Heliococcus bohemicus Šulc. Virus infectivity experiments were performed with this species to study the transmission ability of natural populations living in infected vineyards in Alsace, France. Mealybugs were sampled on vines infected by grapevine leafroll-associated viruses  and by grapevine virus A (GVA), either alone or in combinations. Out of six natural populations tested, only one, located at Bennwihr, was able to transmit GLRaV-1 and -3 to healthy vines, though with low transmission rates . Mealybugs from Bennwihr were also able to transmit GLRaV-3 from grapevines of another location where H. bohemicus was not a vector. Conversely, mealybugs from two other locations did not transmit any virus acquired from infected grapevines at Bennwihr. These results suggest differences in vector ability between H. bohemicus populations. Moreover, laboratory experiments were developed to estimate the minimal acquisition and inoculation access periods  for virus transmission of GLRaV-1 and -3, and GVA. First instar nymphs transmitted GLRaV-1 after 6 h AAP, GLRaV-3 and GVA together after 1 h AAP, and the three viruses after only 1 h IAP, supporting a semi-persistent mode of transmission. Second instar nymphs fed on multi-infected grapevine for 72 h then starved or fed on potatoes tested positive by RT-PCR for GLRaV-1 and -3 after up to 35 and 40 days, respectively, contrasting with the short retention times generally observed for mealybugs. These findings provide new knowledge of the vector ability of H. bohemicus.Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug,  Closteroviridae species, designated as Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, and -4, are associated with GLD, among which three  are naturally transmitted by mealybugs (Hemiptera: Pseudococcidae) and soft scales (Hemiptera: Coccidae) [Grapevine virus A (GVA), GVB, and GVE  associated with the “rugose wood” (RW) complex, are also transmissible by some of these vector species [Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases and occurs in all major grapevine-growing areas. At least four serologically distinct  species . RW may  species . Grapevi species ,8,9,10.  species , Tsai et species , and Le  species  have sugHeliococcus bohemicus Šulc, is a highly polyphagous species, widely distributed in the Palaearctic region from Western Europe to China [H. bohemicus has been identified as a vector of GLRaV-1 and -3, and GVA [Parthenolecanium corni (Bouché), Parthenolecanium persicae (Fabricius), and Pulvinaria vitis (L.), and two of mealybugs, H. bohemicus and Phenacoccus aceris (Signoret), can be found [Pa. corni, H. bohemicus is the most common species. However, it seems more localised and is usually not considered a pest, though populations can sometimes reach high numbers, partly due to reductions in insecticide treatments. The backs of nymphs and adult females have thin and long glassy wax filaments, which differentiate them from Ph. aceris. H. bohemicus is bisexual and develops only one generation per year in our region (monovoltinism) compared with two in Italy [H. bohemicus in north-eastern France is little-documented [The Bohemian mealybug, to China . It occuto China ,20,21,22 and GVA ,24. In tbe found ,21,25. Ain Italy ,20. Secoin Italy . After min Italy . Virus tcumented . In otheH. bohemicus are schematised in The various experimental approaches used in studying the vector biology of Inoculation experiments A were coOrigin of insects and virusesH. bohemicus populations originated from commercial vine plots at six locations in Alsace (north-eastern France): Bennwihr, Colmar, Kientzheim, Nothalten, Ribeauvillé, and Turckheim, where it was the only mealybug species present. Heavily infested grapevines were first tested by enzyme-linked immunosorbent assay (ELISA) for GLRaV-1, -2, and -3, and GVA  was used to detect GLRaV-1, -2, and -3, and GVA in insect samples (1 to 74 L2) before infectivity experiments, using the following primers for GLRaV-1 (‘LR1-H70F1’ GTTGGTGAATTCTCCGTTCGT and ‘LR1-H70R1’ ACTTCGCTTGAACGAGTTATAC), GLRaV-2 (‘P19qtF’ ATGGAGTATTGTTTGAAGCAGGTAC and ‘P24qtR’ AGAATGTCTTCAGCTTCATAAGGAG), GLRaV-3 (‘LR3-POLF1’ ACGTAACGGGGCAGAATATAGT and ‘LR3-POLR1’ TATCAACACCAAGTGTCAAGAGTA), and GVA (‘GVA-CPF1’ GGCTACGACCGAAATATGTAC and ‘GVA-CPR1’ AGAAACGATGGGTCATCCATC), following the protocol developed by Beuve et al. . Nymphs Recipient grapevinesVitis vinifera cv. Pinot noir, frequently planted in vineyards of Alsace, Champagne, and Burgundy (clones P114 and mainly P115), or from germinated pips of Pinot noir and Pinot blanc. Mother plants of the cuttings were tested with ELISA and RT-PCR for the absence of leafroll and rugose wood viruses . Plants were grown in individual pots under greenhouse conditions until the 6–12 leaf stage and then used in transmission experiments. They were sprayed bi-monthly with an insecticide  to ensure the absence of insects; this spraying was stopped at least one month before insect inoculation. A sub-sample of ten recipient plants was tested to determine their virus-free status by ELISA prior to transmission experiments.Virus-free grapevines were obtained from rooted cuttings of Transmission to healthy recipient grapevinesSince viruses are unevenly distributed on grapevines, several leaf pieces were cut from infected grapevines bearing first instars nymphs (L1)  and attached with hairclips onto the leaves of virus-free recipient grapevines (3–4 leaf pieces per plant). Insects crawled off as the leaf fragments dried. As for the L2 nymphs, these were gently stimulated to move, then transferred with a fine paintbrush inside small cages . The cages were then attached with hairclips to leaves of the recipient plants. The number of recipient plants used according to the origin and cultivar of the virus combinations is given in Virus detection in grapevinesThe infection of recipient vines was assessed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The grapevines were checked by ELISA 4–6 and 8–12 months after IAP, and up to 18–24 months for surviving plants that had previously remained negative. The plants inoculated in late September could not be tested before cold storage and were therefore tested first about 6–7 months after IAP. Regardless of the recipient grapevine, these were systematically tested for the presence of GLRaV-1 and -3 and GVA. GLRaV-2 was only tested for when the source grapevine had been infected with this virus. The accession Y258 , multi-infected by GLRaV-1 and -3, and GVA, was used as a positive control for the three viruses. The accession Chardonnay V38, infected by GLRaV-2 and -3, was used as the positive control for GLRaV-2. Healthy P115 cuttings were used as negative controls. Tissue extracts were obtained from pooled fragments of three leaves. Leaf fragments were ground (1 g