Geminiviridae, Genus: Begomovirus) by the whitefly vector Bemisia tabaci MEAM1 (Gennadius) in tomato. The aim of this research was to determine if the manner in which viruses are co-acquired and co-transmitted changes the probability of acquisition, transmission and new host infections. Whiteflies acquired virus by feeding on singly infected plants, co-infected plants, or by sequential feeding on singly infected plants. Viral titers were also quantified by qPCR in vector cohorts, in artificial diet, and plants after exposure to viruliferous vectors. Differences in transmission, infection status of plants, and titers of TYLCV and ToMoV were observed among treatments. All vector cohorts acquired both viruses, but co-acquisition/co-inoculation generally reduced transmission of both viruses as single and mixed infections. Co-inoculation of viruses by the vector also altered virus accumulation in plants regardless of whether one or both viruses were propagated in new hosts. These findings highlight the complex nature of vector-virus-plant interactions that influence the spread and replication of viruses as single and co-infections.This study investigated the role of vector acquisition and transmission on the propagation of single and co-infections of tomato yellow leaf curl virus  and tomato mottle virus (ToMoV) (Family:  Fitness of the co-infecting viruses may be altered by virus-virus and virus-plant interactions that change virus replication, accumulation, movement, and tissue tropism in the plant8. These can influence foliar symptom severity, growth, development, and chemical and physiological profiles of the infected plants9 that can alter vector behavior in ways that affect virus acquisition and transmission22. Most research to-date has been conducted on single pathogen systems, and knowledge of more complex co-infection dynamics is limited. A majority of studies on mixed infections examine within plant-host dynamics of two viruses and their outcomes on disease severity and viral fitness8. Comparatively few studies use insect vector-transmission or examine vector-related effects41. This study was conducted to examine how the manner of acquisition and inoculation of two viruses by their common vector affects propagation of co-infections in their plant hosts.Concurrent infections of two or more plant viruses, referred to as co-infections or mixed infections, are common in nature and are known to influence disease severity, spread, and the emergence of novel variants46. Plant hosts are spatial environments that viruses must effectively exploit to replicate and localize in tissue so that virions are acquired by their vectors47. Co-infections of plant viruses can be propagated by vectors that acquired virions from co-infected plants, sequentially from singly infected plants, or by multiple vector individuals that acquired different viruses. Vector acquisition of two viruses does not ensure both will be transmitted, and the presence of two viruses can alter transmission rates of one or both viruses51. Once acquired, two viruses may compete for binding sites required for retention in and transmission from their vectors52. Order of acquisition sometimes has an antagonistic effect on virus transmission38 and does not always benefit the virus acquired first36. Vector-plant interactions that occur during probing and feeding events can also induce host plant defenses that mediate infection dynamics.Co-infections have both spatial and temporal dimensions based on when and where plant inoculation occursGeminiviridae, Genus: Begomovirus) are members of the largest plant virus genus for which the importance of co-infections has been documented as a key feature of the emergence, severity and evolution of economically important species55. Both are transmitted in a persistent manner by whiteflies in the Bemisia tabaci (Gennadius) cryptic species complex and occur in single and co-infections in tomato production systems in the southeastern U.S.A57. Begomovirus genomes are circular single-stranded DNA (ssDNA) molecules of approximately 2800 nucleotides each that are encapsulated separately in a 22 × 38 nm geminate particles60. ToMoV has a bipartite genome comprised of two ssDNA components, DNA-A and DNA-B, whereas TYLCV is monopartite with all of its genes encoded on one ssDNA molecule. Both components of bipartite begomoviruses are required to initiate an infection because DNA-A encodes proteins necessary for viral replication, encapsidation, transcription and plant defense neutralization, while DNA-B encodes the viral movement proteins62.This study was undertaken to better understand how vector acquisition and transmission of two viruses influences virus persistence. Tomato yellow leaf curl virus (TYLCY) and tomato mottle virus (ToMoV)  probability of virus acquisition by the vector, (2) virus titers in the vector after acquisition, (3) probability of virus transmission, (4) titers of virus transmitted, (5) probability of systemic infection in inoculated host plants, and (6) virus titers of newly infected host plants ; these plants were used for virus acquisition during the AAPs. There were significant differences in the titers of TYLCV and ToMoV DNA-components in rhizobacterium-inoculated plants, but the titers of each viral component reached similar levels in singly and co-infected plants , both viruses (TYLCV & ToMoV), or the total of single and co-acquisitions  among treatment scenarios Table . There w4,65 = 1.76, P = 0.1476), ToMoV-A , or ToMoV-B . An additional analysis on virus titers included only whitefly cohorts that had acquired both viruses from co-acquisition scenario treatments (no single infection treatments or samples that tested positive for only one virus) to compare the main effects of treatment, virus/viral component, and their interaction. There were no significant differences among treatments  containing only one virus (TYLCV only or ToMoV only), both viruses (TYLCV & ToMoV), and the sum of single and co-transmissions  in separate analyses Table . Althoug4,61 = 2.38, P = 0.0612), ToMoV-A , and ToMoV-B . An analysis of virus titers that included only cohorts that transmitted both viruses from co-acquisition scenario treatments revealed the main effect of acquisition scenario treatment was not significant , both viruses (TYLCV & ToMoV), and the total of single and co-infections  Table . There w4,20 = 2.90, P = 0.0481), ToMoV-A , and ToMoV-B  , but there were differences in ToMoV-A  and ToMoV-B . Accumulation of ToMoV components were lower when ToMoV was acquired first (ToMoV»TYLCV) or separately from TYLCV (TYLCV + ToMoV) compared to the ToMoV treatment. Accumulation was not statistically reduced in the TYLCV»ToMoV treatment, but the sample size was low (only one ToMoV-VCSIP was observed in this treatment). No ToMoV-VCSIPs were observed in the Mixed treatment.Differences in virus or viral component titers among treatments were analyzed separately for TYLCV, ToMoV-A and ToMoV-B in VCSIPs Fig. D. There Our results provide new insights into the effects of virus co-acquisition and co-inoculation on (co-)infection of new host plants and contribute to the expanding body of research on how viruses interact directly and indirectly with other viruses, their vectors, and hosts to affect acquisition, spread and accumulation. In this study, the order of virus acquisition did not change the probability of acquisition by vector cohorts Table  but did 66, can be influenced by where viruses localize in plant tissues15, and the titers of virus that accumulate in those tissues67. Titers of TYLCV were higher than ToMoV in single and co-infected rhizobacterium-inoculated plants used in the AAPs , should be transmitted first because it had 24 h to circulate in the vector before acquisition of virus acquired second (virus2). All else being equal, this is expected to result in the predominance of virus1 at initial inoculation sites in plants during the first 24 h of the IAP. If the viruses compete within the vectors for binding sites required for retention and circulation, then sequential acquisition scenarios should also favor virus1. This effect would be greater if initial vector interactions with virus1 produce an antagonistic effect on circulation of virus2. It is also possible, however, that vector interactions with virus2 may result in antagonism on virus1, or that vector-virus1-virus2 interactions may produce antagonistic effects for both viruses. In the Mixed treatment the order of acquisition from the co-infected plant presumably varied among individual whiteflies within a cohort. The acquisition of both viruses reduced the transmission of TYLCV, but not ToMoV to artificial diet  because individual whiteflies could inoculate only one virus and it is highly unlikely that any individuals shared a specific feeding site. In this scenario, temporal differences are also expected due to variation in whitefly settling behaviors that affect the amount of time before feeding and transmission begins.Virus-virus interactions occur in specific plant tissues and at specific time points during the infection cycle, but these interactions have both spatial and temporal dimensions that can be altered by virus-plant, vector-plant and vector-virus-plant interactions54. Our findings raise questions about the direction of diffusible signals across different cell types and between locations where