Statistics (ABS) and forms part of the 2011–2013 Australian Health Survey. Detailed dietary information and physical activity data were collected for the NNPAS during face-to-face interviews by trained interviewers from the ABS. The data were collected from 12,153 participants aged 2 years and over, 7735 of whom provided dietary data for a second day of recall by telephone interview. Participants were categorised as children  .The total fibre intakes, in grams per day, was determined for all respondents. The children were categorised into the age groups 2–3 years, 4–8 years, 9–13 years, and 14–18 years, and the adults were categorised in the age groups 19–30 years, 31–50 years, 51–70 years, or 71+ years. The AI is defined as an adequate intake based on the appearance or disappearance of gastrointestinal function and adequate laxation and is derived from the median intakes from national dietary surveys in Australia and New Zealand, where laxation problems are considered uncommon . The SDTThe Socio-Economic Indexes for Areas (SEIFA)  was used2) was calculated using the measured height and weight. The respondents were classified as: underweight (<18.5 kg/m2), normal weight (≥18.5 kg/m2 to <25 kg/m2), overweight (≥25 kg/m2 to <30 kg/m2), or obese (≥30 kg/m2) [Physical measurements including weight, height, and waist circumference were taken for all respondents by trained interviewers during the face-to-face interviews. For children, the body mass index (BMI) z-score was utilised, also known as BMI standard deviation (SD) score, which is a measure of relative weight adjusted for age and sex. The BMI z-score was calculated using the child’s age, sex, height, and weight, and the World Health Organization growth reference standards for 2–4- and 5–9-year-old children . The sta0 kg/m2) .The basal metabolic rate (BMR) is the amount of energy needed for an individual’s minimum set of body functions necessary for life over a defined period of time. The BMR was measured in kJ per 24 h and calculated using age, sex, and weight (kg) as variables with no adjustment for activity levels. The energy intake-to-basal metabolic rate ratio (EI:BMR) was used to calculate under-reporters, i.e., participants with implausibly low intakes. Participants aged 10 years and over were classified as under-reporters or not under-reporters on the basis of the Goldberg  cut-off p-values < 0.001 were treated as significant. General linear models were used to adjust the mean fibre intake for energy intake, sex, age group, the interaction of sex and age group, SES quintile, BMI (BMI z-score for children), and waist circumference (waist:height ratio for children). Tables of marginal means, standard errors, and 95% confidence intervals were obtained from the analyses. Post hoc pairwise comparisons using the Bonferroni Correction showed pairwise significance between consumer categories. Logistic regression models were used to determine the likelihood of meeting the fibre AI and SDT, adjusted for energy intake, sex, age group, the interaction of sex and age group, SES quintile, BMI (BMI z-score for children), and waist circumference (waist:height ratio for children).The statistical package IBM SPSS version 23.0  was used for all analyses. Because of the large sample sizes, p < 0.001).In children, the median fibre intake was 18.2 g (IQR 13.2–25.0 g), and less than half of the children  met the AI . Boys weIn a general linear model for fibre intake adjusted for energy intake, demographic, and anthropometric measures, 14–18-year-olds and children in the lowest SES quintile had the lowest fibre intakes . There wIn a binary logistic regression, 2–3-year olds were 39 times more likely to meet the AI than adolescents (14–18-year olds) . ChildreIn adults, the median fibre intake was 20.7 g (IQR 14.3–28.7 g), and less than a third met the AI, with fewer than one in five meeting the SDT  . More feIn a general linear model for fibre intake adjusted for energy intake, demographic, and anthropometric measures, young adults (19–30 year olds) had the lowest fibre intake, and those aged 51 years and older had the highest intake . Adults In logistic regression models for the likelihood of meeting the AI and SDT, adult females were 2.2 times more likely to meet the AI and 5.3 times more likely to meet the SDT than males . Young ap < 0.001). Among adults, 21.5% were under-reporters, and significantly fewer under-reporters met the fibre AI  and fibre SDT  compared to adults who did not under-report (p < 0.001).Among children aged 10 years and over, 17.2% were under-reporters, and significantly fewer under-reporters met the fibre AI compared to those who did not under-report (6.4% vs. 40.8%, respectively, The current analysis of the most recent Australian nationally representative nutrition survey shows that most Australians did not meet fibre targets, with more than one in two children and more than seven in 10 adults not meeting the AI, and more than four in five adults with intakes less than the SDT. The likelihood of meeting the fibre targets varied by age, sex, and socio-economic status, showing that 14–18 year olds were the least likely to meet the AI among children, and 19–30 year-olds and men were the least likely to meet the SDT among adults. The lower socio-economic groups were less likely to meet the recommendations for both children and adults. Fibre intake was not related to anthropometric measures. To our knowledge, this study is the first comprehensive analysis of total fibre intake in the Australian population based on recent nationally representative data.Our findings demonstrate that fibre is a nutrient of concern in the Australian diet, in line with global nationally representative nutrition survey data. A review of 47 studies in children and 29 studies in adults found that fibre intakes were lower than the recommended intakes for most countries, and the mean intakes were generally around 20 g per day for men and 18–20 g per day for women . Few stuThe energy-adjusted results showed that adolescents, young adults, and men had the lowest fibre intakes, and this finding confirms that age and sex are important predictors of the likelihood of meeting fibre targets in Australia. The association between fibre intake and age are in line with studies from the U.S., Germany, and Ireland, which found that adolescents consumed less fibre than younger children ,32, and The finding that the socioeconomic status was associated with fibre intake in both children and adults suggests that public health initiatives that address low fibre intakes could prioritise lower SES groups. It is widely established that lower SES groups have poorer dietary habits ,53,54 alThe relationship between fibre and energy intake raises questions about the effects that recommendations to increase fibre intake could have on increasing total energy intake. This concern is supported by intervention evidence, with a large randomised controlled trial showing that an increased total fibre intake achieved by increasing whole grain food consumption also increased the total energy intake . Whilst We found nil or inverse associations between fibre intake and anthropometric measures. There is considerable evidence that a greater fibre intake is associated with a lower obesity risk in adults ,73,74,75The strengths of our study include the use of a large nationally representative sample of children and adults, making our results generalisable to the Australian population. Further, our analysis is adjusted for energy intake wherever relevant, a parameter that was strongly associated with total fibre intake. Despite the lack of an estimated average requirement for fibre, we have included analyses using the Suggested Dietary Target for adults. The limitations of this study should also be considered. As with other nutrition surveys, fibre intake from the 2011–2012 NNPAS may have been affected by underreporting . We founThe majority of Australian children and adults did not meet fibre targets. The greatest fibre shortfalls were observed among adolescents, younger adults, men, those of lower socio-economic status, and those with lower daily energy intakes. Given the costs of chronic disease in Australia and the role that fibre plays in preventing these diseases, our findings strengthen the need for interventions and initiatives that aim to increase fibre intakes in line with the Australian Dietary Guidelines. Further research should focus on the food sources of fibres to help inform dietary recommendations for such initiatives.