leaves for 5 mL buffer) inside extraction bags with a bullet blender . Polyclonal antibodies raised against GLRaV-1, -2, or -3, or GVA produced in the laboratory were used in a biotine-streptavidine procedure . AbsorbaStatistical analysisp-value < 0.05 was considered as the threshold for significance. Statistical analyses were performed with R, version 2.10.Chi-square tests were used to compare the detection rates in L2 nymphs, according to virus species and life cycle. A Firstly, the rate of virus acquisition by L1 nymphs was investigated according to feeding time and to nymph numbers B. SecondOrigin of insects and virus sourcesH. bohemicus culture originated from the Riesling plot at Colmar. L2 nymphs were collected in April from new leaf buds and raised on potato sprouts inside glass jars, closed with a 28 μm mesh tissue to enable airflow, and kept in the dark at 20–23 °C. When they became adults and gave birth to larval clusters, L1 nymphs were collected to determine their rate of virus acquisition according to feeding time and optimal AAP and IAP. L2 nymphs used to evaluate the virus acquisition rate according to feeding time were collected on the same plot and reared for at least one month on potato. Those used for optimal AAP/IAP and retention time were born on potato.The Pa. corni [Phenacoccus aceris (Signoret) [Virus source cuttings from accessions of our reference collection of grapevine viruses  were rooa. corni  and Phenignoret) . The virVirus acquisitionTM to retain the water supply during AAP. Mealybugs were collected with a fine paintbrush from sprouted potatoes and transferred onto the underside of source grapevines leaves for AAP.Leaves of virus source grapevines were collected and placed individually into tight and round polystyrene crystal boxes. A small wet cotton piece was wrapped around the cut petiole of each leaf, then tightly swathed inside ParafilmRate of virus acquisition according to time of feeding: AAP of L1 nymphs on Y258 leaves was tested for 24, 48, and 72 h. For each time, virus detection was conducted by RT-PCR on five samples of 5, 10, and 20 L1. For L2, AAP of 1, 3, 6, 24 h, and from 2 up to 7 days, was tested on leaves of Y258. Ten mealybugs per batch were chosen based on the results obtained with L1, and virus detection by RT-PCR was performed on ten batches of L2 for each AAP. The presence of viruses was checked in fragments of source leaves before AAP. A batch of ten mealybugs collected from potato rearing was used as a healthy control.Minimal time for virus acquisition and inoculation: After 1, 6, or 24 h AAP on Y258 leaves for L1, and on P70 leaves for L2, nymphs were transferred into small cages, which were clipped onto the leaves of five virus-free recipient cuttings (P115) for 1, 6, or 24 h for IAP (30 nymphs per plant distributed on two leaves), allowing for each growth stage a total of 9 AAP × IAP pairs. Each recipient plant was enclosed in a bread bag, as previously described. Plants were placed at 20–23 °C, 16 h/8 h (L/D), under artificial light. After IAP, the recipient plants were immediately sprayed with an insecticide. Grapevine cuttings of the same age, without nymphs and kept in the same conditions under a glasshouse, were used as negative controls.Virus detection in recipient grapevinesLeaves of the recipient vines were tested by ELISA, as described above. First detection tests were conducted on leaves ca. 4 months after the transmission experiments. After a dormancy period under an unheated glasshouse, plants were tested again 10–12 months after IAP. Virus source grapevines and healthy cuttings were used as positive and negative controls, respectively.Comparison between populationsH. bohemicus populations, L2 nymphs were collected in April from three plots located at Bennwihr, Turckheim, and Ribeauvillé, and reared separately on potato tubers. Inoculation experiments were conducted with these mealybugs  and their progeny  in June–July and October. Mealybugs from Bennwihr were placed on leaves of infected grapevines from Nothalten (see Virus acquisition above), whereas those from Turckheim and Ribeauvillé were placed on leaves of infected grapevines from Bennwihr for an AAP of 7 days . After AAP, mealybugs were transferred onto healthy P115 cuttings for an IAP of 7 days.To compare inoculation capacities between L2 nymphs from potato rearing were allowed to feed on leaves of the multi-infected accession Y258 for a 72 h AAP. Then, they were collected, and one half thereof was starved inside an empty box, while the other half was transferred onto potato tubers. Virus content was then tested by RT-PCR, as described above, on five batches of ten mealybugs at times spanning daily from 7 to 15 days after transfer, then every ten days until 60 days for those on potato tubers and every five days until 35 days for those starving. Potatoes used as food supply were simultaneously tested by RT-PCR for virus content.2 = 16.3, df = 3, p = 0.001) due to the lower rates for GLRaV-2 and GVA. The proportion of overwintering samples that tested positive for at least one virus was around 78% . Winged adult male samples collected in April (n = 4 batches) tested negative, as expected, due to their inability to feed and the absence of transstadial virus transmission.For a set of source vines harbouring various virus combinations, virus content was tested by RT-PCR in the L2 nymphs, either active on leaves in spring or overwintering under stock bark. Positive detection was obtained in mealybug samples for the four viruses being tested, with smaller detection rates for GVA. Detection rates of GLRaV-1 (respectively 79 and 62%) were close to those of GLRaV-3 (respectively 68 and 63%) . For theound 78%  and variound 78% . The proVineyard-sampled mealybugs were transferred onto healthy recipient plants. After completing the IAP and the insecticide treatment, no living insects were found on recipient plants. Healthy control plants, with or without non-viruliferous mealybugs, were all negative in ELISA . TransmiWith the aim of comparing the vector ability of various populations, adult females sampled at Bennwihr, Turckheim, and Ribeauvillé were reared on potato to allow them to give birth to nymphs. Thereafter, the L2 nymphs were placed for AAP on excised leaves of infected grapevines from Nothalten or Bennwihr . Only BeAfter various AAPs , GLRaV-1 and GVA were detected by RT-PCR in batches of five L1 or more, whereas GLRaV-3 was detected in ten or 20 L1 per batch . GloballGLRaV-1 and GVA were detected by RT-PCR in batches of L2 tested after 1 h and 3 h of AAP on leaves of the accession Y258, whereas GLRaV-3 was detected only after 6 h AAP . The maxTransmission experiments were performed by combining various AAP and IAP . With thAfter a 3-day AAP on accession Y258, the L2 nymphs were tested by