initial infection occurs.Our results show that the ways in which two viruses are acquired can influence both the probability that they are transmitted Table  and the 65. Vector and virus induced changes can alter viral fitness and can cause chemical and physiological modifications that may influence vector behavior and fitness87.A vector-mediated induction of plant responses may also have influenced virus accumulation because co-acquisition and co-inoculation did not similarly affect the probabilities of systemic infection or virus titers across treatments. Vector and virus induced changes in affected plants may also produce additive, synergistic or antagonistic effects on one another. Transcriptional and proteomic differences are induced by virus infection and vector settling and feeding; a recent study of ToMoV showed that over time virus infection may reverse vector induced changes in the proteome of Lanai tomatoes3. No unexpected differences in visual symptoms were observed and symptoms followed expectations based on infection status  , a dwarf eggplant variety that is non-host for ToMoV or TYLCV. Whiteflies were identified as B. tabaci MEAM1 by sequencing a partial mitochondrial COI gene89 . New whitefly cohorts were initiated every 7–14 days to provide the four-to-seven-day-old adult whiteflies used in the experiment.In 2016, a whitefly colony was established from a population in Auburn, AL and reared in a greenhouse on R. radiobacter63. All virus inoculations (rhizobacterium and whitefly) were conducted using two true-leaf S. lycopersicum L., variety ‘Florida Lanai’ planted in 11.5 cm2 pots64. Twenty plants were inoculated for each infection status: TYCLV, ToMoV and co-infections. Five plants were mock inoculated with the empty vector as a negative control. Rhizobacterium-inoculated plants were used for vector acquisition at 28 days after inoculation (dpi) because previous reports showed that TYLCV and ToMoV titers are highest at 10–24 dpi and decline by 31 dpi in ‘Lanai’ tomatoes90. All experiments were conducted in a growth chamber  equipped with supplemental lighting from two strips of plant Grow LED lights (Litever®) and set at 25 ± 3 °C and a 16:8 h light/dark cycle.To standardize the virus populations of TYLCV, ToMoV, and co-infections, infections were initiated using clones corresponding to partial tandem dimers of ToMoV A (pNSB1906), ToMoV B (pNSB1877), TYLCV (pNSB1736), or pMON721 (empty vector) transformed into 91. Each treatment was replicated six times with each replicate using a different inoculated source plant. Each replicate of treatments 1–3 used one singly or doubly infected source plant, and each replicate of treatments 4–6 used two singly infected plants during the AAPs  stretched and folded thinly over the lid that contained 20 µL of a 20% sucrose solution. The lids were colored with a green Sharpie®  to artificially simulate the color of plant leaves and encourage whitefly feeding. After 48 h, the 1.7-mL tubes containing the whitefly cohorts and sucrose sachets were stored at −80 °C  until further analysis. Due to low numbers of whiteflies during the first replications, sucrose sachet samples were only available from the second experimental replicate. Five sucrose sachets were inoculated in each of the three replicates per treatment . To measure transmission to tomato plants post-AAP, whitefly cohorts were aspirated into clip cages to confine them on the largest leaf of a healthy two-true-leaf tomato seedling. After the 48-h IAP, adult whiteflies were collected from the leaves and killed by freezing. At 14 dpi, a soil drench of imidacloprid  was applied to kill whitefly nymphs. Plants were sampled at 28 dpi and virus infection was confirmed by PCR (described below). Ten plants were inoculated in each of the six replicates per treatment.Overall lower survival of whiteflies on the co-infected plants reduced the sample sizes in the Mixed treatment. We note, however, that whitefly survival on rhizobacterium-inoculated plants coinfected with TYLCV and ToMoV was lower than on single virus-infected plants and the coinfected plants had an unusually strong plant odor . To accommodate this, we infested the virus source plants in the Mixed treatment with a greater number of whiteflies to avoid confounding effects of vector mortality on observed transmission rates.Leaf samples were collected from rhizobacterium-inoculated and whitefly-inoculated tomatoes at 28 dpi. A sterilized one-Hole Punch (6 mm in diameter) (Staples®) was used to take a composite sample comprised of two leaf discs from the terminal leaflet of the first fully expanded leaf below the apex of the plant, one leaf disc from terminal leaflet of the second fully expanded leaf, and one leaf disc from terminal leaflet of third fully expanded leaf. Samples from each plant were placed together in a 1.5-mL microcentrifuge tube with 3 mm glass beads (Millipore Sigma Darmstadt), flash frozen in liquid nitrogen, and stored at −80 °C. Before DNA extraction, frozen plant tissue samples were transported in liquid nitrogen to and from a Mini-BeadBeater™ , where the samples were ground into a fine powder. DNA extractions were performed using the DNeasy® Plant Mini Kit (QIAGEN) following manufacturer’s instructions with minor revisions for eluting DNA using Buffer AE in a final volume of 60 µL.Total DNA was extracted from cohorts of six whiteflies that fed on sucrose sachets using Qiagen® DNeasy Blood and Tissue Kit (QIAGEN) with the following modifications. Whiteflies were placed into 1.5-mL microcentrifuge tubes and 60 µL of Buffer ATL and 7 µL Proteinase K. Whiteflies were homologized into the liquid using a sterile 10-µL pipet tip to grind them against the sides of the tube. Volumes of the solutions were adjusted for low-tissue extractions using following volumes in the order in which they appear in the protocol: 100 µL Buffer AL, 100 µL 100% ethanol, 250 µL Buffer AW1, 250 µL Buffer AW2, and 30 µL Buffer AE repeated twice to yield a total volume of 60 µL.64 using primer pairs TYLCV15-for/TYLCV15-rev (ca. 257 bp) and ToMoV pNSB1/ToMoV pNSB2 for ToMoV-A (ca. 239). Methods of Gong90 were used to amplify ToMoV-B-Fw/ToMoV-B-Rv (ca. 205). Amplified products and a 100-Kb DNA ladder (New England Biolabs Inc.) were visualized using agarose gel electrophoresis.Virus detection in plant samples was conducted using PCR according to the methods of Rajabu90. Total plant DNA and whitefly DNA were measured using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc.) and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc), respectively, following manufacturers’ protocols for dsDNA and ssDNA HS (High Sensitivity) DNA Quantification. Plant DNA was standardized to 25 ng/5 µL of total DNA per qPCR reaction, and total DNA of whitefly cohorts was standardized to 1 ng/5 µL. Sucrose solution from sachets were added without modification in 5-µL volumes to each reaction. The amplification reactions had a total volume of 20 µL containing 5 µL of template DNA, 10 µL iTaq™ Universal SYBR® Green Supermix , 1 µL of 10-µM virus forward primer, 1 µL 10-µM virus reverse primer, and 3 µL sterile H2O. The qPCR analyses were performed using C1000 Touch™ thermocycler  under conditions described in Gong90. Absolute quantification of titers was determined for each sample by comparison to a standard curve as described in Rajabu64. All samples and standards were run in triplicate.Titers of TYLCV, ToMoV-A and ToMoV-B were quantified from virus source plants, whitefly cohorts, sucrose sachets, and whitefly-inoculated plants. Aforementioned primers were used for quantification of TYLCV and ToMoV-A, while ToMoV-B was quantified using the primer pair, ToMoV-B-Fw and ToMoV-B-RvViral titers were quantified from five whitefly cohorts and their respective sucrose sachets from each replicate. Co-inoculation of TYLCV and ToMoV by whiteflies in the co-infection scenario treatments resulted in both singly and co-infected plants. Titers were quantified in at least one infected plant from each replicate and infection status .P < 0.05. There were no significant differences among experimental replicates, so data were pooled for analysis. The proportion of samples testing positive for single infections of TYLCV, single infections of ToMoV, co-infections, and the total of single and co-infections for each virus were compared among treatments in separate analyses using a binary distribution. Presence or absence of the viruses was determined from PCR data from plant samples and qPCR data from whitefly and sucrose sachets. Three separate analyses compared titers of TYLCV, ToMoV-A and ToMoV-B among treatments. A fourth analysis only included samples that contained both viruses. These analyses were conducted separately for whitefly cohorts, sucrose sachets and plants using the main effects of treatment , and virus/viral DNA component , and their interaction term . Titer data were log10 transformed and analyzed using a Gaussian distribution. Analyses of titer data for whitefly inoculated plants were conducted in three separate analyses based on infection status: TYLCV, ToMoV, and co-infection.Statistical analyses were performed using SAS PROC GLIMMIX version 9.4 . Tukey’s tests were used to perform LS means comparisons using a significance value of The plant collection and use was in accordance with all the relevant guidelines.Supplementary Information.


