Daucus carota L.), among the most important root vegetables in the Apiaceae family, are cultivated worldwide. The storage root is widely utilized due to its richness in carotenoids, anthocyanins, dietary fiber, vitamins and other nutrients. Carrot extracts, which serve as sources of antioxidants, have important functions in preventing many diseases. The biosynthesis, metabolism, and medicinal properties of carotenoids in carrots have been widely studied. Research on hormone regulation in the growth and development of carrots has also been widely performed. Recently, with the development of high-throughput sequencing technology, many efficient tools have been adopted in carrot research. A large amount of sequence data has been produced and applied to improve carrot breeding. A genome editing system based on CRISPR/Cas9 was also constructed for carrot research. In this review, we will briefly summarize the origins, genetic breeding, resistance breeding, genome editing, omics research, hormone regulation, and nutritional composition of carrots. Perspectives about future research work on carrots are also briefly provided.Carrots ( China accounts for nearly half of the total global production of carrots and advances in genetic technologies are contributing to improve both crop quality and yield. Cultivated carrot is the second most popular vegetable in the world after potato; this can be largely attributed to their taste and their nutritional benefits. Ai-Sheng Xiong and colleagues at Nanjing Agricultural University, China, review the latest studies on the origin and breeding of carrots. Understanding the genetic sequence and gene expression patterns in carrot plants provides valuable insights into their evolution as well as into the mechanisms underlying their resistance to disease, production of healthy carotenoids and environmental stress tolerance. Future application of gene editing technologies will help to further improve crop production and the nutritional value of carrots. Daucus carota L.), a biennial herbaceous species, is a member of the Apiaceae family1. The cultivated carrots are mainly classified into eastern carrots and western carrots based on pigmentation in the carrot roots2. Eastern carrots are thought to originate from Afghanistan, while the origin of western carrots is still uncertain3. The roots of most eastern carrots are purple, and some are yellow. They have slightly dissected leaves and branched roots. The roots of most western carrots are orange, red or white. The leaves of western carrots are highly dissected, and the roots are unbranched4. Currently, orange carrots are becoming more popular and more widely cultivated in the world.Carrot . The average length of the carrot chromosome is 2.34 μm12. In 2011, Iovene integrated the linkage groups with pachytene chromosomes of carrot through fluorescent in situ hybridization. In the report, the lengths of carrot chromosomes were only 2–4 μm, and the nine chromosomes were classified into three groups. Chromosomes 1, 6, and 8 with subterminal centromeres were a group; chromosomes 2 and 4 with terminal centromeres were a group; and chromosomes 3, 5, 7, and 9 with nearly median centromeres were a group. Among the nine chromosomes, chromosome 1 was the longest chromosome and had a small heterochromatic knob13.The carrot is a biennial herbaceous species in the Apiaceae family. Carrot roots, which develop from the hypocotyls, have good storage ability. A large amount of carbohydrates are stored in the enlarged taproots for the carrot plant flowering in the second year. The flower of the carrot is a flattened umbrella-shaped umbel Fig. . The umb14. Carrots are biennial plants. Vegetative growth is the main process of the first year of the life cycle to store material for reproductive growth. The flesh taproot collected for eating or selling is the root produced in the first year. Carrots will flower or bolt after vernalization when the roots are left in the ground. The time for vernalization must be at least 6 weeks. However, some wild carrots will flower or bolt with little or no vernalization16.Carrot is a cool climate crop that can be sown in spring in temperate climate zones or in the autumn or winter in subtropical climate zones17. In the Stolarczyk and Janickan report, Afghanistan was thought to be the first center of carrot diversity, and Turkey was the second10. With the development of sequencing technology, many molecular markers have been used in research on plant evolution. In Orizzo and his group’s research, single nucleotide polymorphisms (SNPs) were adopted to analyze the structure and phylogeny of wild and cultivated carrots. A clear separation was found between wild and cultivated carrots. Based on historical documents and experimental results, central Asia was thought to be one origin of cultivated carrot18. All of the above research supports the idea that central Asia was an origin of cultivated carrots.The time frame and geographical location(s) of the earliest cultivated carrots are still uncertain. In Vavilov’s opinion, Asia Minor and the inner Asiatic regions were the origin centers of cultivated carrots. In addition, regions including Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan were the basic centers of cultivated carrots in Asia19. The cultivated carrot can be mainly classified into the anthocyanin, or eastern-type, carrot  and the carotene, or western-type, carrot  based on the pigmentation in the roots21. Western carrots are always cylindrical or tapered cylindrical in shape and have less pubescent leaves, higher provitamin A carotenoid content, and higher sugar content than eastern carrots1,22. Eastern carrots always have thicker, shorter, narrow, conical roots, have pubescent leaves, flower early, and are poor in provitamin A carotenoid content1.Shape, color and flavor, etc. were surmised as selection criteria in the domestication of carrot4. A recent study based on the transcriptome data analysis also supports the hypothesis that the eastern-type cultivated carrot originated in Western Asia3. However, there are still some different viewpoints on the origin of western-type cultivated carrots. The western-type cultivated carrot was thought to originate from eastern-type carrots directly, based on the earliest molecular study about carrot domestication18. In contrast, Heywood held the idea that western-type cultivated carrots did not originate directly from the eastern-type carrot2. He summarized the hypothesis that there was a secondary domestication event in the domestication of western-type cultivated carrot2. According to a recent study, western-type orange carrots may also originate from eastern carrots by introgression from wild carrots3. Areas around Afghanistan are generally agreed to be the geographic regions of the first cultivation of eastern-type carrots. To determine the geographic regions of the first cultivation of western-type carrots, more genetic sequencing studies are needed.The viewpoint that the eastern-type cultivated carrot was domesticated from the wild carrots in the area around Afghanistan is generally agreed upon25. The rate of natural hybrids in carrots is very high, but the value of seed production by natural hybrids is uncertain26. Male sterile lines have been used in the hybridization breeding of many crops and have made hybridization breeding easier for many plants that are difficult to breed using artificial emasculation27. In heterosis breeding, male sterile lines were also widely used. F1-hybrid breeding based on cytoplasmic male sterility is the main method of carrot breeding28.Carrot is an allogamous plant. The stamens usually mature earlier than pistils in carrots32. The stamens of the “brown anther” type are deformed and brown-colored. Morelock has identified this type in many cultivated and wild carrots33. The stamens of the “petaloid” type are transformed into a petal-like shape34. This type was found by Thompson31 and Mccollum32 in wild carrot. The anthesis of the “brown anther” type is blocked at a late stage of meiosis, and the stamens of the “petaloid” type produce no pollen. In carrot breeding, the “petaloid” type of male sterility is used more often. In 2018, Tan et al. found a wild petaloid male sterile carrot line (Wuye-BY). The male sterile carrot line has no swollen storage root, petal or anther. The advantage of the F1 hybrid is obvious35. In their subsequent experiments, they found that the sequence length of ATP synthase subunit 6 (atp6) was shorter in the male sterile line (Wuye-D) and longer in the fertile line (Wuye-L). The atp6 gene is related to the fertility of plants. They thought that the longer atp6 gene was associated with carrot fertility and that the short atp6 gene was associated with carrot male sterility36. In a study by Szklarczyk et al., the sequence of the carrot atp9 gene (atp9-1) in petaloid carrots (Sp-cytoplasm) was shorter than the sequence of atp9-3 in N-cytoplasmic plants. In Sp-cytoplasm carrot, the enhanced expression level of atp9-1 was thought to be responsible for the enhanced ATP9 accumulation37. However, male sterility is a genocytoplasmic system, and the mutation in the atp6 gene sequence is not enough to determine plant male sterility. More genetic evidence is needed to determine plant male sterility.In carrot, “brown anther” type and “petaloid” type are the two types of male sterility38. In genetic diversity analysis, molecular markers are also useful39. In basic research and breeding of carrots, molecular markers have been widely used.Molecular markers are a new way to identify germplasm resources based on DNA and mRNA polymorphisms. They can be used in identifying core collections and examining the genetic relationship between parents in breeding research40. In the characterization of genetic diversity in Daucus varieties, 26 accessions of Daucus were discriminated into five Daucus species and seven D. carota subspecies by RAPD and amplified fragment length polymorphism (AFLP)41. Lian et al. classified 34 carrot resources into 5 groups and 8 subgroups with 20 random primers by RAPD42. In the research of Grzebelus and colleagues, RAPD and AFLP were used to analyze the genetic diversity of carrots. Four inbred lines and eight F1 hybrids were identified through AFLP technology43. Six sequence-tagged site (STS) primer combinations were used to identify the carrot petaloid type of cytoplasmic male sterility (CMS). Five CMS lines were classified into two groups, and eight fertile carrots were classified into six groups44.In a study by Briard and colleagues, random amplified polymorphic DNA (RAPD) was found to perform better than morphological or isoenzymatic markers in the varietal identification of carrotAlternaria dauci45. The QTLs for the synthesis of α-carotenes, β-carotenes, carotene lycopene, and precursors have also been found46. Among the molecular markers, simple sequence repeat (SSR) markers are an important way to analyze genetic diversity. In the research of Baranski et al., 88 carrot accessions mainly from Europe, North America, and Asia were collected. Thirty SSR loci were fully characterized in these carrot accessions. As a result, the genetic diversity of the Western gene pool was lower than that of the Asian gene pool47.In breeding carrots for resistance to leaf blight, quantitative trait locus (QTL) mapping technology was used to identify QTLs in carrots with new genetic backgrounds. Eleven QTLs were found in the two carrots with new genetic backgrounds of resistance to In these studies, many molecular markers have been identified. Among these markers, some are disease resistance-related, some are agricultural trait-related and some reflect genetic diversity. All these markers will play important roles in carrot breeding with the carrot genome sequence database.48. The transformation protocols of carrots have also been established over decades. Many transformation methods for carrots have been established. Among the diverse techniques, Agrobacterium-based systems are the most common methods49. Agrobacterium includes A. tumefaciens and A. rhizogenes, and