RT-PCR after various periods spent either starving or confined on potato. In batches of starved nymphs, GLRaV-1 and -3, and GVA RNAs were detected until 35 days after the start of starvation . Those fH. bohemicus distributed in infected vineyards of Alsace (France), only one, located at Bennwihr, was able to transmit GLRaV-1 and -3 to healthy grapevines in infectivity experiments. In addition, this population could transmit GLRV-3 when fed on infected vines from Nothalten, while the mealybug population from this location was non-vector. Conversely, non-vector mealybugs from populations from two other locations did not transmit viruses from the infected vines from Bennwihr. However, in nymphs living on mixed-infected grapevines from the six locations, all the viruses present could be detected, showing that nymphs fed well of phloem sap. Virus detection in mealybugs was thus not linked to their transmission ability. Moreover, the handling of nymphs might have affected their proneness to inoculate the virus. As expected, GLRaV-2 was detected in nymphs but was not transmitted. No species among soft scales is known to transmit GLRaV-2 [Closteroviridae family, assigned to the genus Closterovirus, of which the only known vectors are aphids [H. bohemicus L1 or L2 nymphs per test plant.Among six natural populations of the mealybug e aphids . The repe aphids  obtainedH. bohemicus could favour geographical differences between populations even though our sampling sites are located within a distance of ca. 40 km. In this context, it would be interesting to study whether different the transmission ability between H. bohemicus populations could result from genetic differences. To support this assumption, it would be interesting to precisely localise receptors where virions are retained before being released for inoculation, and compare their affinity capacities in vector and non-vector populations.A possible explanation for this differential vector ability between populations is that the receptors, which retain virions in the insect, may vary in their viral affinity. Geographic intraspecific genetic variations exist among mealybugs . The lowPu. vitis transmits GLRaV-3, Hommay et al. [Pu. vitis nymphs sampled on grapevines infected with various virus associations. In Europe, Pa. corni was shown to transmit GLRaV-1 but not GLRaV-3 [Virus species also present genetically and geographically distinct variants that can influence their transmission efficiency. The L1 from Colmar populations transmitted viruses from the accession Y258, while the L2 from the same population were unable to transmit viruses from the accession P70. In addition, mixed infections of viral variants within a single plant are common and differential transmission of variants may occur . For exay et al.  recorded GLRaV-3 ,32,39, t GLRaV-3 . Variati GLRaV-3 .H. bohemicus specimens sampled in spring from the Colmar population showed that the L1 were able to transmit all three viruses, GLRaV-1 and -3, and GVA, after acquisition on the multi-infected accession Y258. However, the summer L2 from the same location were unable to transmit GLRaV-1 and GVA after AAP on the accession P70. These experiments were performed with the same infected and recipient plant accessions as in the transmission experiments with Pa. corni [Ph. aceris [Pu. vitis [H. bohemicus L2 were unable to do so remains unclear.The L1 and summer L2 nymphs from the Bennwihr population transmitted viruses, but no results were obtained with the L2 sampled during winter or in early spring before maturation into adults. The early spring L2 were probably disturbed when transferred for infectivity experiments, which could have reduced their feeding activity and anticipated moulting into adulthood as some gave birth to crawlers during experiments. Our transmission experiments with a. corni , Ph. ace. aceris , and Pu.u. vitis , where bH. bohemicus nymphs that acquired at least one virus was high (88 to 100%), virus transmission occurred at low rates (26%). Zorloni et al. [H. bohemicus nymphs transmitted GLRaV-3 to 2 out of 77 recipient plants and GVA to 1 out of 38, whereas GLRaV-1 was not transmitted. Therefore, this species was not considered a threat to grapevines in northern Italy. Similarly, in our infectivity experiments, the rate of active H. bohemicus positive for at least one of the viruses reached 88%, while nymphs from Bennwihr population were able to transmit GLRaV-3 to 12% of recipient vines at most. Comparatively, for the other mealybug species Ph. aceris present in our region, the maximal rates of GLRaV-1 and GVA transmission, using only 20 L1 per recipient plant, reached 90% and 100% with a 48 h AAP followed by a 48 h IAP [Sforza et al.  reportedi et al.  showed t48 h IAP .H. bohemicus L1 nymphs were able to acquire and inoculate GLRaV-3 and GVA after as little as 1 h AAP or 1 h IAP, while at least 6 h AAP and 1 h IAP were necessary for GLRaV-1 transmission. GVA and GLRaV-1 RNAs were already detected in batches of the L2 tested after 1 h AAP. Such short durations are similar to results obtained with other vector species and add further evidence for a semi-persistent mode of transmission of these viruses by mealybugs. For Pseudococcus longispinus (Targioni Tozzetti), La Notte et al. [Planococcus ficus (Signoret) and GLRaV-3, acquisition and inoculation could occur within 1 h [Phenacoccus manihoti Matile-Ferrero, the minimal time to reach the phloem was 1.5 h on a favourable host, and 2.8 h on an unfavourable host [Ph. aceris L3 nymphs reached the phloem of infected plants in a mean time of 2.6 h [Ps. longispinus could be due to species difference and to the higher number of mealybugs used, which increased the probability of acquisition by the fastest individual in the batch.Our transmission experiments with varying AAP and IAP showed that the e et al.  found the et al.  obtainedthin 1 h . These rthin 1 h ,45,46,47thin 1 h ). For Phble host . Ph. aceof 2.6 h  for Ps. H. bohemicus L2 removed from a GLRaV-1 and -3, and GVA infected source grapevines tested positive for viruses until at least 40 days after their transfer onto potatoes, and for 35 days when starved. These results contrast with the few days of retention generally reported in mealybugs [Ph. aceris lost virus and infectivity for GVA and GLRaV-1 at 5 and 7 days, respectively, after leaving an infected source [Pl. ficus nymphs retained GLRaV-3 RNA for at least eight days when feeding on a virus non-host and for two days when starving, and were then capable of transmitting it successfully to healthy grapevines [Pseudococcus calceolariae (Maskell) up to 16 days on non-Vitis plant hosts, but not after 20 days [H. bohemicus L2 nymphs overwintering on infected grapevines tested positive for viral RNA from December to mid-April. Yet, these nymphs were