Moreover, SLC26A9 does not secrete but probably supports reabsorption of fluid particularly in the alveolar space, which explains early death by neonatal distress in Slc26a9-knockout animals. While the novel SLC26A9 inhibitor S9-A13 helped to unmask the role of SLC26A9 in the airways, it also provided evidence for an additional role in acid secretion by gastric parietal cells. Here we discuss recent data on the function of SLC26A9 in airways and gut, and how S9-A13 may be useful in unraveling the physiological role of SLC26A9.SLC26A9 is one out of 11 proteins that belong to the SLC26A family of anion transporters. Apart from expression in the gastrointestinal tract, SLC26A9 is also found in the respiratory system, in male tissues and in the skin. SLC26A9 has gained attention because of its modifier role in the gastrointestinal manifestation of cystic fibrosis (CF). SLC26A9 appears to have an impact on the extent of intestinal obstruction caused by meconium ileus. SLC26A9 supports duodenal bicarbonate secretion, but was assumed to provide a basal Cl- secretory pathway in airways. However, recent results show that basal airway Cl- secretion is due to cystic fibrosis conductance regulator (CFTR), while SLC26A9 may rather secrete HCO The mammalian SLC26 family comprises 11 transport proteins with different physiological functions and variable expression in the human body. SLC26 proteins exchange anions and function as electroneutral or electrogenic transporters. A number of diseases are connected to mutations in SLC26 genes, like chondrodysplasia (SLC26A2), congenital chloride-losing diarrhea SLC26A3), or Pendred Syndrome (SLC26A4) , or Pend. While t2+ channels /[HCO3-] ~ 4) is much higher. Determination of ASL pH is technically demanding and expression levels and location of bicarbonate transporters vary significantly between airways of different species. Nevertheless, an overall picture emerged connecting lack of CFTR with airway acidification, mucus abnormalities, and attenuation of airway defense, finally leading to a CF lung phenotype  that activates the Cl- sensitive kinase WNK1 (with-no-lysine kinase), OSR1 (oxidative stress-responsive kinase 1) and SPAK  [The contribution of additional apical HCO 4 or 13 ,45. In rfibrosis ,47. Althosis [3- . Thus, P by CFTR ,50. The  kinase) .3- in exchange with Cl- ions, it may even serve in fluid reabsorption rather than secretion (in vitro.If SLC26A9 truly secretes HCOecretion . Homozyghttps://www.proteinatlas.org/ENSG00000174502-SLC26A9/single+cell+type). It is currently not known whether these transcript data correspond to protein expression. SLC26A9 is clearly labeled in airways from individuals expressing wtCFTR, and no artefactual staining of cilia was detected. Most important, tissues expressing F508del-CFTR stained negative for apical SLC26A9. Nevertheless, there is always a possibility for false positive stainings.Apart from Pendrin, also SLC26A9 is expressed in airway epithelial cells. Using immunohistochemistry, we demonstrated location of SLC26A9 in the apical membrane of ciliated epithelial cells of human, mouse, and piglet airways, as well as ciliated Bci-NS1 and CFBE airway epithelial cells ,56. In cAlong this line, a nonspecific binding was detected for anti-CFTR antibodies in ciliated cells and single cell RNA analysis (scRNAseq) now suggests predominant expression of CFTR in ionocytes and secretory cells . It shou3--cotransporter NBCe1 by S0859 completely abolished S9-A13 induced transport. None of this was observed in the absence of CFTR (CFTR-/-). While the underlying mechanisms still remain obscure, we speculate that in the presence of CFTR, SLC26A9 can operate as a transporter for HCO3-. Accordingly, acute inhibition of SLC26A9 would inhibit HCO3- secretion and cause intracellular alkalosis, which may activate pH-sensitive K+ channels [3- via Pendrin (Our recent data demonstrate acidification of the ASL pH induced by the SLC26A9 inhibitor S9-A13 . These dchannels  and poss Pendrin .Figure 3- transport by SLC26A9 [3- permeability of SLC26A9 [3- transport [3- permeability of SLC26A9 may require co-localization with CFTR in the apical membrane of polarized epithelia. A recent study on perfused mouse bronchioles supports the concept of HCO3- secretion by SLC26A9 in the airway epithelium [Several previous studies reported HCO SLC26A9 ,71–75. H SLC26A9 ,14,16,17ransport . We hypoithelium . This, hAdult SLC26A9-/- mice do not show a pulmonary phenotype and basal airway transport assessed in Ussing chamber experiments appeared undisturbed . However3- secretion was also found in the proximal duodenum, which shows pronounced expression of CFTR, in contrast to low levels of CFTR in the stomach [3- secretion in the GI-tract should be further investigated, ideally in inducible tissue-specific Slc26a9-knockout animals. In these studies, the inhibitor S9-A13 could be useful. Moreover, improved immunohistochemistry of SLC26A9 in the stomach is required, which allows a more precise cellular localization of SLC26A9, under both resting conditions and after stimulation with secretagogues (SLC26A9 is highly expressed in stomach, less in duodenum, and not detected in the distal GI tract. Early work suggested that SLC26A9 expressed in surface epithelial cells of mouse stomach, mediates bicarbonate secretion, and protects the gastric mucosa ,77. A ro stomach ,78. Acid stomach ,80, CFTR stomach , or intr stomach . An earl stomach . It shou stomach . Neverth stomach  (Figure tagogues .Figure nCl- -HCO3- exchanger, Cl- channel, and Na+-anion cotransporter, enabled by a tissue-speciﬁc regulation [In our recent report we propose that SLC26A9 may operate differentially, depending on coexpression of CFTR . In factgulation . CFTR cogulation ,83–85. T


Cryptococcus gattii species complex (CGSC) is a basidiomycete haploid yeast and globally distributed mammalian pathogen. CGSC is comprised of six distinct lineages ; however, the geographical distribution and population structure of these lineages is incompletely described. In this study, we analyze published multi-locus sequence data at seven loci for 566 previously recorded sequence types (STs) encompassing four distinct lineages  within the CGSC. We investigate indicators of both clonal dispersal and recombination. Population genetic analyses of the 375 STs representing 1202 isolates with geographic information and 188 STs representing 788 isolates with ecological source data suggested historically differentiated geographic populations with infrequent long-distance gene flow. Phylogenetic analyses of sequences at the individual locus and of the concatenated sequences at all seven loci among all 566 STs revealed distinct clusters largely congruent with four major distinct lineages. However, 23 of the 566 STs (4%) each contained alleles at the seven loci belonging to two or more lineages, consistent with their hybrid origins among lineages. Within each of the four major lineages, phylogenetic incompatibility analyses revealed evidence for recombination. However, linkage disequilibrium analyses rejected the hypothesis of random recombination across all samples. Together, our results suggest evidence for historical geographical differentiation, sexual recombination, hybridization, and both long-distance and localized clonal expansion in the global CGSC population. Cryptococcus (HPC) from the environment. The opportunistic haploid yeast initially establishes within the lungs, and without treatment or in the case of long, asymptomatic latent periods, it can enter the bloodstream and into the central nervous system, leading to meningoencephalitis and potentially death [Cryptococcus gattii species complex (CGSC) and the Cryptococcus neoformans species complex (CNSC) [Cryptococcosis is an infection caused by the inhalation of the human pathogenic ly death . Due to ly death , HPC wasly death ,4,5,6. Hx (CNSC) .C. neoformans var. gattii and treated as a variety of C. neoformans [C. gattii has often been overlooked and is most likely underrepresented in the available historical literature. It was Dr. June Kwon-Chung who emphasized the difference between isolates of C. neoformans and C. gattii and presented that C. gattii was predominantly found in tropical and subtropical climates, while C. neoformans was shown to be distributed globally [C. gattii came from regions of Australia and Papua New Guinea [C. gattii favored immunocompetent hosts, contrasting C. neoformans, which predominantly infects immunocompromised hosts [For over 100 years, CGSC was referred to as oformans . Becauseglobally ,10. Consw Guinea ,13,14,15C. gattii began attracting broader attention after an unusual outbreak started in the early 2000s on Vancouver Island, a large island off the coast of British Columbia, Canada. The outbreak has since spread to mainland BC and the Pacific Northwest in the U.S. [C. gattii infection and colonization has increased dramatically in humans, animals, and the environment across these temperate regions [C. gattii has been isolated from soil, air, water, and >10 tree species native to Canada’s west coast [C. gattii in the temperate region as well as an increase in human populations have generated several hypotheses about its emergence and mechanism of dispersal. Surveys of air samples have found particles indictive of C. gattii spores [C. gattii in nature is likely uncommon [C. gattii, both of which could have contributed to basidiospore production [C. gattii [C. gattii call for greater efforts to understanding the epidemiology, pathogenesis, and evolution.the U.S. ,17. Inde regions ,19,20. P regions ,22, C. gst coast . The proi spores , likely i spores . In addii spores ,26. Howeuncommon . In contoduction ,28,29. I. gattii . This abC. gattii remains unknown.Although