A. tumefaciens is the most common strain in Agrobacterium-based systems. The first carrot transformation based on A. tumefaciens was reported in 1987 50. According to many optimized transformation protocols of carrot transgenesis systems, explant type, variety, and bacterial strain were found to be the main factors affecting the transformation frequency51. In carrots, roots, cotyledons, hypocotyls, and petioles can all be used as explants. In Pawlicki and his group’s study, the transformation frequency was higher when petioles were used as the explants52. The time of cocultivation was also important. Compared with a cocultivation time of 1 or 7 days, the transformation frequency at a cocultivation time of 2 or 3 days was higher51. Following the development of the carrot transformation system, the media was also changed from MS to B5 53.Carrot is known as one of the pioneer species in the research of plant tissue cultureOsPOC1, OsPrx114, and AtNPR1) from other species were overexpressed in carrots to enhance fungal and disease resistance. The transgenic lines overexpressing OsPrx114 displayed high disease resistance compared with the control54. In addition to resistance breeding, genetic engineering was also used to accumulate some special components by overexpressing characteristic genes in carrots. In the research of Luchakivskaya et al., HuINFα-2b was overexpressed in carrots to enhance the accumulation of human interferon alpha-2b protein. The transgenic lines expressing HuINFα-2b were thought to be useful in curing various viral diseases55.In the breeding research on carrots, the genetic engineering method was also adopted. The approach based on overexpressing functional genes was widely used. In the research of Wally et al., functional genes  and Cercospora carotae (Pass.) Solheim around the world64. A. dauci lesions always occur on older leaves, and C. carotae lesions always occur on new leaves in carrots67. Fungal leaf blights are found to cause yield loss by reducing leaf photosynthetic area and breaking carrot petioles. The A. dauci and C. carotae lesions can break carrot seedlings by girdling their petioles68. Breeding for leaf blight resistance has been performed for many years. The less-susceptible cultivars are found to have characteristics that delay the spread rate of pathogens. According to Gugino’s report, “Bolero”, “Carson”, “Calgary”, “Ithaca”, and “Fullback” were the cultivars that were less susceptible to A. dauci, and “Bolero”, “Carson”, and “Bergen” were the cultivars that were less susceptible to C. carotae68. In the research of Le Clerc et al., QTL mapping technology was adopted for the resistance breeding of leaf blight, and 11 QTLs related to resistance to A. dauci were found45.Fungal leaf blight is a kind of foliar disease in the cultivation of carrots. Fungal leaf blights are mainly caused by Psila rosae F. (Diptera: Psilidae), is the most serious and widespread pest in carrot production69. Carrot leaves turn red, orange or rust- colored, and roots present rusty brown scars and tunnels when infected by carrot flies. Carrots infected by carrot flies are inedible and unmarketable70. Reports about carrot fly resistance breeding are available from more than 100 years ago69. From 1977 to 1978, Ellis and his group performed carrot fly resistance breeding at 12 different locations in England. A total of eight varieties with different resistances were cultivated, including “Clause’s Sytan Original”, “Gelbe Rheinische”, “Vertou LD”, “Clause’s Jaune Obtuse de Doubs”, “Royal Chantenay Elite (Rota) No.275”, “Long Chantenay”, “Danvers Half Long 126”, and “St. Valery”. Among the eight varieties, “Sytan”, cultivated in Nantes, showed the highest resistances to carrot fly71. To compare the difference between carrots with different resistances to carrot flies, “Sytan” (most resistant) and “Danvers” (least resistant) were chosen for comparison. The results of the comparative experiment suggested that carrots with high resistance to carrot flies reduce fly damage by delaying the development of larvae70. To overcome the effect of environmental factors in variety selection, Ellis and his group developed inbred carrots by using a single seed descent program. A total of nine carrot lines with moderate resistance to carrot fly were selected and seeded72. Many other reports about carrot fly resistance breeding have shown that “Sytan” is the variety with the highest resistance. The resistance of “Sytan” to carrot flies has been demonstrated in Canada, Germany, Ireland, New Zealand, and the UK75. During breeding progress, it was realized that varieties with high carrot fly resistance have lower levels of chlorogenic acid than varieties with low resistance76. The concentration of chlorogenic acid may be used as a selection criterion to select a variety with high resistance to carrot flies. However, reports about genetic research into the resistance to carrot flies in carrots are still scarce.The carrot fly, 77. In many carrot-producing regions, RKNs are major pests that limit carrot production78. In cooler producing regions, Meloidogyne hapla is the most prevalent. In warmer producing areas, Meloidogyne javanica and Meloidogyne incognita are the predominant RKN species79. The characteristics of RKNs (present in soil and having a broad host range) and the limited use of nematicides lead to difficulty in controlling the pathogen80. To guarantee carrot quality, it is necessary to perform root-knot nematode resistance breeding in carrots. Huang et al. found that “Brasilia” and “Tropical” are resistant to M. javanica82. “Brasilia” is the most promising source of resistance to M. javanica84. Through the use of molecular markers (RPAD and QTL), a locus named Mj-1 that imparts resistance to M. javanica was found in the “Brasilia” cultivar85. The Mj-1 locus is mapped on chromosome 8 79. In 2014, another locus that imparts resistance to M. javanica was mapped in the cultivar “PI652188” and named Mj-2. Mj-2 is also on chromosome 8 but does not map to the same locus as Mj-1 86. The discovery of Mj-1 and Mj-2 is meaningful for the root-knot nematode resistance breeding of carrots.Root-knot nematodes (RKNs) are significant pests that are widely present in plants7. As the most important vegetable in the Apiaceae family, the carrot genome was also sequenced. In 2014, a genomic database for the carrot was released. This database provides de novo assembled whole-genome sequences and classified transcription factor families of carrots, which is helpful for further research on carrots87. Two years later, a high-quality carrot genome (421.5 Mb) was released. In this study, the evolution of the carrot genome was analyzed, and two new whole-genome duplications (WGDs) were identified. The two WGD possibilities occurred ~43 and ~70 million years ago, respectively. Furthermore, DCAR_032551 was hypothesized to play roles in regulating photomorphogenesis and root de-etiolation of carrots. Based on the whole-genome sequencing of carrots, 32,113 genes were predicted. Among the 32,113 genes, 10,530 genes unique to carrots were found88. In 2018, the genome sequence data for “Kurodagosun”, a major carrot cultivar in Japan and China, was also released89. All of these genome sequence data will significantly promote research on carrot evolution, carotenoid synthesis, and many other important projects in carrots. The genome in Iorizzo’s research (421.5 Mb) is obviously larger than that in Wang’s research (371.6 Mb), which suggests a difference between the sequencing quality in the two studies. The genome data in Iorizzo’s research have higher quality and were analyzed more deeply. The database published by Xu provides a tool for researchers to download gene sequences from Wang’s research. In future research, the two carrot genome datasets should be combined in carrot studies.Over the past few decades, sequencing technology has developed rapidly, and more than 100 plant genomes have been sequencedDaucus and Panax were all 100% in the Maximum parsimony (MP) tree and Maximum likelihood (ML) tree. The results strongly supported the sister relationship between Daucus and Panax90.The plastid genome of carrots has also been sequenced. In 2006, the plastid genome of carrot was sequenced by Ruhlman et al. The length of the carrot plastid genome is 155,911 bp with 115 unique genes. The results from that research provided a valuable resource for phylogenetic analysis among different angiosperms. In the phylogenetic analysis, the bootstrap values between 92. In carrot research, transcriptomics was also widely adopted. In 2011, to build a molecular resource for revealing new markers and novel genes, the carrot transcriptome was de novo assembled and characterized by Simon’s group. To our knowledge, this transcriptome is the first transcriptome of carrot. Based on transcriptome sequencing, 114 computationally polymorphic SSRs and 20,058 SNPs were identified. In addition, polymorphisms were found predominantly between inbred lines93. Xiong’s group established the transcriptomic database (CarrotDB) in 2014. The database was established based on transcriptomic sequences from 14 carrot genotypes87.Transcriptomics is an approach to studying gene expression by measuring all mRNA transcripts in one cell or tissue. The transcriptome sequence dataset is widely used in analyzing gene expression, discovering gene functions, and developing molecular markersD. carota subspecies should also be involved in the research of carrot domestication. In cultivated carrots, the expression of the water-channel-protein gene and carotenoid-binding-protein gene was upregulated, and the expression of allergen-protein-like genes was silenced3. All these results revealed the potential role of regulators of gene expression in domestication. The western carrots were thought to originate from eastern carrots based on the analysis of transcriptome data from different cultivated and wild carrots.To further investigate the domestication of carrots, the root transcriptomes of six cultivated carrots and five wild carrots were sequenced. Rong et al. thought that some other wild 94. The transcriptome analysis of the key genes involved in the biosynthesis and signaling pathway of phytohormones helped to clarify the roles of hormones during root development.Phytohormones play important roles in controlling plant root growth and development. As a root vegetable, the effect of hormones on carrot root growth should be investigated. To investigate the molecular mechanisms of hormones on carrot root growth, the transcriptomes of four different developmental stages were sequenced. A total of 4818 unigenes with differential expression between the four stages were identified. Among the 4818 unigenes, 87 genes were found to be involved in the hormone-related pathway DcLBCY, DcLECY, and DcZEP1 may be responsible for the differences in carotene and xanthophyll levels between carrot leaves and roots95.To investigate the biosynthesis of carotenoids in carrot leaves and roots, Ma et al. sequenced the transcriptome of carrot leaves and roots. Based on the transcriptome data, DcPSY1 was thought to be the crucial factor responsible for the higher carotenoid content in carrot leaves. Depending on the demands of different studies, various transcriptome sequence datasets have been generated. All the datasets are the sequences of genes that are expressed under certain conditions. Transcriptomics promotes research on carrots, and many single important genes were found through this method. On the other hand, research on the relationship among different genes is also interesting. In the future, more research on the interaction among genes based on transcriptome sequence datasets should be performed in carrots.96. Numerous studies have found that plant miRNAs preferentially target transcription factors and play important roles in regulating plant development97. miRNAs are also important participants in responses to abiotic and biotic stresses 98. As a consensus, miRNA may be an important research object in research on improving the agronomic characteristics of crops.microRNAs (miRNAs) are a type of noncoding small RNA ~20–24 nucleotides in length99. The microRNA database and the findings are valuable resources for further research in improving the agronomic characteristics of carrots. Unfortunately, research about the microRNA in carrots is still scarce.The first carrot microRNA database was reported in 2013. Seventeen microRNAs were identified from the research. The 17 microRNAs came from 12 different families . In analyzing the potential targets of the 17 microRNAs, 24 targets were determined. Among the 24 potential targets, 8 were transcription factors, 6 were stress related, 5 were involved in metabolism and 4 were related to plant growth. Most targets identified in the research have also been reported as microRNA targets in other plants101. Through proteomics, numerous proteins involved in the complex signaling and metabolic network of plants can be qualitatively and quantitatively analyzed100. In stress-response research in plants, proteomics has proven to be a powerful method and has been used in studying drought, flooding, and nutritional stress responses102. Furthermore, proteomics has also been widely used in studying the mechanisms and biological processes of plants, such as fruit ripening, seed germination, and floral development103.Proteomics is an important technology for studying the growth, development, and stress responses of plants by systematically analyzing the plant proteome. This technology is an important complement to the genome104. In the research of Wang et al., isobaric tags for relative and absolute quantification (iTRAQ) were used to investigate the impact of elevated carbon dioxide (CO2) on carrot growth and development. Through proteome sequencing, they found a potential molecular mechanism for the altered lignin content in carrot roots induced by elevated CO2105. Proteomic technology provides a powerful way to understand the complex signaling and metabolic network of plant physiological progress and can contribute to further investigation of improving carrot yield and nutrition.In the research of Louarn et al., proteomics was used to analyze changes in the proteome of carrots during cold storage. Carrots cultivated in two different cropping systems were selected as the research object. A total of 15 proteins were found to change in levels in the first month of storage. Between the two different cropping systems, the change in protein level was small. Among the 15 proteins, three were related to the stress response, and three were related to the cytoskeleton. The research indicated that carrot roots have an adaptation to the low temperature within the first month of cold storage106. This is a novel approach to performing qualitative and quantitative studies on plant biochemistry at a global level. Metabolomics has been widely adopted to study plant metabolism and food quality107.Metabolomics is a kind of functional genomics that measures all small molecules (molecular masses ≤ 1500 Da) in a cell or a tissue. The cell or the tissue measured is always in a particular physiological or developmental state108.To investigate the difference between wild and cultivated carrots, Grebenstein et al. constructed the metabolic fingerprinting of wild (“Dutch”) and cultivated (western orange) carrots through nuclear magnetic resonance spectroscopy (NMR). Differences between the two kinds of carrots only appeared at the quantitative level in the metabolic content. The metabolome of the hybrid of wild and cultivated carrots showed high similarity to that of the maternal carrot. The maternal characteristics and maternal environment may be the factors leading to the similar metabolic content between hybrid and maternal carrots109.In carrot breeding research, metabolomics is also used as a powerful tool. In Leiss and his group’s research, metabolomics was adopted to investigate the resistance of different carrot varieties to Western flower thrips. A cultivated carrot (Ingot) was found to have the highest resistance. The wild and biofortified carrots did not show distinct resistance to the thrips. The contents of the flavanoid luteolin, the phenylpropanoid sinapic acid and the amino acid balanine were more abundant in the resistant carrots. These results were useful in the research of thrip resistance breeding in carrots110.In 2014, the metabolomes of five carrot varieties were measured by NMR to study the effect of genotype on the metabolite components of carrot. In this study, orange carrot varieties were found to be more abundant in the contents of sucrose and β-carotene and to be lacking in the contents of fructose and glucose. The difference between yellow and white carrot varieties was unclear. Genetic differences, growing strategies, and soil types were finally determined to be the factors that affect the composition of different carrot varieties. The results are useful in breeding to improve carrot quality112. Cytokinins (CK), abscisic acid (ABA), auxins (Aux), ethylene (ET), gibberellins (GA), brassinosteroids (BR) and jasmonates (JA) are the most studied hormones in plants111. Among these hormones, Aux, GA, BR and CK are mainly related to plant development; JA and ET are mainly related to plant defense; and ABA is related to the abiotic stress response113. Research on the roles of ABA, GA, BR, CK, Aux, and JA in growth and development of carrots has been performed.Phytohormones are important regulators of plant growth and environmental responses. They are involved in almost all physiological processes during the growth and development of plants, such as cell division, growth and differentiation, flowering, seed development, and senescence115. Among the different kinds of naturally active auxins, IAA is the best studied116. In the research of Wu et al., contents of IAA changed obviously at different growth stages and showed distinct discrepancies in different organs. In addition, 18 genes involved in the biosynthesis and signaling pathway of IAA were identified. The way that IAA regulates carrot growth and development may be tissue-specific117. However, there are few reports on the effect of exogenous IAA on the field production of carrots.Auxin is a pivotal plant hormone whose cellular level is important for regulating plant growth and development. Fruit formation, leaf abscission, cell division, and cell elongation were all reported to be regulated by auxin118. In cellular growth, promoting the elongation and expansion of cells are the main functions of GAs. Mutants that are defective in GA biosynthesis are always dwarfs119.Gibberellins are diterpenoid compounds. Through the whole life cycle of plants, GAs play pivotal roles in regulating growth and development, including seed germination, stem elongation, flowering, and fruit development120. Through foliar application, exogenous GA3 promotes the growth of the aboveground part and inhibits the growth of roots119. At higher temperatures, GA spray application can influence carrot flowering121. The impact of GAs on carrot roots promotes the development of secondary xylem and decreases the proportion of secondary phloem122. In a study by Wang et al., exogenous GA3 was shown to enhance the lignification of carrot roots123. In addition, GA was involved in the regulation of the differentiation of embryogenic cells in carrot124.During the growth and development of carrots, GAs play pivotal roles. In carrots, the content levels in roots are lower than those in petioles and leaf blades125. To date, more than 70 brassinosteroid compounds have been isolated, and brassinolide is the most bioactive compound126. During plant growth and development, BRs are involved in various biological processes, such as the formation of stomata and lateral roots, flowering, and fruit maturation. BRs also play important roles in promoting cell division, vascular differentiation and cell elongation, and enhancing the tolerance of the plant128. The spatiotemporal distribution of brassinosteroid activity is a decisive factor that influences the function of BRs129. However, the precise spatial and subcellular distribution of BRs in plant organs is still unclear. Regarding the biosynthesis and signal transduction of BRs, the related genes have been determined by using BR-related mutants in Arabidopsis and many other plants125. In carrots, genes involved in the biosynthesis and signal transduction pathways have been identified via transcriptomic research. Foliar application of 24-epibrassinolide was proven to promote the elongation of carrot petioles. The aboveground part of the carrot treated with 24-epibrassinolide was taller and heavier than that without treatment130.Brassinosteroids (BRs) are steroid hormones, an important kind of plant regulator131. ABA plays important roles in regulating almost all physiological processes during plant growth and development, including seed dormancy, seed germination, and fruit maturity132. In the presence of environmental stress, ABA plays important roles in inducing stomatal closure to respond to water deficiency133. The concentration of ABA within plants determines the roles of ABA in response to changing physiology. In carrots, most reports about ABA are related to somatic embryogenesis. Kiyosue et al. measured the concentration of endogenous ABA in the embryogenic cells, nonembryogenic cells and somatic embryos of carrot. They found that embryogenic cells had the highest level of ABA134. In the research of Nishiwaki et al., ABA was found to be the signal substance in the process of carrot somatic embryogenesis induced by stress135. In addition, ABA plays an important role in inducing the secondary embryogenesis of carrot somatic embryos136. Huang et al. found that genes involved in the biosynthesis and signal pathway of ABA may be affected by carotenogenesis in carrot137. The expression level of DcPSY2 (phytoene synthase) can be induced by salt stress and ABA138. In Arabidopsis, salt stress was found to induce carotenoid synthesis to contribute to ABA production139. These results suggest a critical relationship between carotenoids and ABA.Abscisic acid (ABA) was first recognized as a plant hormone in the early 1960s140. Regulating cell division and differentiation is the main function of cytokinins. In some plant physiology processes, CK plays critical roles such as promoting shoot growth, inhibiting root growth, and regulating female gametophyte development141. CK also plays important roles in enhancing the tolerance of plants to drought, salt stress, and heat142. In carrots, 2-isopentenyladenine and 2-isopentenyladenosine are the major cytokinins, and 2-isopentenyladenine was only detected in carrot roots. The diameter of carrot roots is positively related to the level of CK. The concentration of endogenous CK was found to have a circadian rhythm143. In Chen et al., cambium tissues were found to be the cytokinin biosynthetic site in carrots. In the field production of carrots, foliar application of CK could promote the growth of carrots on nitrogen- and phosphate-depleted soil. The application of exogenous CK promotes the synthesis of endogenous CK and stimulates the growth of the cortex in carrots144. In the study of Wang et al., CK was thought to serve an important function in the second stage of carrot growth (40 days after sowing)145.Cytokinin (CK) is one of the five classic plant hormones and is widely used in agriculture, industry, and research146. This lipid-derived hormone plays critical roles in plant defense against various abiotic and biotic stresses such as pathogen infection, wounding, ultraviolet radiation, and freezing147. In some physiological processes of plant growth, JA also plays roles such as inhibiting root growth, and inducing leaf senescence and flowering148. The levels of JA in senescent leaves are significantly higher than those in nonsenescent leaves. The application of exogenous JA was reported to enhance the tolerance of Arabidopsis to low temperature149. In carrot, application of MeJA increased the total phenolics in Parano carrot. However, the concentrations of mono- and sesquiterpenes in the leaf were not influenced