embedded alone or together inside a powdery wax cocoon under bark layers and could not reach the phloem. Similarly, Le Maguet [Ph. aceris nymphs from December to February. As in the case of H. bohemicus, no virus transmission occurred after infectivity experiments conducted with overwintering Ph. aceris, which calls into question their infectiousness despite the detection of viral RNA sequences.Finally, in our retention experiments, virus detections using RT-PCR showed that the ealybugs ,10,24,50d source . Pl. fic 20 days . Moreovee Maguet  detectedTo conclude, we provide further findings on the interaction between H. bohemicus and grapevine ampelo- and vitiviruses. We observed that its vector ability can vary locally between populations. The duration of virus acquisition and inoculation were found to be only a few hours, in agreement with published evidence of a semi-persistent transmission mode. Mealybugs removed from infected grapevines, then placed on non-host plants or starved, tested positive for viral RNA until 40 and 35 days, respectively, much longer times than those reported for other mealybug species, but this does not imply that they were still infective. Moreover, as with Ph. aceris, viral RNA remained detectable in most H. bohemicus nymphs overwintering under the trunk bark of infected grapevines, without any feeding opportunity. Further research is needed to characterise the variability in vector efficiency between mealybug populations by clarifying virus–vector interactions and factors affecting their respective virus transmission efficiencies, including genetics.


Hepadnaviridae and demonstrate comparable infection outcomes and progression to hepatocellular carcinoma. North American woodchucks (Marmota monax) infected with WHV present a natural infection model for the assessment of acute and chronic hepadnaviral infections. We hypothesize that KC absence in acute viral hepatitis leads to chronic infections by impacting downstream virus processing and immune responses.Hepatitis B virus (HBV) infections are one of the largest global infectious disease burdens, despite the availability of a preventative vaccine. The virus infects the liver which is a vital organ that plays a critical role in homeostasis due to its metabolic and immunoregulatory functions. Kupffer cells (KCs) are considered to be a first line of immune defense for protection of hepatocytes. Both woodchuck hepatitis virus (WHV) and HBV are members of We aim to uncover the early host-virus interactions in acute WHV infections to facilitate our understanding of the risk of progression to chronic infection and the immunopathogenic mechanisms involved in chronic hepatitis.10VGE copies/ml), we imaged host cell-hepadnaviral interactions and characterized immunological and virological differences between KC-depleted animals and non-depleted animals.We designed and optimized in-house WHV and woodchuck-specific assays to assess intrahepatic and systemic virus presence and anti-viral immune responses which occur following WHV invasion (<24 hours) and infection (4-6 weeks). A live imaging protocol was developed for capturing real-time events in a living host. Next, we optimized macrophage depletion in woodchucks to evaluate virus invasion and acute infection outcomes in the presence and absence of KCs. Using purified and fluorescently labelled WHV virions  revealed that the virus traffics to, and accumulates within, the liver 10 seconds following intravenous administration, demonstrating direct binding and capture by liver macrophages. In contrast, virus only begins to accumulate in the KC-non-depleted control minutes after infection in a randomized manner. Preliminary invasion results indicate that in both the KC-depleted and non-depleted animals, virus is most readily detectable in the blood within the first hour following viral administration. By 24-hours post-virus injection the highest viral load in non-depleted woodchucks was identified in the liver whereas in KC-depleted woodchucks, the highest viral loads were found in the spleen 24-hours post-injection.We have successfully captured the early host-virus interactions and continue to uncover the role of KCs in viral hepatitis infections. Tracking the initial interactions allows us to understand the events promoting viral dissemination and development of chronic hepatitis.None Declared


Multimorbidity is a rising global phenomenon, placing strains on countries’ population health and finances. This systematic review provides insight into the costs of multimorbidity through addressing the following primary and secondary research questions: What evidence exists on the costs of multimorbidity? How do costs of specific disease combinations vary across countries? How do multimorbidity costs vary across disease combinations? What “cost ingredients” are most commonly included in these multimorbidity studies?We conducted a systematic review (PROSPERO: CRD42020204871) of studies published from January 2010 to January 2022, which reported on costs associated with combinations of at least two specified conditions. Systematic string-based searches were conducted in MEDLINE, The Cochrane Library, SCOPUS, Global Health, Web of Science, and Business Source Complete. We explored the association between costs of multimorbidity and country Gross Domestic Product (GDP) per capita using a linear mixed model with random intercept. Annual mean direct medical costs per capita were pooled in fixed-effects meta-analyses for each of the frequently reported dyads. Costs are reported in 2021 International Dollars (I$).Fifty-nine studies were included in the review, the majority of which were from high-income countries, particularly the United States. (1) Reported annual costs of multimorbidity per person ranged from I$800 to I$150,000, depending on disease combination, country, cost ingredients, and other study characteristics. (2) Our results further demonstrated that increased country GDP per capita was associated with higher costs of multimorbidity. (3) Meta-analyses of 15 studies showed that on average, dyads which featured Hypertension were among the least expensive to manage, with the most expensive dyads being Respiratory and Mental Health condition , Diabetes and Heart/vascular condition , and Cancer and Mental Health condition in the first year after cancer diagnosis . (4) Most studies reported only direct medical costs, such as costs of hospitalization, outpatient care, emergency care, and drugs.Multimorbidity imposes a large economic burden on both the health system and society, most notably for patients with cancer and mental health condition in the first year after cancer diagnosis. Whether the cost of a disease combination is more or less than the additive costs of the component diseases needs to be further