both CNSC and CGSC have a well-defined sexual phase , historiCryptococcus, including PCR fingerprinting [C. gattii isolates and established that C. gattii is a species complex containing multiple distinct lineages. These lineages are frequently referred to as molecular types and increasingly more as species [C. gattii species complex is divided into six distinct lineages  [International Fungal Multi Locus Sequence Typing (IFMLST) database. Evidence for hybridization has been reported between some of the lineages within CGSC [Since the 1990s, a variety of molecular methods has been used to identify the genotypes of strains of the human pathogenic printing , amplifiprinting , multi-lprinting , and whoprinting . These s species . Formallnd VGVI) ,43,44. Ihin CGSC  as well hin CGSC . The extCAP59, GPD1, LAC1, PLB1, SOD1, URA5, and IGS1) [Cryptococcus neoformans species complex revealed evidence for recombination and statistically significant geographic structuring of the two species within CNSC: C. neoformans and C. deneoformans [To standardize genotyping and enhance data sharing and communication, in 2009, the International Society for Human and Animal Mycology (ISHAM) implemented the multi-locus sequence typing (MLST) scheme for global genotyping of CNSC and CGSC isolates. The proposed scheme recommends the use of partial DNA sequences from seven unlinked nuclear loci (nd IGS1) . For eacnd IGS1) . This aloformans .In this study, we examined the global population structure of both the total CGSC samples and the individual lineages within CGSC by analyzing the published MLST data of this species complex. We extracted information from a total of 1202 non-redundant isolates from 31 published studies. We evaluated the overall geographical pattern of genetic variation as well as investigated potential evidence for recombination within individual lineages and hybridization among the lineages.PubMed for the term “Cryptococcus MLST”. All papers were examined for relevant MLST data associated with isolates of CGSC, including C. bacillisporus, C. gattii, C. deuterogattii, and C. tetragattii, and all reported data regarding CGSC were retrieved and consolidated. All MLST data for strains of CGSC published by November 2022 were collected from the publicly available International Fungal Multi Locus Sequence Typing database. For each unique sequence type, the database stores its allelic profile and DNA sequences across seven loci. A total of 1202 isolates from 31 studies were included in our meta-analyses of population geographic structures. Protocols for DNA extraction, amplification, sequencing, and allelic profiling as well as detailed descriptions of these isolates and their genotype data can be found in the original reports [We carried out a literature search through  reports ,73,74,75To determine the relationships among STs within CGSC, DNA sequences at the seven MLST loci were concatenated for each sequence type. The concatenated sequences were then imported into MEGA X and aligned through Muscle ,77 to reCAP59 locus) and (AT43 and AT44 at the PLB1 locus). MEGA X software was used to reconstruct phylogenetic trees for the seven loci with neighbor-joining method based on muscle alignment and the K2P distance model (Kimura 2-parameter model) [To investigate the partitioning of ATs among lineages, single-locus phylogenetic trees were reconstructed for all seven MLST loci using the DNA sequences for each AT in the IFMLST database. Three of the ATs associated with STs were excluded from the analysis because they did not have corresponding DNA sequences on the MLST database (AT91 at the r model) ,77. The r model) . In addiPopulation genetic analyses were performed to determine if the total CGSC and individual lineages within CGSC were genetically subdivided at the continental and country levels using GenAlEx V.6.5. . StrainsST values between pairs of geographical populations were determined. To prevent potential biases due to small sample sizes, only subpopulations with >five isolates were included in these analyses. Statistical significance was determined by comparing the observed results with 1000 permutated datasets established with a null hypothesis of no genetic differentiations among geographic populations within each analyzed dataset.An analysis of molecular variance (AMOVA) was conducted for each of the ten datasets (five taxonomic CC datasets and five taxonomic NCC datasets) at the country and continental levels. Additionally, Wright’s FPhylogenetic compatibility and linkage disequilibrium analyses were then conducted using the clone-corrected allelic profiles at the seven loci across 566 STs to test for evidence of recombination within and among individual lineages. Tests were performed across 17 population samples: the total CGSC sample, the four CGSC lineages  at the global level, the CGSC sample divided by each continent , as well as six population samples of individual lineage by continent combinations for samples with more than 20 recorded STs . Information regarding the underlying concepts of these analyses and how they were conducted can be found in the MultiLocus V1.3 manual .As of November 2022, there were 566 sequence types (STs) with affiliated DNA sequence data for all seven loci stored on the Cryptococcus MLST database for CGSC. Geographical data were available for 375 STs (66%) representing 1202 isolates collected from 31 published reports along with associated metadata. However, the remaining 191 STs (33%) were not associated with any geographic information. Our population genetic analyses focused on the 375 STs with geographic information. However, for recombination and hybridization analyses, all STs were included. Below, we outline the collected data regarding the 1202 isolates and illustrate the results of our analyses.The 375 STs with geographic information representing 1202 isolates were subdivided into four previously proposed lineages  as indicated by original authors. Among these four lineages, VGII was the most prevalent , followed by VGI, VGIII, and VGIV, containing 225 (19%), 221 (18%), and 34 (3%) isolates, respectively. The CGSC isolates as well as lineages VGI and VGII were distributed across all six continents. However, among the isolates in the MLST database, VGIV was not represented in North America, and VGIII was not recorded in Africa or Asia. In total, the CGSC isolates were distributed across 45 countries. Among the four lineages, VGI was recorded from the most countries (31), followed by VGII (25), VGIII (12), and VGIV (9). Out of the 1202 isolates with geographical data, only 788 isolates (65.5%) included ecological source data, while the remaining 414 isolates (34.5%) were not associated with any source information. Here, we broadly categorize the isolates into three ecological sources: clinical, environmental, and veterinary. Among the 788 isolates with ecological source information, 416 were collected from clinical sources (53%), followed by environmental  and veterinary  sources . The totThe geographical distribution of the non-redundant 1202 CGSC isolates as well as the isolates subdivided into the four lineages are shown in p < 0.001 level, and the VGIV population was statistically significant at the p < 0.05 level  were statistically significant at the 05 level . In the ectively . The paip < 0.001 level, representing 22%, 11%, 10%, 13%, and 10% of genetic variation coming from among ecological niches in Australia, Brazil, Colombia, the USA, and Taiwan, respectively. These results are summarized in Further AMOVA analyses were conducted to determine the amount of genetic variation contributed by ecological niche separation within five countries/region each with a sample size greater than 100 . Both the non-clone-corrected and clone-corrected populations were analyzed and divided into clinical, environmental, and veterinary sample populations. Only ecological populations with >9 isolates were considered. Because of the small sample size, the clone-corrected Australia and clone-corrected Taiwan populations were not analyzed. The remaining three clone-corrected sample populations  failed to reject the null hypothesis that ecological niche separations contributed significantly to the total observed genetic variation within each country. However, all five non-clone-corrected population samples rejected the null hypothesis of no genetic differentiation among ecological samples at the CAP59, GPD1, LAC1, IGS1, PLB1, SOD1, and URA5) for each ST were retrieved from the IFMLST database. A summary of the total reported ATs for each of the seven loci as well as the proportion of shared ATs across the four VG lineages is shown in PLB1 locus to 136 at the SOD1 locus. In addition, the proportion of alleles shared among the four VG lineages ranged from 1 to 6, with the GPD1 locus containing the most shared alleles (6) and loci CAP59, PLB1, and URA5 each having only one shared allele. The differences in length (of base pair) among ATs at each locus ranged from 2 bp for CAP59 to 82 bp for IGS1. The range of occurrence of each AT within each of the four lineages is shown in The allelic profiles of all 566 STs, including the allele type (AT) number at each of the seven MLST loci  were grouped inconsistently to the lineages that they were originally assigned in the published reports. In addition, two STs with previously undefined lineages (ST2 and ST71) were clearly grouped into VGI and VGIV, respectively .CAP59 locus, AT63 and AT64 at the GPD1 locus, AT64 at the LAC1 locus, AT46 and AT47 at the PLB1 locus, AT113 at the IGS1 locus, AT133 and AT134 at the SOD1 locus, and AT59 at the URA5 locus. Single-locus phylogenetic trees including all ATs can be seen in GPD1) and CAP59, LAC1, PLB1 IGS1, and SOD1) showed