after MeJA treatment150. In Heredia and Cisneros-Zevallos’ research, MeJA application increased the accumulation of phenolics in wounded carrot tissues151. In the research of Wang et al., the levels of JA at different growth stages and expression profiles of genes related to JA were measured. They thought that the regulation of JA in carrots was stage-dependent and organ-specific152.Jasmonic acid (JA) and its derivatives originate from lipids of chloroplast membranes153. According to previous reports, carotenoids function in preventing cancer, cerebrovascular disease (CVD), human immunodeficiency virus (HIV) and cataracts. Their antioxidant properties were thought to be the main factor for the abundant functions of carotenoids154. In Krinsky’s research, eating fruit and vegetables that are rich in carotenoids was found to prevent many diseases, such as disorders related to the eye155.Carotenoids are natural pigments and were found to be present in all photosynthetic organisms. Additionally, carotenoids are known to be good for human health, especially in disease prevention157. Carrot is a kind of root vegetable that has abundant carotenoids accumulating in the root. In the research of Perrin et al., the accumulation of carotenoids was different in the carrots with different root colors. The accumulation in different root tissues was also different. In orange and purple carrot roots, the content of carotenoids in phloem was obviously higher than that in xylem. In the red genotype, the contents of carotenoids in phloem and xylem were similar. The difference in the accumulation in different root tissues was thought to be the result of the different expression patterns of carotenoid biosynthesis genes in specific tissues158.In horticultural crops, studies on carotenoids have been widely performed. Tomato, pepper, and carrot were found to be carotenoid-rich vegetables46. In 2007, 24 potential carotenoid biosynthesis structural genes were identified by Just et al. Two phytoene synthase (PSY) genes (DQ192186 and DQ192187) were found in carrots159. PSY is an important gene that encodes the rate-limiting enzyme in the biosynthesis pathway of carotenoids. The contents of carotenoids in the PSY-overexpressing lines of some vegetables were obviously enhanced162. In the sequencing of the carrot genome, a central gene (DCAR_032551) involved in regulating carotenoid accumulation was found. Furthermore, authors of the genome paper hypothesized that root de-etiolation may be responsible for carotenoid accumulation in carrot roots163. In the research of Ma et al., carotenoid contents in six different carrot cultivars were measured. They thought that carotene hydroxylase genes were involved in α-carotene accumulation and xanthophyll formation164. Although homologs of all known genes involved in carotenoid biosynthesis have been identified in carrots, there are still some questions that need answers, such as the accumulation of carotenoids in nonphotosynthetic carrot roots.The biosynthesis of carotenoids has been widely studied in plants. In 2002, Santos and Simon identified some putative QTLs that are associated with the accumulation of ξ-carotene, α-carotene, β-carotene, lycopene and phytoene in carrot165. Anthocyanins play critical roles in the pigmentation of flowers and fruits166. In many colored vegetables and fruits such as red cabbage, eggplant, purple corn, purple potato, grape, peach, plum, and pomegranate, anthocyanins were discovered167. In plants, anthocyanins can protect plants from strong light by forming light-absorbing screens and scavenging reactive oxygen species when suffering from light stress168. Anthocyanins have also been reported to have many health benefits, including prevention of cardiovascular diseases, anticarcinogenic activity, control of diabetes, and improvement of vision165. Providing antioxidants was determined to be the main mechanism for anthocyanins to prevent diseases in humans.Anthocyanins are widely distributed natural pigments and flavonoids169. The accumulation of anthocyanins in the carrot roots was affected by many factors, including temperature, nutrients, and light170. At the molecular level, many studies on the biosynthesis pathway of anthocyanins have been performed in carrots. Phenylalanine ammonialyase (PAL), flavanone 3-hydroxylase (F3H), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin dioxygenase (LDOX) are members of the biosynthesis pathway and have been identified in carrots171. According to previous reports, the genes DcUCGalT1, DcMYB6, and DcUSAGT1 of carrot were involved in the biosynthesis of anthocyanins174. In Yildiz and his group’s research, the expression profiles of six anthocyanin biosynthesis-related genes  were measured. CHS1, DFR2, F3H, LDOX2 and PAL3 have the highest expression level in solid purple carrots175.Among the cultivars of carrot, the taproot color variety is abundant. The main colors include orange, purple, yellow, red, and white. In the research of Xu et al., the taproot of purple carrots had more anthocyanin accumulation than that of yellow and orange carrots. Only a few anthocyanins were measured in the taproots of orange carrots, and no anthocyanins were measured in yellow carrot roots177. The dietary fibers are further classified into soluble and insoluble fibers. Pectins, gums, and hemicelluloses are soluble fibers, and cellulose is the main component of insoluble fiber. In most foods, insoluble fiber is the main dietary fiber, and the content of soluble fibers is small or negligible. The sources of soluble fibers are limited and include citrus fruits, barley, legumes, oats, avocado, and rye. The sources of insoluble fibers are abundant and include most vegetables and cereal grains178.Dietary fiber is a class of compounds that mainly includes carbohydrates, polysaccharides, and lignin179. Another way that insoluble fiber protects the colon is through enhancing the proliferation of microbes178. In addition, many other functions of dietary fiber have also been reported, such as moderating the postprandial insulin response, reducing cholesterol, regulating appetite, and reducing the risk of coronary heart disease. Sufficient intake of dietary fiber is good for humans to stay healthy180.Dietary fiber is well known and popular as a healthy substance within society. The widely known benefit of dietary fiber is its role in improving gastrointestinal function. Insoluble fiber can improve gastrointestinal function by enhancing the peristalsis of the intestine and increasing the bulk of feces181. Carrot is a good material for producing juice, but the carrot pomace is wasted. According to previous reports, the dietary fiber of the carrot pomace was abundant and was higher than that of some other agricultural byproducts, including asparagus and onion. The insoluble dietary fiber in the carrot pomace are mainly composed of pectic polysaccharides, hemicelluloses and cellulose and have desirable functions177. As a byproduct, carrot pomace is very abundant and could be chosen as a source of dietary fiber. In the research of Chou et al., reducing particle sizes of the carrot insoluble fiber-rich fraction (IFF) can clearly enhance the function of fiber in gastrointestinal function. Furthermore, the ability of micronized IFF to reduce the concentrations of serum triglycerides, serum total cholesterol, and liver lipids was substantially enhanced182.In the storage root of carrot, 1.2–6.44% of the mass is dietary fiber, and 80.94% of the dietary fiber is cellulose183. Hypoxia was also found to enhance the lignification of carrot roots184. As a promising source of dietary fiber, carrot should be further studied.In the research of Wang et al., most lignin in the carrot root was deposited in the xylem. The proportions of lignin in carrot roots decreased with root development185. According to Li et al., sugars, glucose, fructose and starch are the main types of carbohydrates in carrot storage roots181. The biosynthesis of the sources is thought to be regulated by DcSus genes in carrot186. There are also many minerals in carrot roots, such as potassium, magnesium, calcium, sodium, and iron. In the research of Nicolle et al., potassium was found to be the most abundant mineral in carrots6. Among these minerals, the content of iron, sodium and magnesium is highly dependent on the carrot variety, whereas the content of potassium and calcium is not187. In addition, carrot roots are a good source of vitamin E and ascorbic acid. The concentrations of vitamin E and ascorbic acid in carrots are approximately 191−703 μg and 1.4−5.8 mg per 100 g fresh weight, respectively6.Carrot is well known as being a good carotenoid provider. Moreover, carrot roots also contain many other beneficial contents, including vitamins, carbohydrates and mineralsVegetables are good sources of nutritious substances such as vitamins, minerals, and dietary fiber. With the increasing requirement for healthy diets, vegetables are becoming increasingly popular. Scientific studies on vegetables have become more necessary. Studies on vegetables have been substantially promoted by the use of many new technologies. As a nutritious root vegetable, carrots are popular and cultivated around the world. Many studies on the germplasm resource, breeding, tissue culture, and molecular research of carrots have been reported. Future research on carrots may focus on the following aspects.Arabidopsis thaliana, soybean, maize, and liverwort. This system is an efficient technology for targeting and modifying DNA sequences of interest and is a useful way to study gene functions and crop improvement. However, this efficient tool has not been widely used in carrot research. Improving the CRISPR/Cas9 system in carrots and employing it for carrot improvement should be a focus in the future.The CRISPR/Cas9 system is a highly efficient genome editing method developed in recent years. This system was first used in editing the human genome in 2013 and was then widely employed in editing the genomes of other species. The CRISPR/Cas9 system has been successfully applied in many plant species, including rice, The carrot genome has been sequenced, and the genome sequence data have been released. With the rapid development of sequencing technology, many omics technologies, such as transcriptomics, proteomics, and metabolomics, have been employed in carrot research. A large amount of data generated from these studies provides a potential reference for mining carrot functional genes. Genes controlling root genotype, disease resistance, and cytoplasmic male sterility are candidates to be further identified and determined in future research work. Mining functional genes will guide and promote the breeding work of carrots. The genes that control the swelling progress of carrot roots are still unidentified. With the development of sequencing technology, many opportunities will present themselves for research on the swelling of carrot roots.Carrot storage roots contain abundant biologically active substances. Many of these substances are important for human health. Carrots are used to make juice and dietary fiber in the food industry. In medicinal use, there has been little research on the pharmacological mechanism of many active substances in carrots. Many antioxidants, such as anthocyanins, carotenoids, and polyacetylene, provided by carrots play important roles in preventing disease. With increasing health awareness, the active substances in carrots have good research value and prospects for medicinal use. More future work may focus on developing the medicinal uses of carrots.189. However, there are few clinical studies on the application of other hormones. More future work may focus on the use of hormones in carrot field production, including application method and efficiency. At the same time, more clinical studies should be performed to investigate the interactions between human health and hormone application.The human population is increasing rapidly around the world, and a substantial increase in agricultural productivity is needed. Through promoting plant growth and enhancing tolerance to biotic and abiotic stresses, plant hormones provide an opportunity to improve crop production. However, prohibitive costs restrict the widespread and continuous application of hormones in yield production. In carrots, there are almost no reports about the use of hormones in field production. In our opinion, plant hormone application in carrot yield production is worthy of further study. The interaction between human health and hormone application also needs to be studied. In the research on brassinosteroids, the application of brassinosteroids was reported to have no negative impact on human health. In clinical studies, only a very high concentration of brassinosteroids (1000 mg/kg) led to some undesirable side effects in rats