explored. Multimorbidity costing studies typically consider only a limited number of disease combinations, and few have been conducted in low- and middle-income countries and Europe. Rigorous and standardized methods of data collection and costing for multimorbidity should be developed to provide more comprehensive and comparable evidence for the costs of multimorbidity.The online version contains supplementary material available at 10.1186/s12916-022-02427-9. Across the world, the prevalence of multimorbidity is increasing, especially among older populations. Studies reporting the prevalence of multimorbidity have given rates ranging from 32% of patients attending general practices in the Netherlands  to 99% oBeyond the prevalence of the individual “combiners” — conditions found in a multimorbity combination — the prevalence of a disease combination is typically linked to both the age structure of the population  and behaTreating multimorbidity is generally more complex than treating single diseases, thereby increasing the demand for healthcare resources. Such treatment often requires additional customization for the patient and often lasts longer , 15, 16.Charlson Comorbidity Index . The 95% CI of the two mean cost estimates do not overlap; hence, the difference is statistically significant.Kidney disease when comorbid with hypertension resulted in a lower mean cost than when comorbid with diabetes  — the difference is statistically significant. However, when comorbid with heart/vascular condition, diabetes incurred higher mean cost than respiratory condition  — the difference is statistically significant.Mental health condition when comorbid with diabetes incurred lower mean cost than when comorbid with a respiratory condition [I$20,750  average cost of treating a patient with Diabetes + Kidney Disease  is observed to be larger than the summed cost of treating both {a patient with Hypertension + Diabetes  and a patient with Hypertension + Kidney Disease }; however, this result is not statistically significant.Our results further demonstrate that the costs of multimorbidity may not be additive of individual diseases. For example, the n = 57), outpatient care (n = 56), emergency care (n = 50), and drugs (n = 40). Other cost ingredients which were less common were those for consultations, diagnostics, surgery, medical equipment, specialist services, and therapy. For studies that reported direct non-medical costs (n = 2), food and transport costs were reported frequently, while costs of social care were rarely reported [n = 7) principally focused on productivity loss, including wage and/or time loss, with a single study additionally including the interest from debts/loans [Studies reporting total direct medical costs most frequently included costs of hospitalization  and a few pre-selected comorbidities —for example , 69, 95 Moreover, the majority of studies obtained their costs from standalone data sources. Such an approach cannot provide a holistic view of healthcare costs including aspects such as non-medical and indirect costs . A “bottFurthermore, with costs markedly higher in the 6-month windows before death and after diagnosis, further studies taking a longitudinal design would provide a clearer understanding of how costs and drivers evolve along the disease trajectory . UnderstThe clear lack of agreement on the scope of health services to include, or which ingredients should be costed, along with a diverse array of methodologies and definitions contribute to heterogeneity between studies. A recommended guideline to collecting and estimating costs of multimorbidity may help in standardizing the definitions, process, and components and reducing the level of variability between studies , 48.Finally, the scarcity of studies on the cost of multimorbidity from LMICs raises concern over equity. LMICs have become the new hub for NCDs and more research is imperative to setting out priority health agendas and informing the re-organization of healthcare delivery to support patients with multiple needs .vs integrated care, may play an important factor, though was not reported in the studies.To contextualize the possible drivers of the difference in costs among different country settings, we used GDP per capita as a determinant. While the data available for these analyses were again skewed by the predominance of American studies (14 out of 28 data points from the US), our regression showed that costs were generally higher in countries with higher GDP per capita . Other sThe high in-group variability of the costs of dyads  is double that of another US study included in this review, which reported the cost of cancer and mental health not specific to any stage of the cascade of care  . Indeed,or diabetes is cheaper than kidney disease or heart/vascular condition. A study found that based on the number of episodes that occurred within a period, and the frequency of transition from primary to specialist or emergency care, conditions such as hypertension may indicate “low severity of healthcare impact” and conditions such as kidney disease, heart/vascular disease, and respiratory disease may indicate “high severity”; thus the effect on costs [Dyads involving hypertension were among the least expensive to manage. For patients with kidney disease, having a comorbidity of hypertension costs less than half of having a comorbidity of diabetes. Similarly, in the case of patients with a heart/vascular condition, having hypertension costs around half of having a respiratory condition. Specifically among hypertensive patients, treatment for musculoskeletal disorder on costs . Many chAlthough all studies included in the meta-analyses originated from the US, our findings provide valuable data on the differences in costs for treating different disease combinations within the American healthcare system. Though healthcare systems vary considerably between countries, the difference in costs between these 11 dyads in the US provides an indication of how they may be in other countries; however, more research in this area is needed.Our review highlights several missing pieces of the multimorbidity burden puzzle, notably that non-medical and indirect costs of multimorbidity are often not costed for. Particularly, social care is considered an important element of care for multimorbid patients given the long-term spectrum and complexity of illness, which may have lasting effects on their care needs . In someThe most frequently reported cost ingredients identified were outpatient, inpatient, emergency care, and drugs. However, across studies, certain cost ingredients may overlap with one another . Due to the ambiguous use of terms, it is difficult to clearly pinpoint the definition of the specific cost ingredients and what they entail.To the best of our