no inconsistencies between expected and observed VG lineage clustering. However, the GPD1 and URA5 single-locus trees contain one and two instances of inconsistent VG lineage grouping. At the GPD1 locus, AT52 (as part of ST400 and ST409) was expected to be grouped within the VGII cluster based on their original assignment in the database but instead was placed within the VGI cluster. At the URA5 locus, AT35 (as part of ST336) and AT34 (as part of ST419) were both grouped within the VGII cluster but were expected to be within the VGIII and VGIV clusters, respectively.Phylogenetic analyses of the single-locus trees were overall consistent with the phylogenetic pattern based on concatenated gene sequences. However, there was several interesting observations. First, 1–2 allele types at each of the seven loci showed great divergence from their expected clusters. These included AT96 at the Interestingly, each of the seven single-locus trees included at least one occurrence of shared alleles among VG lineages representing potential signatures of recombination or loss of heterozygosity after hybridization. In total, of the 566 STs, 23 (4%) contained an unexpected allele shared with a VG lineage not associated with their originally proposed lineage. Interestingly, ST402 and ST351, which were inconsistently grouped in We examined potential indicators of recombination by investigating two standard signatures: phylogenetic incompatibility and linkage equilibrium. Here, we analyzed a total of 17 population samples including the total CGSC population and the four major VG lineages  at the global level, the total CGSC sample separated by continent , as well as individual lineages separated by continent for populations >20 STs . In the phylogenetic incompatibility test, we found that none of the 17 population samples showed 100% phylogenetic compatibility . Five ofCAP59 or IGS1 loci. Similarly, in the global VGIV sample, 5 of the 21 loci pairs had observed genotype frequencies not significantly different from random recombination, all involving the GPD1 locus. In comparison, no evidence for linkage equilibrium across all 21 loci pairs was observed within the total global CGSC sample, the global VGII sample, and the global VGIII sample. The complete allelic associations among loci for all 17 samples are shown in At the global level, linkage disequilibrium analyses revealed that in all five samples, the null hypothesis of random recombination was rejected . HoweverThis study extracted MLST data of CGSC from 31 peer-reviewed publications and the IFMLST database. The IFMLST database currently consists of 566 established sequence types (STs) grouped into four VG lineages  representing the recognized MLST genotypes of CGSC. The extracted MLST data were used to investigate the population structure of the total CGSC and each of the four lineages within CGSC. Below, we discuss the results of our analyses.As reported in the original literature ,73,74,75C. neoformans species complex [Our global population genetic analyses included 1202 isolates representing 375 STs from 45 countries across six continents. Several STs were broadly distributed, consistent with long-distance clonal expansion of these STs across countries and continents. However, of the 375 STs, 341 STs (90%) were sampled from a single continent, and none were recorded from all six or a combination of five continents. This pattern is different from what was observed in the sister-species complex CNSC, where several STs (ST5 and ST93) were broadly and abundantly distributed . While a complex .Ecological data were available for 788 isolates represented by 188 STs. Across the whole CGSC population, VGI lineage, VGII lineage, VGIII lineage, and VGIV lineage, most isolates  were sampled from a clinical source, followed by  from environmental sources, and the remaining  from veterinary sources. The overall prevalence of isolates recorded from clinical settings is likely due to a greater effort in obtaining and analyzing clinical specimens than from environmental and veterinary sources. Because patient infection by CGSC is from environmental sources, it is important that we increase our efforts to understand environmental populations of this pathogen and identify the potential factors that may contribute to the differences between clinical and environmental samples and how factors in the environment such as fungicide usage may impact clinical antifungal resistance in CGSC . Indeed,C. gattii species complex using distance-based models. Overall, the phylogenetic results were consistent with the four main lineages as revealed based on PCR fingerprinting, AFLP, and/or PCR-RFLP of the URA5 gene fragment. There were two cases of inconsistent observed grouping: ST351 and ST402 observed in CAP59, IGS1, and URA5 loci within VGI. However, the concatenated sequence of ST351 was grouped within the VGII branch and with ATs at the remaining four loci GPD1, LAC1, PLB1, and SOD1 clustered within the VGII lineage. ST402 was previously designated within the VGIV lineage [URA5 locus within the VGIV cluster. Interestingly, ST402 contains AT at the PLB1 locus being grouped within the VGII lineage, while alleles at the remaining five loci were clustered within the VGI lineage. The dominance of VGI alleles (5/7) within ST402 led to the clustering of ST402 within the VGI branch. In most cases, lineage designation of strains of human pathogenic Cryptococcus was derived based on PCR-restriction fragment length polymorphism at the URA5 locus [URA5 locus for lineage assignment.We performed phylogenetic analyses using the published 566 STs from the IFMLST database to examine the overall population structure of  lineage  but contA5 locus . Aside fA5 locus  and betwA5 locus . In addiA5 locus ,33,37. TIn addition to hybridizations among lineages, we identified evidence for recombination within each of the four lineages. The results are consistent with sexual reproduction playing a role in natural populations of CGSC. However, some of the observed phylogenetic incompatibility could be due to reverse mutations, convergent evolution, and/or incomplete lineage sorting. Additional investigations are needed to determine whether some of the nucleotide substitutions at the seven MLST loci have been positively selected and potentially contributed to the observed phylogenetic incompatibilities (and hence recombination). Similar to those observed for CNSC , evidencSeveral theories have been brought forward to illustrate the potential origin(s) of the Vancouver Island outbreak thought to involve ST20 and ST7 ,72, withC. gattii species complex has a known sexual phase with two mating types, aMAT and MATα, defined at the mating type (MAT) locus [MATα-dominant population within CGSC and the sister species CNSC. However, some populations, specifically in Australia, have been described as predominantly aMAT [MAT locus is not included within the MLST scheme, this study did not account for the distribution of aMAT versus MATα strains in our analyses. Nonetheless, it seems uncommon to observe an equal distribution of both mating types in nature. Evidence suggesting sexual recombination within populations with a bias mating type can be explained by same sex mating [T) locus ,96. In tT) locus . Reportstly MATa . Since tx mating ,27,96. TC. neoformans and C. deneoformans) of CNSC [Sexual mating and reproduction among lineages have been observed in the lab. However, most hybrid progeny in laboratory crosses were diploid/aneuploid, with evidence of heterozygosity across most chromosomes . In cont of CNSC ,100. DueC. gattii species complex. Our analyses illustrated geographic structuring of the CGSC population. While evidence for clonal dispersal was found, there is evidence for significant genetic differentiations in all four lineages at both the continental and country levels, with localized clonal expansion contributing to the observed geographic differences within most lineages. However, evidence for 19 incidences of ATs shared across VG lineages and significant evidence for recombination within all lineages demonstrates that genetic exchanges within and among lineages are ongoing. Continued population monitoring of CGSC, especially from environmental sources, is needed in order to effectively cope with the threats caused by this deadly pathogen. In conclusion, our meta-analyses of the published MLST data allowed us to determine the genotype distribution and population structure of the