Overall, 48 subjects were enrolled in the study . All subjects received a single oral dose of 372 mg of isavuconazonium sulfate . PK samples were collected for analysis of isavuconazole plasma concentrations from the predose time point up to 336 h postdose. Data were analyzed using population pharmacokinetic (PPK) analysis. The resulting PPK model included two compartments with Weibull absorption function as well as interindividual variability with respect to clearance, intercompartment clearance, volumes of central and peripheral compartments, and two Weibull absorption parameters, RA and KAMAX. The PPK analysis showed that elderly females had the highest exposure versus males  and versus nonelderly females . Higher exposures in elderly females were not associated with significant toxicity or treatment-emergent adverse events, as measured in this study. No dose adjustments appear to be necessary based on either age group or sex even with an increase in exposure for elderly females. This phase 1, open-label, single-dose, parallel-group study evaluated the pharmacokinetics (PK) of isavuconazole after a single oral dose of the prodrug isavuconazonium sulfate in healthy nonelderly  and elderly  males and females. Overall, 48 subjects were enrolled in the study ( Pharmacotherapy can be a challenging aspect of the care of elderly patients, as drug distribution, metabolism, and renal elimination can all be affected by age group . There iTriazole antifungal agents are frequently first-line agents for the prevention and treatment of IFD in older adults or in immunocompromised patients with cancer or those requiring transplantation . IsavucoIt is crucial for the optimal clinical use of isavuconazole to understand its pharmacokinetics (PK) in various patient populations –13, incl–A total of 48 healthy adults were enrolled in the study, including 12 female and 12 male nonelderly adults and 12 female and 12 male elderly adults . The proV2 and V3, respectively), and two Weibull absorption parameters (RA and KAMAX). Modeling of isavuconazole PPK data then proceeded with an exploratory graphical inspection of potential covariate parameter relationships for the primary covariate of interest.The plasma concentrations of isavuconazole showed a biexponential decline after the peak plasma concentration was achieved, with a prolonged terminal elimination phase . The iniV3 was weight:4 is the typical value of volume of distribution, θ9 is the value of volume of distribution associated with changing weight, and WTKG is weight in kilograms. The diagnostics of the model fit  indicated an overall good fit of the model to the data. Typical population parameters, including covariate effects as well as most of the random variance parameters, were estimated with good precision. Plots of the normalized prediction distribution error  demonstrated that the normality assumption was met, and plots of NPDE versus time (independent variable) did not show any trend. The parameters for the best covariate model are presented in Following the development of the modified base model, covariates of interest were added in a stepwise manner using the forward-inclusion/backward-elimination procedure. The only covariate statistically significant on Area under the concentration-time curve (AUC) data were calculated using empirical Bayes estimates. Similar isavuconazole CL values were evident for the male and nonelderly female subjects . The eldOverall, 15 subjects (31.3%) reported 19 treatment-emergent adverse events (TEAEs) during the study . ElderlyThe present study was designed to determine the effect of age group or sex on the PK profile of isavuconazole after a single 200-mg oral dose in healthy nonelderly and elderly male and female subjects. Data were analyzed using PPK. PPK analysis was performed to see if there was any potential bias in sampling time points that might affect the exposures of subjects and also to determine the effects of age and sex on the PK of isavuconazole. The PPK modeling showed that the elderly female subjects had lower isavuconazole clearance values and higher exposure levels than the other groups in the study. However, the PK values determined for the elderly females differed by less than 1.5-fold from those seen with other groups, and the differences were not considered clinically meaningful. One possible explanation for higher exposures in elderly females might be the predominance of Asian subjects. Data analyzed from phase 3 studies showed that Asian subjects have higher exposures than Caucasian subjects . Since tCmax (maximum concentration of drug in serum) and AUC values for voriconazole were higher in young healthy females aged 18 to 45 years than in young healthy men, but no significant differences were observed between healthy elderly men and elderly women (≥65 years) in the same study ,14–me study . Furtherme study , 21. NonIsavuconazole given as a single dose was safe and well tolerated in the elderly subjects as well as in the nonelderly subjects. TEAEs were reported more frequently in the elderly female subject group; however, all TEAEs reported were mild in intensity and no clinically relevant effects with respect to vital signs or physical examination findings were observed in this study. Although PPK analysis showed that elderly female subjects had lower CL values and higher levels of exposure than the other groups of subjects investigated, higher exposures in elderly females were not associated with significant toxicity, as measured in this study. Therefore, no dose adjustment of isavuconazole appears to be necessary based on either the age group or sex of individuals.This was a phase 1, open-label, parallel group, single-dose study to assess the PK of isavuconazole in healthy male and female adults . The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonisation Guidelines for Good Clinical Practice. An institutional review board at the study center approved the protocol and all amendments. All subjects provided written informed consent prior to enrollment.2, respectively, and the 12-lead electrocardiograph (ECG) test results were required to be normal at screening and on day –1 or, if abnormal, not clinically significantly abnormal, as determined by the investigator. Aspartate aminotransferase, alanine aminotransferase, and total bilirubin levels were to be within the normal reference range.Subjects were eligible for the study if they were healthy nonelderly adults aged between 18 and 45 years or were healthy elderly adults aged ≥65 years or had a calculated creatinine clearance value that was within the age-appropriate normal range or that, if abnormal, was not clinically significantly abnormal. Additionally, the body weight and body mass index (BMI) of eligible subjects were required to be at least 45 kg and between 18 and 32 kg/mExclusion criteria included nonelderly subjects with any clinically significant disease history of a pulmonary, gastrointestinal, cardiovascular, hepatic, respiratory, neurological, psychiatric, renal, genitourinary, endocrine, metabolic, dermatologic, immunological, hematological, inflammatory, or malignant condition(s) (excluding nonmelanoma skin cancer). Other exclusion criteria included the following: history of cardiac disorders; viral, bacterial, or fungal infection 7 days prior to baseline visit or vaccination 30 days prior to baseline visit; hepatitis B, hepatitis C, or HIV positivity; and/or a medical or surgical condition that might have interfered with the absorption, distribution, metabolism, or excretion of isavuconazole.All subjects received a single dose of 372 mg of isavuconazonium sulfate, corresponding to 200 mg of isavuconazole, on day 1, after fasting for approximately 10 h prior to dosing. Subjects attended the study center from day –1 to day 4 and returned to the center for outpatient assessments on days 6, 8, 11, 13, and 15. Serial blood samples for isavuconazole PK were collected predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 72, 120, 168, 228, 240, and 336 h postdose.The safety analysis set consisted of all participants who had one oral dose of isavuconazole. Safety assessments included an evaluation of the incidence, nature, and severity of adverse events, TEAEs, drug-related TEAEs, and ECG and vital sign measurements, including 12-lead ECG.Noncompartmental analysis was performed on the PK data, which are not presented in this article. However, some of the parameters might not have been reliable due to insufficient sampling time in the elimination phase. Therefore, PPK was performed to estimate parameters with reliability. A total of 882 concentrations from 48 subjects were used for modeling. PPK modeling was performed using the concentration-time data in both elderly male and female subjects and nonelderly male and female subjects using nonlinear mixed-effects modeling with the software program NONMEM . The first-order conditional estimation methods in NONMEM were employed for all model runs. Model selection was driven by data and based on various goodness-of-fit criteria, which included visual inspection of diagnostic scatter plots , successful convergence of the minimization routine with at least two significant digits in parameter estimates, precision of parameter estimates, and the minimum objective function value and the number of estimated parameters.A variety of linear compartmental models were explored to describe total isavuconazole concentration-time data. The base PPK model included two compartments, with either simple first-order absorption models or the Weibull absorption model. All random effects were treated as log-normally distributed. The ln-ln transformations of both the model and the data were used to stabilize the residual variance. The residual variance was finally modeled as additive in nature. The models were coded using NONMEM subroutines for prediction of PPK parameters. Initially, different age groups/sex groups  were built directly into the base model by allowing each group to have their own unique population mean CL values since the aim was to determine if PK values differed based on age or sex.https://uupharmacometrics.github.io/PsN/) by the use of forward-inclusion and backward-elimination steps. In the forward-inclusion step, covariates  were added to the model one at a time, using a P value of 0.01 for entry into the model. Univariate analysis of all specified covariate-parameter relationships was explored. The best covariate was added to the model, and the univariate step was repeated with the remaining covariates. This process was continued until no significant covariates were left to be added to the model. In the backward-elimination step, covariates were removed one at a time using a P value of 0.01 for retention in the model. The process was continued until all remaining covariates were statistically significant. The total AUC at steady state for individual subjects was calculated using the standard formula  based on the individual parameter estimates from the best covariate model.Following development of the base structural PPK model, a covariate analysis conducted to determine stepwise covariate modeling was performed in PSN  were generated. Nonparametric bootstrapping was used with 500 replications to provide validation of the model parameter estimated. The NPDE data were also plotted to evaluate the best model.