knowledge, our review is the first to systematically collect and quantitatively synthesize costs for disease combinations with comparability. We screened a large number of articles having conducted an extensive search. Beyond answering our research questions, we have additionally provided valuable insights into commonalities and inconsistencies in underlying methodologies between studies, and the ways in which these studies reported costs.hypertension separately from the heart/vascular group as it is a major risk factor for the latter. Most studies also report hypertension separately; therefore, following this approach allows for cross-comparison between studies.Our study also has several limitations. Firstly, our categorizations of diseases were made in line with health system organizational considerations, grouping conditions at an organ system level within medical specialties. This enabled us to reduce the number of combinations enabling meaningful interpretations; however, alternate groupings would have been possible, and those following a more treatment-focused perspective may have led to variation in results. On the other hand, we categorized I2 values were estimated for all disease combinations indicating a high degree of between-studies heterogeneity. Despite this, these results may still provide the best available insights though need to be interpreted in light of the highlighted methodological challenges.In the meta-analyses, the degree of heterogeneity and variation of the underlying studies limited the number of studies included, all of which were from the US. Several sub-group analyses  comprised only two data points. This may have limited the plausibility of the assumed approximated normality of mean costs. Furthermore, high The limited availability of studies from other countries led to the country comparison being dominated by studies from the US, with variation in these costs. More data from other countries are required to further examine this relationship.vs private, primary vs secondary care), and study-level factors . These details, however, were frequently unavailable, or not available to a sufficient level  to allow for structural reporting. Therefore, we were unable to control for some of these factors in the analysis. Despite these limitations, our review has revealed important findings.Overall, we acknowledge that costs of multimorbidity may depend on many different factors including and beyond those that have been discussed in this paper. These may include individual-level factors , system-level factors (e.g., the country, health system financing structure, level of care — e.g., public This review provides valuable insights into the costs across multimorbid health profiles, highlighting where priorities should be aligned to combat the escalating and complex economic burden of multimorbidity. These timely findings are essential for informing both the implementation of health interventions and the restructuring of healthcare delivery for multimorbid patients.Multimorbidity imposes a large economic burden on both the health system and society, most notably for patients with cancer and mental health condition in the first year after cancer diagnosis. Whether the cost of a disease combination is more or less than the additive costs of the component diseases needs to be further explored.The scope of research on the costs of multimorbidity is still narrow. Previous studies have typically only considered a narrow range of disease combinations, with data often obtained from single sources. Research to date has originated from a small pool of countries, with a striking lack of costing studies on multimorbidity from LMICs and Europe. Further rigorous and standardized methods of data collection and costing are essential to provide more comprehensive and comparable evidence of the cost of multimorbidity.Additional file 1. Search stringsAdditional file 2. Screening checklistsAdditional file 3. Data cleaning flow chartAdditional file 4. Definition of terminologiesAdditional file 5. Charting of study characteristicsAdditional file 6. The cost conversion tableAdditional file 7: Table S2. Evidence on the costs of multimorbidityAdditional file 8. Checklist and data used for the country comparisonAdditional file 9. Homogeneity checklist and data used for the meta-analysesAdditional file 10. Charting of cost ingredients


Cryptococcus gattii species complex is an increasingly important mycological disease in humans and other mammals. In Australia, cases of C. gattii-related cryptococcosis are more prevalent in the koala (Phascolarctos cinereus) compared to humans and other animals, likely due to the close association that both C. gattii and koalas have with Eucalyptus species. This provides a cogent opportunity to investigate the epidemiology of spontaneous C. gattii infections in a free-living mammalian host, thereby offering insights into similar infections in humans. This study aimed to establish a link between nasal colonisation by C. gattii in free-ranging koalas and the tree hollows of Eucalyptus species, the key environmental source of the pathogen. We (i) detected and genotyped C. gattii from nine out of 169 free-ranging koalas and representative tree hollows within their home range in the Liverpool Plains, New South Wales, and (ii) examined potential environmental predictors of nasal colonisation in koalas and the presence of C. gattii in tree hollows. Phylogenetic analyses based on multi-locus sequence typing (MLST) revealed that the koalas were most likely colonised by the most abundant C. gattii genotypes found in the Eucalyptus species, or closely related genotypes. Importantly, the likelihood of the presence of C. gattii in tree hollows was correlated with increasing hollow size.Cryptococcosis caused by yeasts of the  Cryptococcus gattii and Cryptococcus neoformans species complexes. In this paper, we will adopt the “species complex” nomenclature suggested by Kwon-Chung et al. [C. gattii species complex containing six molecular types—VGI, VGII, VGIII, VGIV, and the recently established VGV and VGVI [C. neoformans species complex containing five molecular types: VNI, VNB, VNII, VNIII, and VNIV [C. gattii species complex will be referred to as ‘C. gattii’ and the C. neoformans species complex will be referred to as ‘C. neoformans’ unless the molecular type is denoted.Cryptococcosis is a mycotic disease of humans and other mammalian species. It has become increasingly prevalent over the last two decades . The disg et al. . Each ofand VGVI —and the and VNIV ,4. SubseC. neoformans predominantly affects immunocompromised patients, and there are an estimated 1 million cases of C. neoformans-related cryptococcosis reported globally per year, closely reflecting the distribution and number of people living with