Foodservices in residential aged-care homes (RACHs) play a vital role in providing meals and maintaining residents’ health through good nutrition. However, foodservices are often required to work within a budget, and the costs involved in foodservices are often misunderstood and underestimated. The aim of this work was to design a costing tool that included all relevant costs of a foodservice. A systems approach was used to inform the development of the Foodservice Costing Tool (FCT). Eight domains were identified, including costs that are both directly and indirectly associated with foodservices. The tool was piloted and trialled in the Australian aged-care setting and compared to currently available national estimates of costs. Through four pilots and subsequent trials, the FCT was able to capture the costs of a foodservice system in a small sample of RACHs, although the low response rate may have biased the sample toward those homes that had fewer problems with the FCT. The results highlighted the limitations of currently reported estimates, which underestimate total costs, as they fail to encompass the complexity of foodservices and to recognise that costs extend beyond the kitchen. The FCT is a useful tool and has the potential to be used by RACHs to both measure and understand their costs at a more granular level to ensure cost effectiveness and accountability. Further research is required to validate the tool and investigate the implementation of the FCT on a larger scale. Foodservices provide the sole source of nutrition for the majority of residents in residential aged care, and therefore they should be designed and operated in a way that meets the needs and expectations of residents. However, there are often organisational structures and budgets that impact how foodservices are provided. Foodservice managers play an important role in reconciling the needs and desires of residents with organisational budgets. Complex systems, such as those found in RACHs, can be difficult to cost; however, understanding the costs involved in these complex systems is vital to ensure efficiency and quality.Nationally, there has been an increasing focus on costs and the level of care provided to residents in RACHs, of which the food expenditure has been highly publicised and criticised . This inWith current tools unable to accurately depict costs, there is little known about what the true costs are in running a foodservice in residential (RAC). This has repercussions for management, including difficulties in accurately measuring and understanding costs, the potential for systems to run at a higher cost than anticipated and difficulties in planning or implementing systems due to unmeasured costs . In addiA search of published and grey literature was conducted to identify existing tools that measured costs of hospital or RAC foodservices. It was identified that there were very few tools available to calculate foodservice costs in these settings, with most focused on hospital foodservice and on food and staff costs alone.The Meal Unit Methodology Costing Formula created by the Institute of Hospital Catering Limited was the most comprehensive tool available, and included food, labour and kitchen supplies in its calculation . As it wThe systems approach has long been applied to the management of complex organisations, and was first applied to foodservice by Vaden . When foThe initial tool was tested for feasibility in calculating costs and useability by aged-care staff in three volunteer aged-care homes, located in two major capital cities in Australia between January 2019 and January 2020. Homes were both of small  or medium sizes  and were operated by either not-for-profit or for-profit organisations. A convenience sample was used, with homes known to researchers approached to participate in the study. One home piloted the tool twice, with the first pilot measuring from January 2018 to January 2019 and the second pilot including January 2019–January 2020. The remaining two homes completed one pilot of the tool each for the 2018–2019 financial year. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of The University of Queensland . Informed consent was obtained from all participants involved in the study.There were 4 iterations within the pilot, with feedback incorporated after each iteration. Field notes were kept by the main researcher to record issues that arose with the usage of the tool. This included questions from managers to clarify components of the tool, errors that had been made in the form and missing data. Several changes were made to the content, format, and process of using the tool over the four iterations. These are outlined in After piloting the tool, researchers aimed to validate the FCT using a number of homes of varying size, location and ownership type. Snowballing was used to recruit homes. The study was advertised through multiple channels including through personal networks, through advertisement in newsletters of three national aged-care or foodservice organisations and through social media. No restrictions were placed on the type or number of homes that could participate. While all homes were eligible to participate and were going to be provided with an individualised cost report, a subgroup of homes was going to be selected to be included in the final analysis. This subgroup was to include an appropriate mix of homes that considered their location, bed number and ownership type to ensure the final group would be representative of aged-care homes within Australia. Due to the low participation of providers, there was an inadequate number of homes to complete analysis in this way.n = 26 homes) provided consent to participate in the first recruitment phase; however, due to the rise of COVID-19 in early 2020, a number of participating homes withdrew or paused their enrolment in the study. One provider withdrew 10 homes, and another requested a pause on data collection in its 15 homes. Only one home continued with data collection from this phase. Recruitment was paused during this time due to the large focus on pandemic management for RACHs and anticipated low levels of expression of interest in participating.Three RAC providers (n = 16 homes) expressed interest in participating in the study and seven provided written consent (n = 14 homes). Fourteen homes were emailed the FCT forms. Following subsequent reminders, two providers (n= 3 homes) did not return their forms. This was considered as withdrawing from the study. Five providers (n= 11 homes) partially completed the forms, and only one home completed the entire foodservice costing tool and received a report of their foodservice from phase two of recruitment. Across both phases of recruitment, only two homes completed the FCT. Demographics for homes in the pilot and validation studies are listed in Recruitment recommenced in January 2021, through the previously mentioned channels. Nine providers  . The ranThe allocation of activities to non-foodservice staff varied between homes, with most hours spent on mealtime assistance. On average, mealtime assistance was provided for 45 min at the three main meals. Smaller mealtimes, including morning tea, afternoon tea and supper, took on average 15 min. Activities such as setting and packing up the dining room, taking residents’ orders, meal delivery and meal ordering were regularly undertaken by non-foodservice staff in each home, with an associated cost ranging from AUD $2.38 to $8.65 prpd . This coAn analysis of foodservice staff workflows was completed to understand how staff time was spent in foodservices. Results showed that for each home, the most time was spent on activities relating to kitchen clean up. The second highest was meal production, followed by meal-service preparation and kitchen administration. Activity related to texture-modified meals consumed the least amount of time. Collecting workflow data was difficult for each home, with workflows either non-existent or not reported in enough detail to allow for granular analysis. In order to obtain this information, significant time was spent by researchers creating personalised forms for each home.Two homes completed the FCT  due to the pandemic, multiple snap or prolonged lockdowns across multiple states, a sector under stress due to a national Royal Commission into Aged Care Quality and Safety, increased scrutiny from the media and changes to funding, reporting requirements and accreditation (increased unannounced visits). All of these external events placed significant strain on the aged-care system, and provide potential reasons as to why RACHs may not have engaged in research. It is also noted that Stewart Brown Chartered Accountants completes annual financial benchmarking for 1200 RACHs , which includes an estimation of food and foodservice staff costs. With this information already provided to a large percentage of RACHs, it is possible that there was not a perceived need for the FCT.While five providers failed to complete the FCT, all provided some data. To further understand whether there were areas of the tool that were more difficult to complete than others, an analysis of the submitted data was completed. When mapped across areas of the tool, it became clear that costs for utilities, supplements and non-foodservice staff were most commonly not included. To further explore these costs, an influence diagram was created to understand what determines the total cost of foodservices within RAC. From this analysis, it became evident that while these categories contribute to the cost of the foodservice, they traditionally sit outside the foodservice itself, potentially increasing the difficulty of obtaining this information. An example is staff allocation for nursing and care staff, who may assist in taking residents’ orders, or assist at mealtimes or supplement expenditure. These costs traditionally sit within nursing; however, they are interrelated with foodservice costs. Difficulty in obtaining information from other parts of the system was also evident in communication with homes. Most cited a lack of access to data or difficulty in obtaining data from other people outside the foodservice.n = 6), the average spend on foodservices was AUD $42.65 per resident per day (range $27.91–$54.85) Food expenditure ranged from AUD $9.37 to $11.36 per resident per day, which is consistent with the costs published by Stewart Brown for the same year [The FCT was developed to calculate the total cost of foodservices in RACH due to the limited scope of current tools. Across all trials  and contract catering ($33.14). The average amount spent on food, supplements and cooking ingredients was AUD $12.92 prpd . This wa and contn = 4) are similar to the Stewart Brown figures (AUD $30.66–$39.08). This provides a degree of confidence in the tool to correctly account for costs, and highlights the additional costs that are included in the FCT.Costs reported by the FCT are higher than currently reported costs, which is to be expected due to the