Taenia solium and Taenia saginata are food-borne parasites of global importance. In eastern Europe only fragmented information is available on the epidemiology of these zoonotic parasites in humans and animal populations. In particular for T. solium, on-going transmission is suspected. The aim of this systematic review was to collect the available data and describe the current knowledge on the epidemiology of T. solium and T. saginata in eastern Europe.Literature published in international databases from 1990 to 2017 was systematically reviewed. Furthermore, local sources and unpublished data from national databases were retrieved from local eastern European experts. The study area included 22 countries.Researchers from 18 out of the 22 countries provided data from local and unpublished sources, while no contacts could be established with researchers from Belarus, Kosovo, Malta and Ukraine. Taeniosis and human cysticercosis cases were reported in 14 and 15 out of the 22 countries, respectively. Estonia, the Former Yugoslav Republic of Macedonia, Lithuania, Moldova, Poland, Romania, Serbia, and Slovakia reported cases of porcine cysticercosis. Croatia, Czech Republic, Estonia, Former Yugoslav Republic of Macedonia, Moldova, Poland, Romania, Serbia, Slovakia, and Ukraine reported bovine cysticercosis.T. solium and T. saginata across the region remains incomplete. Available data are scarce and species identification is in most cases absent. Given the public health impact of T. solium and the potential economic and trade implications due to T. saginata, notification of taeniosis and human cysticercosis should be implemented and surveillance and notification systems in animals should be improved.There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of The online version of this article (10.1186/s13071-018-3153-5) contains supplementary material, which is available to authorized users. Taenia solium and Taenia saginata, also referred to as the pork and beef tapeworm, respectively, are food-borne parasites of global importance. In 2014, T. solium, for which humans act as final hosts after consumption of undercooked pork, was ranked by an international panel of experts as the food-borne parasite of greatest global concern, affecting millions of individuals every year and causing a substantial economic impact , and 0.0% (95% CI: 0–0.3%) using the ELISA and a Western Blot IgG  in series [In total, five epidemiological studies reporting taeniosis prevalence data published between 1990n series , while tRelevant information on human cysticercosis was obtained from 63 sources. In total, 40 reports reported individual, and 23 reports reported aggregated cysticercosis cases. Information on human cysticercosis cases was available for 15 out of 22 eastern European countries  reported from Romania. Figure T. solium cysticercosis prevalence  was of 0.0% in samples from the general Estonian population, children, veterinarians and hunters [Only two serological studies using ELISA and/or western blot techniques were identified. One of the studies was in Croatia, where a prevalence of 1.5% was recorded among people with epilepsy . The oth hunters .cysticercus cellulosae” (name used for T. solium cysticercus) in two publications  was recorded in Poland in 2003; however, in the report species were not specified and porcine cysticercosis could refer to “cysticercus cellulosae” or cysticerci of other species like “cysticercus tenuicollis” (name used for Taenia hydatigena cysticercus). Of the porcine cysticercosis cases reported, a molecular method was reportedly used only in Estonia, and T. solium could not be confirmed [Relevant information on porcine cysticercosis was obtained from 53 sources, which reported the occurrence of the infection in 8 countries. In Poland the causative agent was specified as being “onfirmed . No caseonfirmed  and varionfirmed .In Latvia, according to the Food and Veterinary Service, the main slaughtering monitoring authority, in the last 10 years no suspicious cysts have been found in pigs .Relevant information on bovine cysticercosis was obtained from 41 sources reporting data for 15 out of 22 countries  the cases being T. hydatigena cannot be excluded. Also, for most other porcine cysticercosis cases reported in eastern Europe, species identification was never performed, nor molecularly confirmed. Only in one study from Estonia, molecular techniques were applied to confirm a false positive case [Cases of porcine cysticercosis were reported in a number of countries, and as for the human cases, the number of cases and reports per country varied significantly. Notably, in Poland over 4000 cases of porcine cysticercosis were recorded by the veterinary inspection services in 2003; however, no information on the ive case . Lack ofBovine cysticercosis was reported in over two thirds of the countries in the region. The highest number of cases was reported in Poland, where 4718 bovine cysticercosis cases were recorded in 1994. General meat inspection has a very low sensitivity, hence slaughterhouse figures, even when well-recorded will always underestimate the prevalence . False pT. saginata which has been shown to be significant in Belgium (858 €/heavily infected carcass and 586 €/lightly infected carcass) [T. saginata and T. solium, the possibility to molecularly confirm T. solium findings should be available. Recently, the laboratories across Europe that are running tests for the larval and adult stages of the parasites have been summarised and mapped, facilitating the process when suspected lesions are detected [Better, cost-effective and more sensitive diagnostic tools should be developed, and data recording systems should be improved to assure that data from individual slaughterhouses are centralised. Increased availability of data will further allow estimating the overall economic impact of carcass) , but lesdetected .The early species-level identification of the tapeworm and subsequent adapted management are crucial to avoid not only human-to-human transmission, but also human-to-pig/cattle transmission.T. saginata and T. solium in these countries cannot be excluded. For western Europe information and a contact was established with all counties of the region, and the amount of published and unpublished literature was more than double that of eastern Europe, which helped to provide a better and more complete overview of the epidemiological situation [Finally, for nearly all the countries, at least some data and research on the topic could be retrieved and both published and unpublished data sources were available. Particularly the unpublished sources were of added value and contributed to an improved picture of the epidemiological situation in eastern Europe, highlighting the importance of data access and collaborations through networks such as CYSTINET. Nevertheless, for Belarus, Kosovo, Malta and Ukraine a contact could not be established and no information could be retrieved leaving information gaps. In the absence of data the presence of ituation . The twoT. solium and T. saginata across the region remains fragmented. For a number of countries, no information was available and for some, establishing a contact with experts within the field proved challenging. Species identification is not performed in most countries and the findings are not confirmed using molecular methods , making the endemicity status in the region still unclear. Attention should be paid to: (i) suspected autochthonous human cysticercosis cases to rule out ongoing transmission; (ii) notification of taeniosis and human cysticercosis as these should be implemented to get better data and a clearer overview of the epidemiological situation; (iii) surveillance and reporting systems in animals; and (iv) methods for confirming findings.There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of Additional file 1:PRISMA checklist. (PDF 68 kb)Additional file 2:Country sheets template. (PDF 81 kb)Additional file 3:Table S1. List of references included in the review retrieved through online international databases. (XLSX 24 kb)Additional file 4:Table S2. List of references included in the review made available through local sources. (XLSX 71 kb)Additional file 5:Table S3. Individual taeniosis cases identified in case reports in eastern Europe available from 1990 to 2017. Table S4. Aggregated taeniosis cases reported in documents  identified in eastern Europe available from 1990 to 2017. Table S5. Taeniosis prevalence data reported in epidemiological studies published between 1990–2017. Table S6. Individual human cysticercosis cases identified in case reports in eastern Europe (1990–2017). Table S7. Aggregated human cysticercosis cases identified in case reports and publications in eastern Europe (1990–2017). Table S8. Porcine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990–2017). Table S9. Bovine cysticercosis cases identified and reported during meat inspection in case reports and publications in eastern Europe (1990–2017). (DOCX 207 kb)