untreated HIV/AIDS [C. gattii-related cases is substantially lower, accounting for less than 20% of global cases [C. gattii from both immunocompetent and immunocompromised clinical cases with no observed bias [C. gattii VGII in the Pacific-Northwest in the 1990s [C. gattii and its potential to become a globally significant pathogen outside of the ‘classical’ geographical predilections of subtropical and tropical regions.Cryptococcosis caused by HIV/AIDS . On the al cases . Howeverved bias  suggestshe 1990s ,9, and iC. gattii is required for the establishment of invasive disease in humans and animals [C. gattii is extensive, from soil, varying tree species and related debris [C. gattii found in clinical or veterinary cases, or from environmental isolation are many, few studies draw a direct connection between identified case isolates and a possible source of infection. In Australia, C. gattii VGI is the most common molecular type isolated from the environment, and is also the most frequent cause of human and veterinary C. gattii-related cryptococcosis [C. gattii VGII is the most prevalent cause of cryptococcosis, particularly in indigenous Australian populations, with Aboriginal individuals accounting for 26.7% of cases in Australia [C. gattii is strongly associated with a number of Eucalyptus species in Australia [C. gattii, likely due to the abundance of organic detritus, and shelter from weather and sunlight [Eucalyptus species, C. gattii-related cryptococcosis is more common in koalas (Phascolarctos cinereus) than other mammalian species [C. gattii as a spontaneous animal disease model and provide greater insight into the risk posed to humans who may frequently be exposed to Eucalyptus material and nearby environments, such as forestry workers, farmers, and indigenous groups.The environmental, pathogenic, and host factors that predispose patients to infection by these opportunistic pathogens remain largely unclear. The inhalation of infective propagules of  animals . The rand debris , to the d debris  and indod debris . Althougcoccosis ,13. The ustralia ,15, althustralia ,16,17, asunlight ,19,20. P species ,22. As aC. gattii [C. gattii VGI and VGII may occur commonly, whereas clinical disease in koalas and companion species, such as dogs and cats, is rare [Nasal colonisation is the suspected precursor to cryptococcosis, and is the most common outcome of a significant interaction with . gattii . It is d. gattii . Colonis. gattii ,23,24. P. gattii ,23. Prev is rare ,26.C. gattii in the environment may play a major role in the colonisation or infection of humans and other mammals [C. gattii VGI or VGIIb [C. gattii and the prevalence of nasal colonisation in captive koalas. They documented the nasal colonisation of a previously negative koala when it was moved to a new captive facility with a greater environmental presence of the yeast [C. gattii, occurred when they were placed in enclosures with colonized koalas [It is suspected that the load of  mammals ,24. In mor VGIIb , suggesthe yeast . The envd koalas .C. gattii nasal colonisation, which was suspected to have originated from colonised koalas translocated from Australia [C. gattii VGII occurred as the possible result of the introduction of a colonized koala from Western Australia into a group of co-owned wildlife parks in Queensland, in which the movement and exchange of koalas occurs regularly [C. gattii may occur through the contamination of the environment by infected/colonized individuals, followed by the propagation of the pathogen in the new environment, with colonization/infection then occurring in naïve individuals which are exposed to high environmental loads. In the case of captive populations, this in turn can be further aggravated by a high stocking density of colonized koalas, based on the notion that koalas can amplify the environmental presence in tree hollows through the effects of faeces, urine and scarification by their claws [C. gattii may still play a significant role in the colonisation of wild koalas.A similar case occurred in a zoo in Japan where a previously naïve koala later tested positive for ustralia . More reegularly . This suir claws . We predC. gattii becomes important in the establishment of areas of high risk for infection. Interregional factors, such as climatic variation, have been repeatedly examined as limiters of environmental C. gattii. For example, winter temperatures below 0 °C and heavy summer rainfall in the outbreak areas of British Columbia, the Pacific Northwest of the USA, and Europe significantly mitigate the presence of C. gattii in the environment [C. gattii in New South Wales (NSW), Australia, and cannot explain the large variation in the presence of C. gattii between similar habitats within the same region [C. gattii must exist between these habitats.The examination of the environmental factors that affect the presence and load of ironment ,31,32. He region . This suC. gattii has a predisposition to colonise certain tree genera, such as Eucalyptus spp. and Ceratonia spp. [C. gattii presence between habitats or even individual trees.nia spp. ,17,31,33C. gattii within a free-ranging population of koalas and tree hollows of Eucalyptus species within their home ranges in the Liverpool Plains region of northern NSW, Australia. The ISHAM consensus multi-locus sequence typing (MLST) scheme [Eucalyptus species, along transects overlapping known home ranges of GPS-tracked koalas. By doing so, we aimed to compare the environmental load and presence of C. gattii between the home ranges of naïve koalas and koalas with nasal colonisation, and to evaluate the role of different predictors of Eucalyptus trees and hollows that may affect the likelihood of hollow colonization by C. gattii.We investigated the prevalence of ) scheme  was emplA total of 169 free-ranging koalas were captured at two sites approximately 50 km apart, the Dimberoy (31°08′24″ S 150°01′34″ E) and Watermark (31°15′57″ S 150°22′16″ E) sites, located in the Liverpool Plains region of NSW, during half-yearly field trips between 2015 and 2017. The selected koalas were ear-tagged and fitted with a Sirtrack GPS collar  weighing less than 3% of their body weight at capture. In order to determine the nasal colonization status of collared koalas, a sterile saline-moistened cotton swab was inserted approximately 2–2.5 cm into both nasal vestibules (left and right) and thoroughly rotated against the inner mucosal surface. The swabs were stored in Amies Transport Medium in the dark at room temperature during the field collection, and were then transferred to refrigerated storage for a maximum of five days prior to plating.The GPS co-ordinates of the koalas were recorded every 4 h for up to 5 months. Further detailed information on the GPS tracking methods can be found