inclusion of a larger number of foodservice costs in its calculation. However, if these costs, including non-foodservice staff, maintenance, utilities, large equipment and kitchen-related expenses, are removed for comparison, it can be seen that the total foodservice costs for the majority of homes (The Resource Utilisation and Classification Study (RUCS), completed by the University of Wollongong, proposed a new assessment and funding model for residential aged care in Australia. While it did not provide specific costs, it did identify cost drivers  within the system. Costs were classified as structural or fixed costs. Fixed costs are those that are related to the characteristics of the facility rather than individual care needs, such as general supervision at mealtimes. Variable costs are the cost of care that is required to address the individual care needs of residents. This study found that facilities that are remote or small, or that specialise in Indigenous care, had higher fixed care costs . When coA difference between the FCT and the currently reported figures is the inclusion of costs associated with mealtime activities that are completed by non-foodservice staff, such as supervision or providing assistance at mealtimes. The RUCS study identified that mealtimes contribute to both the fixed and individual costs. The Resource Utilisation Groups-Activities of daily living (RUG-ADL) is designed to profile loss of function using, eating, transfers, toileting and mobility. It measures the resources required to carry out respective functional tasks. An analysis of independent variables showed that function, mobility, and activities of daily living (including eating) were the largest cost drivers for residents . It is tSince the commencement of this work, there have been changes in funding related to food and nutrition as a result of the Royal Commission into Aged Care Quality and Safety. The Royal Commission highlighted a number of areas in which the aged-care system required improvement, one of which was food and nutrition. Recommendation 112 proposed immediate changes to the Basic Daily Fee by providing an additional $10 per resident per day . This reWhile this study adds to the literature around costs of foodservices in residential aged care, there are limitations to its findings. The major limitation of this work is the low recruitment and the subsequent small sample used to pilot and test the FCT. Due to external events, such as the Royal Commission and COVID-19, recruitment was difficult. The study also saw high dropout and non-completion rates. Whether this was due to external events or the use of the tool itself is unknown. Due to the low response rate, the sample might not be representative. In particular, homes that had problems with the FCT might have been less willing to complete and return it. Further research is required to investigate the use of the tool in RAC and its applicability to practice.Due to the prolonged impact of COVID-19, data collection spanned multiple years. This may impact the ability to compare different costs between each of the homes, due to potential changes in the prices of goods. The complexity of foodservices and the need to rely on facility staff to report figures does affect the precision and accuracy of results. While the sample of homes used was small and not representative of Australian aged-care homes, the FCT measured costs in the aged-care setting and identified components of foodservices contributing to overall costs. Further studies are needed to validate the tool and recruit a larger, more representative sample of homes to determine the average spend on foodservices in Australia.While the development and testing of the tool faced many barriers, the final FCT was able to capture the costs of RACH foodservice for a small and possibly unrepresentative sample of homes. It is the first tool to identify the complexity of foodservices and recognise that costs are located within different parts of the residential aged-care system. By ensuring homes are accurately reporting and attributing their costs to all parts of the foodservice, the FCT enables services to demonstrate cost-effectiveness and to potentially justify and plan future system changes in order to meet the needs and expectations of consumers now and into the future.


Glycogen storage diseases (GSDs) with liver involvement are classified into types 0, I, III, IV, VI, IX and XI, depending on the affected enzyme. Hypoglycemia and hepatomegaly are hallmarks of disease, but muscular and renal tubular involvement, dyslipidemia and osteopenia can develop. Considering the paucity of literature available, herein we provide a narrative review of these latter forms of GSDs.Diagnosis is based on clinical manifestations and laboratory test results, but molecular analysis is often necessary to distinguish the various forms, whose presentation can be similar. Compared to GSD type I and III, which are characterized by a more severe impact on metabolic and glycemic homeostasis, GSD type 0, VI, IX and XI are usually known to be responsive to the nutritional treatment for achieving a balanced metabolic homeostasis in the pediatric age. However, some patients can exhibit a more severe phenotype and an important progression of the liver and muscular disease. The effects of dietary adjustments in GSD type IV are encouraging, but data are limited.Early diagnosis allows a good metabolic control, with improvement of quality of life and prognosis, therefore we underline the importance of building a proper knowledge among physicians about these rare conditions. Regular monitoring is necessary to restrain disease progression and complications. Glycogen storage diseases (GSDs) are a group of rare inborn disorders of glycogen metabolism , a multiGSDs may show a similar clinical presentation, although they may have a variable expressivity. Their overall incidence is approximately 1:10,000 live births  and theyThe literature search occurred in November 2021. All potentially relevant articles were selected from three electronic databases: Google Scholar, Medline and PubMed. Search terms such as “glycogen storage disease”, “glycogen storage disease type 0”, “GYS2 defects”, “glycogen storage disease type IV”, “GBE1”, “glycogen storage disease type VI”, “PYGL”, “glycogen storage disease type IX”, “PHKA2”, “PHKB”, “PHKG2”, “glycogen storage disease type XI”, “GLUT2 deficiency”, “Fanconi-Bickel syndrome” were used in various combinations and permutations.GYS2 (chromosome 12p12.2), which catalyzes the linear addition of glucose residues to the branching structure of glycogen. Mutations of the muscle-specific isoform of glycogen synthase encoded by GYS1 gene originate GSD type 0b. Clinically, it exhibits weakness, exercise intolerance and arrythmias without liver involvement [GSD type 0a is caused by defects of the hepatic isoform of glycogen synthase, encoded by olvement .A focus on GSD subtype 0a is provided in this review and it will be referred to as “GSD type 0”. The onset of symptoms occurs before 3.5 years old on average . AffecteGBE1 gene, chromosome 3p14) causes the GSD type IV or Andersen disease. The branching enzyme adds a segment of a minimum of six α-1,4 linked glycosyl units into an α-1,6 position; this activity is fundamental for the correct glycogen storage and degradation. Indeed, GBE1 deficiency induces the accumulation of an amylopectin-like molecule called polyglucosan, an insoluble polymer which has fewer branching points and longer outer chains than normal glycogen [The branching enzyme deficiency , three neuromuscular subtypes  and a multisystem form (adult polyglucosan body disease). Indeed, the GSD type IV has a very heterogeneous clinical presentation, ranging between myopathy, cardiomyopathy, neuropathy and liver failure variously combined , 21. OneThe classical progressive hepatic form is characterized by an initially normal phenotype with rapid deterioration in infancy, when hepatomegaly and failure to thrive appear. Prolonged partial thromboplastin time and prothrombin time progressively develop, along with hypoalbuminemia. Linear glycogen molecules can be metabolized, so this prevents severe hypoglycemia. Nevertheless, it may develop in the final stages of liver failure . The finThe non-progressive hepatic disease exhibits different grades of severity, with the milder phenotype showing hepatomegaly and inconstant elevation of transaminases. McConkie-Rosell et al.  in 1996 The neuromuscular variants of GSD type IV are very rare; nevertheless, the perinatal variant should be considered one of the differential diagnoses in neonates with severe hypotonia and in pregnancies complicated by polyhydramnios, fetal hydrops, reduced fetal movements, arthrogryposis, hypoplastic lungs of unknown etiology –21, 32. PYGL gene (chromosome 14q21-q22) mutations are associated to GSD type VI, also known as Hers disease [ disease . Affecte disease , 35. Sym disease , 36. KetGSD type VI was previously considered as a mild disease. However, recent reports highlighted the possibility of a progression to fibrosis and cirrhosis, and a degeneration to hepatocellular carcinoma, so a rigorous long-term monitoring of hepatic function is needed , 37, 38.PHKA2, PHKB and PHKG2 respectively, and are causative of GSD IX subtypes IXa, IXb and IXc [PHKB is expressed in both liver and muscle. Furthermore, the α- and γ-subunits have a muscle-specific isoform, encoded by PHKA1 and PHKG1 respectively. The α-subunit isoforms are inherited in an X-linked fashion, while the other isoforms have an autosomal recessive inheritance [The above mentioned glycogen phosphorylase is functionally activated by the phosphorylase kinase (PhK), a serine/threonine-specific protein kinase. PhK