A retrospective cohort design was used with the objective to evaluate cancer risk among people with type 2 diabetes mellitus (T2DM) in Lithuania. The cohort was established by identifying all patients with the first diagnosis of T2DM in the National Health Insurance Fund database during 2000–2012. Cancer cases were identified by record linkage with the Lithuanian Cancer Registry. Standardized incidence ratios (SIRs) were calculated. Of the 127,290 people that were included, 5959 cases of cancer in men and 6661 cancer cases in women with T2DM were observed. A statistically significant increase in risk for all cancer sites was observed in women, SIR 1.16 (95% CI 1.14–1.19), but not in men, SIR 1.00 (95% CI 0.98–1.03). Among males, a significant increase of liver , pancreas , kidney (SIR 1.46 95% CI 1.31–1.62), thyroid , colorectal , skin melanoma , and non–melanoma skin  cancer was observed. For females with T2DM, a significant increase in risk of cancer of the liver , pancreas , kidney , thyroid , breast , and corpus uteri  was observed. In conclusion, people with T2DM in Lithuania had an increased risk of site-specific cancer. Diabetes mellitus and cancer are complex and growing worldwide health problems, associated with severe acute and chronic complications affecting both the quality of life and survival. According to the International Diabetes Federation, it is estimated that there were 425 million people with diabetes in 2017, and by 2045 the number will rise to 629 million, increasing by 48% . Since tA wide range of epidemiological studies on cancer and diabetes interactions have been conducted and diabetes (primarily T2DM) was shown to be associated with increased risk for some cancer sites , for other sites there appears to be no association or the evidence is inconclusive . There aNeither diabetes, nor cancer patients can be considered a very homogeneous cohort, with great differences in the disease course, not to mention risk factors, medication for the treatment of the diseases itself and comorbidities, nutritional characteristics, nationality, ethnicity, etc., which may impact the course of diabetes and the risk of cancer and provide a basis for study bias. Therefore, this association of the two diseases remains a question that is relevant now and in years to come. Most of the previous studies have focused on specific cancer sites. This study provides detailed evaluation of cancer risk by cancer site in the diabetic patient cohort. The aim of this study was to describe the cancer risk pattern among T2DM patients in the whole population of Lithuania. A retrospective cohort study was conducted to examine the relationship between T2DM and site-specific cancer risk. Due to the design of the study, informed consent of individuals was not obtained. The study was approved by the Vilnius Regional Biomedical Research Ethics Committee.Lithuania has a compulsory health insurance system, therefore, the vast majority of healthcare services are covered by the National Health Insurance Fund (NHIF). NHIF database was created in 1999 for the management, storage, analysis and reporting of the services provided by healthcare organizations. The database includes demographic data and entries on the primary, secondary and tertiary healthcare services provided, hospital admissions, and prescriptions of reimbursed medicine.The cohort was established by identifying all male and female patients with the first entry of T2DM diagnosis  in the NHIF database from 1 January 2000, until 31 December 2012. To minimize the risk of false type 2 diabetes classification, T2DM status was assigned to patients who were reported as T2DM patients and received prescriptions of antidiabetic medications in the NHIF database. To ascertain cancer incidence in the cohort, diabetes records were linked to the Lithuanian National Cancer Registry by use of a personal identification number assigned to all Lithuanian citizens, using data up to and including 31 December 2012. The Cancer Registry is a nationwide population-based cancer registry that contains personal and demographic information on all people diagnosed with cancer in Lithuania since 1978. It is a statutory requirement to notify the registry of all cases of malignant neoplasms. All recorded cancers in the Cancer Registry are coded according to the ICD-10-AM.Patients with T2DM aged 40 years or older were included into the final cohort. We excluded 8230 cases with a cancer diagnosis before the diagnosis of diabetes. The cohort exit date was defined as either the date of death, emigration or 31 December 2012, whichever came first. Overall, 127,290 people  were included into the study, with the observation period of 828,242.3 person years.We calculated standardized incidence ratios (SIRs) for site-specific and overall cancer as a ratio of observed number of cancer cases in people with T2DM to the expected number of cancer cases in the underlying general Lithuanian population. Expected number of cancer cases were calculated by multiplication of the exact person-years under observation in the cohort by sex, calendar year, and 5 year age-group specific national incidence rates . The perAll statistical analyses were carried out using STATA 11 statistical software . The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Vilnius Regional Biomedical Research Ethics Committee (Nr. 158200-17-913-423).During 12 years of follow-up, 5959 cases of cancer in men and 6661 cases of cancer in women with T2DM were diagnosed. The study population’s baseline characteristics are described in Among males with T2DM, significantly increased risk was found for cancer of the liver , pancreas , kidney , and thyroid . Colorectal cancer , skin melanoma , non–melanoma skin cancer , male genital organ , and other endocrine organ  cancer risk was elevated significantly as well.For females with T2DM, similarly to males, significantly increased risk was found for cancer of the liver , pancreas , kidney , and thyroid . Breast  and corpus uteri  cancer risk was significantly increased as well. There was also an inverse association with several cancer sites. A decreased incidence in cancer of the mouth , oesophagus , larynx , lung and trachea  in men, and multiple myeloma  and leukemia  in women was observed .Results of this large population-based retrospective study based on medical service records and the data of the Cancer Registry showed significant increase in site-specific cancer risk among T2DM patients. We found different risk estimates for overall cancer among males and females. The male vs. female cancer risk difference observed in the patient cohort can be partly explained by an increased number of the most common sex-specific, corpus uteri and breast, cancer in the female cohort group. When these cancers are excluded from the calculation for overall cancer risk, a smaller, yet statistically significant increase for overall cancer in women remains . Men, on the other hand, had an increased risk for more types of cancer than women, however, these cancers where not so common and did not affect the overall cancer risk.SIR estimates for overall cancer risk in T2DM patients obtained in our study are comparable to previously performed European cohort studies. A cohort study in Denmark showed a 10% increase in overall cancer for both men and women, with highest risk of hepatic, pancreatic, kidney, and corpus uteri cancer . A cohorAccording to a recent meta-analysis of 121 cohorts  on all-site cancer risk, the risk of cancer among patients with diabetes is shown to be higher: the pooled adjusted relative risk (RR) for all-site cancer associated with diabetes was 1.27 (95% CI 1.21–1.32) in women and 1.19 (95% CI 1.13–1.25) in men . With regards to SIR estimates on site-specific cancer incidence, our results are comparable to previous meta-analyses ,20,21,22We observed a more than 75% higher risk of pancreatic cancer in both men and women with T2DM. A meta-analysis by Song et al., 2015, found that long diabetes duration was associated with a 1.5- to 1.7-fold increased risk of pancreatic cancer . AlthougWe found a 40% increased risk of kidney cancer in both women and men. This finding is similar to a meta-analysis by Bao et al., 2013, which showed a slightly stronger positive relation in women  than in men  . AnalyseIn our study, women with T2DM had a 40% and men had an 80% higher risk for thyroid cancer, although it remained a rare form of cancer in men. The results on thyroid cancer are inconsistent among studies. Our results for women are comparable with a 2014 meta-analysis by Yeo et al., which showed a 30% increased risk in women, but not in men. The risk was higher among women living in areas where thyroid cancer is more common relative to other geographic areas . HoweverOur study showed a 23% increased colorectal cancer risk in men. Several meta-analyses of cohort and case-control studies demonstrated an increased risk of colorectal cancer in both women and men with diabetes ,18,19. ASome significant associations were sex-specific. Women with diabetes had a two-fold increased risk of corpus uteri cancer, which is a well-recognized association and our results are comparable to the latest meta-analyses ,21. Our There was no reduction in prostate cancer risk among men with T2DM in our study, in contrast to a recent meta-analysis, which showed a decreased risk of prostate cancer in diabetic men  . In LithWe found a lower risk of lung, tracheal, oesophageal, mouth, and laryngeal cancer in diabetic men. In comparison, a meta-analysis of 13 studies indicates that DM is positively correlated with oesophageal cancer, although there were limitations on potential clinical confounding factors in each study included in the meta-analysis . CancersOur study has strengths and limitations. The strengths of this study are the evaluation based on real life data from two well established registries. This study is population based with a large sample size, long follow-up time, and the diagnosis of T2DM is well defined. We add more precise risk estimates for some of the less common cancers to the current literature on T2DM and cancer incidence. The limitations of our study are those typical in observational studies, such as lack of confounding factors and the potential interactions of diabetes and cancer, for example, problems involving reverse causality, common risk factors and competing risks. It is possible that we have underestimated the impact since standard for calculation of SIR was given by the whole population, which may also include undiagnosed T2DM patients or those treated outside the national health insurance scheme. In conclusion, our study shows a heightened risk of overall cancer in women and several types of site-specific cancer in patients with T2DM in Lithuania. Significantly increased risk was found for cancers of the liver, pancreas, kidney and thyroid in both men and women. Risk of colorectal cancer, skin melanoma, and non–melanoma skin cancer was elevated among males, and risk of breast and corpus uteri cancers among females.


Thus, participants aged 30 and above tend to neglect 5 more areas of the hand than those aged below 30 .The outbreak of severe acute respiratory syndrome (SARS) claimed the lives of 286 Hong Kong people in 2003. Since then, the Hong Kong government has been promoting the benefits of proper hand hygiene. There are few studies that explore the general quality of handwashing and the hand-hygiene practices of the public of Hong Kong; given this, the aim of this study is to explore this neglected topic. This study is a quantitative study that was conducted in January 2018. The results show that the majority of participants only wash their hands after using the toilet (87%) or handling vomitus or faecal matter (91%). The mean duration of handwashing was 36.54 seconds (SD = 18.57). The areas of the hand most neglected during handwashing were the fingertips (48.1%), medial area (30.5%), and back of the hand (28%). A multiple logistic regression shows that participants who have reached third-level education or higher often tend to be more hand hygienic than those who have not reached third-level education ( In 2003, Hong Kong was profoundly affected by an unknown communicable disease entitled “severe acute respiratory syndrome” (SARS). The outbreak originated in a hospital and then spread to the wider community. The disease took the lives of 286 Hong Kong citizens and 774 people worldwide. In total, there were 8,096 known cases . Given tAfter the SARS outbreak and the development of hand-hygiene guidelines, researchers used various tools to evaluate the effectiveness of handwashing and alcohol-based hand sanitizers. The majority of handwashing and hygiene studies typically focused on healthcare providers and students, while few studies targeted the general public. The common areas of focus for these studies included the length of time spent handwashing, missed areas, common situations where handwashing was warranted, compliance with guidelines, and the relationship between demographic data and hand cleanliness.For instance, in the US, Monk-Tuner et al. reported that only 10% of their 313 participants washed their hands for 15 seconds or longer. Drankiewicz and Dundes observed in their study that only 2% of participating university students washed their hands for 10 seconds or more . Shanks In Turkey, Ergin et al. reported that 27% of 303 university students washed their hands just 4 times per day. They found that the main reason for students not washing their hands on a more regular basis was due to the belief that there was no need to do so. They also found that female participants scored significantly higher in knowledge, skills, and practices of hand hygiene . In KenyAlso, previous studies have identified that gender, age, employment status, and educational level are the factors that affect an individual's hand-hygiene practices. In the US, Anderson et al., Berry et al., Edwards et al., and Vanyolos et al. found that female participants washed their hands significantly more than male participants; in Korea, Park et al. also found this to be true –14. In tRegarding the effectiveness of handwashing, Szilágyi et al. and Vanyolos et al. used fluorescent hand gel with ultraviolet (UV) light to assess the nursing and medical students' quality of handwashing , 19. TheWhat are the most common instances of handwashing among Hong Kong adults?How long do Hong Kong adults spend washing their hands?What areas are most commonly missed in handwashing?How many of the participants have received proper hand-hygiene information, and what are the common information resources?What are the differences between independent variables, common missed areas, and handwashing instances?What is the relationship between independent variables, common missed areas, and handwashing instances?In Hong Kong, the CHP advocates proper handwashing practice to the public aligned with the CDC in the United States, the National Health Service (NHS) in the United Kingdom, the Public Health Agency of Canada, and the Global Handwashing Partnership which includes washing one's hands with water and soap before and after at least eight specific situations; the process comprises six steps over seven areas of both hands for no less than 20 seconds . In 2014This study used a cross-sectional survey with convenience sampling, and behavioural observations were conducted from January to March in 2018. Before conducting this study, ethical approval was obtained from the research ethics committee of a local higher-education institute in Hong Kong. The ethical committee reference number is NUR/SRC/20171220/016.Given that the target population was Hong Kong adults and that public handwashing facilities were required, this study was conducted in several public barbecue sites in three different territories of Hong Kong  in order to broaden the scope of this study. Barbeque sites were chosen as areas to recruit participants because spacious handwashing facilities with touchless faucets and sinks were provided in an outdoor washing area. The participants were the visitors to the barbecue sites whose age ≥18, mentally sound Hong Kong residents, and those who were able to communicate with Cantonese as Cantonese is the mother tongue of Hong Kong people. Participants who had experience working in healthcare settings and those with artificial nails, irremovable hand accessories, or a disability in both hands were excluded from this study.A survey was carried out by face-to-face interviews at the barbecue sites. The participants were required to report their demographic information, instances of handwashing, and their sources of handwashing information.First, information sheets were given to the participants. After obtaining informed consent from them, the participants were asked to rub Glo Germ gel in their hands. After the rater confirmed that both hands were fully covered by the gel, the participants were asked to wash their hands under running water in a way that was typical for them. When the participants were washing their hands, the rater stood far behind them and recorded the time it took them to complete the process. After the participants had washed their hands, a portable, rechargeable black box with 10 watts of UV light was used to assess the residual fluorescent stains on the hands of the participants. The rater then recorded the results.t-test and one-way ANOVA were used. Linear and multiple logistic regressions were used to examine the relationship between variables in demographic data, duration of handwashing, and coverage of missed areas during handwashing. Variables were presented as regression coefficients. The odds ratio was adjusted with corresponding 95% confidence intervals and p values. The statistical significance of p < 0.05 was taken into account. All statistical tests were two-tailed.International Business Machine (IBM) Statistical Product and Service Solutions (SPSS) for Windows version 24.0  was used for data analysis. The situations in which participants would commonly wash their hands, demographic data, and the duration of handwashing were analyzed by using descriptive statistics of frequency count, mean, and standard deviation. To compare the continuous data between the missed areas and variables in demographic data, independent Eventually, 190  valid data were collected from the barbeque sites in three main regions of Hong Kong, comprising 47 (25%) participants from Hong Kong Island, 59 (31%) from Kowloon, and 84 (44%) from the New Territories. The ratio of the number of participants in these three geographical areas was similar to the total population distribution of Hong Kong. The characteristics of the samples are shown in The leaflet devised by the CHP advises Hong Kong people to wash their hands in at least 8 different instances. They are as follows: (1) after handling vomitus or faecal matter, (2) after using the toilet, (3) before and after visiting hospitals or residential care homes or caring for the sick, (4) after contact with animals or pets, (5) before eating or handling food, (6) after coughing or sneezing, (7) after touching public installations or equipment, and (8) before touching eyes, nose, and mouth. Among the 190 participants of this study, half (52.6%) washed their hands in 5 of the 8 instances and only 3 participants (1.6%) washed their hands in all 8 instances. The results showed that more than half of the participants washed their hands after handling vomitus or faecal matter (91.1%), after using the toilet (87.4%), before and after visiting a hospital or a residential care home or caring for the sick (72.6%), after having contact with animals or pets (61.6%), and before eating or handling food (58.4%). In contrast, less than half of the participants washed their hands after coughing or sneezing (48.4%), after touching public installations or equipment (16.8%), and before touching their eyes, nose, and mouth (12.1%) .t-test and an analysis of variance (ANOVA) to compare the demographic data with handwashing instances, the results showed that the mean handwashing instances of participants with third-level education or above were significantly higher than those of participants who had primary- and secondary-level education only (p ≤ 0.001).Using an independent p ≤ 0.001). Thus, the mean of the total missed areas was significantly higher in participants who did not receive information about proper hand hygiene than those who received it (p=0.004).Among the 190 participants, 84 (44.2%) received proper hand-hygiene information. Given that this was an open-ended question, participants could list more than one resource. Thus, 106 resources were reported. After grouping and categorising the resources, the result was revealed that the participants obtained information from the media (45%) and from schools (34%); relatively less information was obtained from hospitals (15%) and the workplace (12%). In addition, nearly two-thirds of the youngest age group obtained information from both schools and the media. However, only 31% of the oldest age group received that same information. Moreover, the instances of handwashing of those participants who received informed hand-hygiene information were significantly higher than those of participants who did not  to wash their hands. The majority of participants (86.8%) washed their hands for longer than 20 seconds, as advocated.t-test and ANOVA to compare the differences between the demographic data and missed areas, we found that unemployed participants had significantly more missed areas than the other participants; the unemployed missed certain areas, including the back of the fingers (p=0.014), palms (p=0.007), back of the hand (p=0.010), and medial areas (p=0.003) (p=0.037). Blue-collar workers had significantly more missed areas, with the medial areas (p=0.003) and the lateral areas (p=0.005) of the hands being neglected. Moreover, the results showed that participants with third-level education or above  had significantly (p=0.004) fewer total missed areas than those with primary- and secondary-level education only . Among the different age groups, the youngest age group performed significantly better than the older age groups over the front of the fingers (p=0.038), the back of the fingers (p=0.015), and the lateral side of the hand (p=0.012  and  0.019). In terms of the total missed areas, the youngest age group missed significantly fewer areas than the oldest (p=0.046).For each participant, a total of 86 anatomical areas were evaluated. The results showed that the fingertips (48.1%), medial area (30.5%), and back of the hand (28%) were the most commonly missed areas in terms of washing. Using an independent p=0.003) . Full-tip=0.001, B = 4.933, 95% CI = 2.119–7.747). Considering the variables affecting the total number of instances of hand hygiene, the only significant predictor among the variables was the educational level of the participants. Those with only primary- and secondary-level education tended to have 1 more missed hand-hygiene instance than those whose educational levels were above the third level .Multiple logistic regression was used to examine the relationship between the demographic variables, total missed areas, and instances of handwashing. The results showed that age is the only significant predictor of the total missed areas. Those aged 30 and above tended to have 5 more missed areas than those aged 30 or below (Although there is scant empirical evidence on the duration of handwashing for the general public, the key public health agencies around the world, such as CDC, NHS, Public Health Agency of Canada, and Global Handwashing Partnership, adopt the WHO's guideline for the healthcare providers as well as for the community use. In our study, almost 90% of the participants completed their handwashing routine in 20 seconds or more. This contrasts with the studies conducted in the United States by Drankiewicz and Dundes, Shanks and Peteroy-Kelly, and Borchgrevink et al. –6.In 2008, the WHO designed a handwashing leaflet, making reference to Taylor, who indicated that the fingertips, interdigital areas, thumbs, and wrists are the most commonly missed areas in handwashing . Pan et In this study, we found that participants with higher educational levels had fewer missed areas, and they performed handwashing on a more regular basis; this is in contrast to the findings of Duggan et al. . HoweverIn relation to the association between age and hand-hygiene practices, we found that as age increases, handwashing becomes more neglected. However, Burnett highlighted that participants aged 55 years and older had good perceptions of hand hygiene, while the age group with the poorest perceptions was those under 26 years of age.  The resuThis study did have its limitations. One of these limitations is the potential unease felt by the participants that evolved by asking for ad hoc handwashing at the venue. Therefore, the researcher did stand far behind the participants, and the participants were not informed that the time spent washing their hands was being recorded. Given the researcher stayed far behind the participants, their compliance with the steps of handwashing could not be evaluated. Besides, although the self-report survey is the fastest way to gather abundant data, this method cannot avoid social-desirability bias in which the participants wanted to “be good,” even though the survey is anonymous. Hence, these two unpreventable conditions may produce potential influences on the results. Furthermore, since most people had barbeque at sunny weekends only, suitable weather and period were quite short which limited the sample size.The results provide insights that will be useful for public health and primary-care professionals when reviewing health-promotion strategies for proper hand hygiene. Although the sample size of this study was not large, this study and Pan et al.'s Taiwanese study found that the fingertips are the most commonly neglected areas . Given tAccording to the World Bank in 2018, the population density of Hong Kong increased from 6,587 people per square kilometre in 2007 to 7,040 in 2017, and it ranks as the fourth highest one in the world. People live more closely together than before, and population will increase in the future. Since the outbreak of seasonal influenza and hand-foot-mouth disease has been attacking Hong Kong frequently in recent years, the high population density of Hong Kong may exacerbate the risk of contracting communicable diseases through common contact surfaces such as door knobs, elevator buttons, handrails of escalators, and public transport. In addition to the effects of globalisation, other developed countries or cities with high population density may also be facing similar challenges. Therefore, the public health and primary-care professionals may consider reviewing the protocol for proper hand hygiene and strategy of education and promotion and examine its effectiveness.