is composed of four different subunits: α, β, γ and δ. Subunits α and β have regulatory functions, the γ-subunit has catalytic function and δ-subunit is a calmodulin protein. The subunits possess tissue-specific isoforms; the liver-specific isoforms of the α-, β- and γ-subunits are encoded by  and IXc . Howevereritance . Furthereritance .PHKA2 mutations [GSD type IX represents the most frequent type of glycogen storage disease, with a prevalence of 1:100,000 births . The pheutations .PHKA2 mutations has been reported [PhK deficiency was generally considered a benign condition, with symptoms of hypoglycemia, hepatomegaly and growth retardation improving after the early introduction of a strict dietary treatment . Neverthreported , 47. FurPHKB mutations is similar to what observed in the milder PHKA2 mutations; symptoms related to muscular involvement may be present [PHKG2 associated phenotypes show a more severe presentation [The phenotype associated to  present . PHKG2 aentation . The clientation , 51. Morentation .SLC2A2 gene, chromosome 3q26.1–26.3), expressed in hepatocytes, pancreatic β-cells, enterocytes and renal tubular cells [GSD type XI is caused by defective glucose and galactose transporter GLUT2 (ar cells . This coar cells . This isHNF4α gene cause hyperinsulinemic hypoglycemia associated to hepatomegaly and renal Fanconi syndrome. These HNF4α mutations might decrease the SLC2A2 expression in both liver and kidney, resulting in nonfunctional GLUT2 and are responsive to therapy with diazoxide [Neonatal screening may show hypergalactosemia but cataract is not present in this condition . Impaireiazoxide , 60.Fasting ketotic hypoglycemia is a hallmark of hepatic GSDs , 61. PatElevated triglyceridemia and cholesterolemia are common findings in GSDs with liver involvement. In these conditions, the dysregulation of glucose metabolism leads to fasting intolerance, enhancing secondary lipolysis and increased mitochondrial fatty acid oxidation . In GDS As previously mentioned, the distinctive element of the glycogen synthase deficiency is the absence of hepatomegaly, since hepatic glycogen storage is impaired , 12, altIn GSD type IV the accumulation of abnormal glycogen, less soluble than normal glycogen, causes a foreign body reaction with consequent osmotic swelling and cell death , leadingPHKG2 mutations [PHKA2 mutations. More recently, early appearance of liver cirrhosis in a 2 years old child with homozygous mutations in PHKB has been reported [Furthermore, cirrhosis has recently been depicted in GSD type VI . Among tutations . Neverthutations  describereported .Tumor degeneration is described in GSD type IV, VI and IX. Hepatocellular adenoma and carcinoma have been described in GSD type IV . GSD typRenal involvement is not described in GSDs types 0 and VI to date.PHKA2 and PHKG2 mutations can display renal tubular acidosis and tubulopathy with secondary development of rickets in a patient with GSD type IXc. The establishment of an adequate nutritional therapy improves tubular acidosis [Conversely, individuals affected by acidosis .In addition, renal involvement represents a hallmark of GSD type XI, in which the renal epithelial cells are damaged by the accumulation of glycogen and monosaccharides; this alteration leads to proximal tubular dysfunction, documented by glycosuria and aminoaciduria. Although this condition is related to a severe phenotype, rare cases of patients with mild renal dysfunction have been described .Normal length and weight at birth are usually observed, suggesting that the metabolic disorders do not interfere with fetal growth , 19, 72,Patients diagnosed with GSD type 0 may show either normal or poor growth with a delayed bone age in early childhood , 17. A cGrowth impairment is not a hallmark of GDS type IV and it may be present or not, depending on the causing mutation and the clinical subtype .In contrast, short stature is a common feature in GSD types VI and IX, with a variability in the degree of improvement of parameters in treated patients reaching the adult age . Most inSevere growth impairment is described in Fanconi-Bickel syndrome. Patients affected by proximal renal tubular dysfunction of variable genetic causes show growth retardation ascribed to renal losses but the short stature observed in Fanconi-Bickel syndrome is more pronounced, suggesting other mechanisms not clearly understood . NewbornAmong all GSDs, bone is mostly affected in GSD type XI, where hypophosphatemic rickets, frequent fractures and bone deformities are described as a result of the renal tubular dysfunction . Limbs dSkeletal muscle and myocardial involvement is not observed in GSD type 0a .Heart failure after orthotropic liver transplantation has been described in patients with the progressive liver form of GSD type IV with no previous history of cardiac involvement , 28. ThiMild to severe myopathy and dilated cardiomyopathy are also described in the neuromuscular forms of GSD type IV , 79. RemMuscular cramps or fatigue after physical exercise have been recorded in a minority of reports of GSD type VI, usually related to undertreatment and protein deficiency . Muscle In a recent case series, asymptomatic left ventricular and septal hypertrophy was reported in a patient with GSD type VI, and interventricular septal hypertrophy was found in a patient with GSD type IXb. The authors recommended echocardiogram every 1–2 years for patients with GSD type VI and IX after 5 years of age . A systeDevelopmental delay has been described in 22% of children with GSD type 0 , whereasMild developmental delay was also reported in GSD types VI, IX and XI , 76. WitWith respect to the motor impairment, GSD type IV, VI, IX and XI deserve to be mentioned.In the progressive hepatic GSD type IV the muscle tone is often normal at the time of diagnosis, but progression to generalized hypotonia may develop within the two years of life . GSD typMild hypotonia was reported in a few GSD type VI patients .PHKB and PHKG2 mutations [PHKA2 mutations, Lau et al. [Hypotonia and motor delay can be rarely associated to utations , 51. Witu et al.  and collu et al. .A summary of the main clinical features of the GSDs is provided in Table PHKA2 and PHKB [A careful clinical history and examination together with laboratory findings may suggest the diagnosis. An OGTT can be realized when GSD types 0, VI and IX are suspected; in all forms elevated lactate will be recorded at 120 min. Patients with GSD type 0 will show hyperglycemia within the first two hours, then hypoglycemia might be observed at a prolonged OGTT, likely due to hyperglycemia-induced hyperinsulinemia . In GSD and PHKB , 47. In and PHKB . Howeverand PHKB , 84. In and PHKB .PHKA2 and PHKG2 associated to a severe phenotype often require overnight feeding to maintain euglycemia [A strict dietary regimen high in proteins and low in simple carbohydrates, which includes frequent intake of complex carbohydrates such as maltodextrin and uncooked cornstarch, is fundamental to prevent hypoglycemia in ketotic GSDs . Indeed,glycemia . Since gglycemia , 37, 61.glycemia . Carbohyglycemia  describeglycemia . AdversePatients with GSD type 0 are treated with frequent feeds of hyperglucidic diet plus cornstarch and protein supplementation. Patients with GSD type IV are managed with hyperglucidic diet plus cornstarch, nocturnal enteral feeding, protein enrichment with the aim to limit the accumulation of glycogen, to prevent catabolism and to improve growth and fasting tolerance. The more severe forms are treated with liver transplantation . For GSDAccording to the available data, universally accepted guidelines for the management of these types of GSDs have not been defined. Nevertheless, an appropriate follow-up should be provided, in order to establish a good metabolic control and monitor the possible complications. Medical and nutritional evaluations and blood assessment, including complete liver and renal function, lipid profile, calcium-phosphate metabolism, serum electrolytes, blood gas analysis and urinalysis, should be fulfilled every 6 months on average; a higher frequency is recommended in younger patients and in those who have not achieved a metabolic balance. A continuous glucose monitoring may be helpful to survey the glycemic fluctuations, especially in younger patients. Alpha-fetoprotein levels along with abdomen ultrasound can be used to screen for hepatocellular carcinoma, even though there are no validated surveillance protocols to date . Liver fGSD type IV patients require a complete cardiac function evaluation, including electrocardiogram and echocardiography. For patients with GSD types VI and IX after 5 years of age a cardiac evaluation is recommended every 1–2 years .Regarding the bone metabolism, a careful assessment of calcium and vitamin D intake and monitoring of 25-OH vitamin D level is recommended. Calcium, phosphate and vitamin D supplementations, along with annual DXA scan evaluation, are required to prevent osteopenia and fractures, particularly in GSD type XI, along with a surveillance of renal function . SkeletaGSDs type 0, IV, VI, IX and XI with liver involvement may have a similar clinical presentation. However, these diseases exhibit a phenotypic continuum, and even in the mildest forms, regular monitoring and dietary adjustments are necessary to restrain disease progression and complications. Some cases may exhibit a clinical burden with severe organ complications. Building a proper knowledge among physicians about these rare conditions is crucial to improve prognosis and quality of life of patients, especially those affected by the most severe forms. Further studies are needed to outline the genotype–phenotype correlation and define personalized therapies and management.