in Crowther et al., 2014  and Rus All of the procedures were undertaken with approval from The University of Sydney Animal Ethics Committee (Project number 2016/955) and under veterinarian supervision.The environmental sampling was conducted during three field trips over four months (February–May 2017) on transects at the two koala capture sites, the Watermark site and the Dimberoy site.From the data obtained from the animal sampling and GPS tracking, 14 transects (Transects 1–14) of 250–600 m in length (dependent on tree density and home range size) and at least 150 m apart were designed using the geographical information software ArcGIS Ver. 10.5.1 , intersecting trees within known home ranges of koalas with and without nasal colonisation. The start and endpoints of the transects were then uploaded on portable GPS devices  and used as a guide to sample trees within 5 m of these transects. An additional four transects were conducted opportunistically without koala GPS tracking data (Transects 15–18) but in areas suspected to be within a koala habitat, or where koalas were previously captured and assigned a GPS collar but had not yet been recaptured. GPS data were later obtained for koalas on two of these transects (Transect 15 and Transect 18). In total, 16/18 of the transects were conducted within known koala home ranges. Of these transects, 5/16 represented koalas with colonisation, and the remaining represented koalas without colonization. The opportunistic environmental sampling of trees where koalas were caught was also undertaken .Eucalyptus species in the form of swabs and bagged debris, as described by Krockenberger et al. [Environmental samples were obtained from tree hollows of r et al. . SterileIndividual measurements of tree height and tree diameter were recorded. Measurements of the tree hollow itself were taken, including the hollow height from the ground and the directional aspect of the hollow opening, as this would affect the exposure of the hollow to sunlight. Observations of the hollow directional aspect were used to generate a sun index, i.e., a relative representation of sun exposure categorised into five levels: north-facing hollows scored the highest value of 5, northeast- and northwest-facing hollows scored 4, east and west facing hollows scored 3, southeast- and southwest facing hollows -scored 2, and south-facing hollows scored 1. The size of the hollow was hypothesised to affect the overall exposure to weather and the availability of organic detritus. The hollow size was divided into three categories, which were dependent on the diameter of the hollow opening: small (0–15 cm), medium (15 cm–30 cm), and large (>30 cm) .Guizotia abyssinica) Extract Agar (BSA), and was incubated for 7 days at 27 °C. BSA was used to identify C. gattii/C. neoformans colonies from those of other environmental micro-organisms by the brown-colour-effect [Each environmental and animal sample was cultured on Staib’s Bird Seed  counts were conducted on plates displaying colonies with the brown-colour-effect, categorised into three categories of colonisation load , and single colonies were subsequently subcultured onto Sabouraud’s dextrose agar (SDA). At least one and up to twelve individual colonies were isolated on separate SDA agar plates from each positive sample, dependent on the level of colonisation on each plate, and were incubated at 37 °C for 48–72 h prior to the DNA extraction.2O) and 5 µL 2-mercaptoethanol for 1 h. In total, 500 µL phenol-chloroform-isoamyl alcohol  was then added to the sample and vigorously vortexed to produce a homogenous suspension. The suspension was then centrifuged at 14,000 rpm for 15 min. In total, 500 µL of the upper aqueous layer of the resulting solution was mixed with an equal volume of isopropanol and precipitated for a minimum of 1 h at −30 °C. The sample was then centrifuged at 14,000 rpm at 4 °C for 15 min before the supernatant was discarded. The resulting pellet was then washed with 70% ethanol and centrifuged again at 14,000 rpm for 15 min. The supernatant was then removed, and the pellet was dried before being resuspended in 50 µL sterile distilled water.The DNA extraction was conducted using a modified version of the Ferrer et al. DNA extraction protocol for fungal cultures . For eacURA5 locus was performed for each isolate to be used in the restriction fragment length polymorphism (RFLP) analysis [2 (50 mmol) , 16.5 µL dH2O, 0.5 µL BioTaq . The target was amplified following the conditions of pre-heating at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 63 °C for 1 min, and extension at 72 °C for 2 min.The PCR amplification of the analysis . Each PCC. gattii/C. neoformans species complexes (WM 148 (VNI), WM 626 (VNII), WM 628 (VNIII), WM 629 (VNIV), WM 179 (VGI), WM 178 (VGII), WM 161 (VGIII), and WM 779 (VGIV)) underwent enzyme digestion by mixing 25.8 µL URA5 PCR product with 4.2 µL RFLP master mix , followed by incubation at 37 °C for 3 h before being visualised on a 2.5% agarose gel run at 80 V for 3 h.The PCR products from each isolate and the standard reference strains for the eight major molecular types of the C. neoformans/C. gattii species complexes for all seven loci required in the scheme (available at https://mlst.mycologylab.org/ (accessed on 31 August 2017)) [https://mlst.mycologylab.org/ (accessed on 31 August 2017)). In the case of new AT combinations, the curators of the MLST database were contacted for the designation of a new ST.The PCRs conducted in this study followed the protocols and conditions provided by the ISHAM consensus MLST scheme for the t 2017)) . The PCRPhylogenetic analysis was conducted using the Molecular Evolutionary Genetics Analysis (MEGA) Ver. 7 software  on the concatenated sequences of all seven MLST loci for each isolate. The concatenated sequences were aligned using the CLUSTALW algorithm. Subsequently, the model selection was completed using the Maximum Likelihood method, and the model with the lowest Bayesian information criterion (BIC) value was selected . A dendrAll of the statistical analyses were conducted using the R Statistical Environment (R Core Team). For the analysis of the predictors of hollow colonisation, only environmental data from transects with known koala home ranges , opportunistic transects (Transects 16 and 17) and opportunistic sampling were used. For the analysis of the predictors of koala colonisation, only environmental data collected from transects with known koala home ranges were used .C. gattii colonisation of tree hollows. Hollows which were only positive for C. neoformans were considered negative for C. gattii in the analysis. We included the random effects of sites and individual trees (nested within sites). The transect was not included in the model as a random effect because many transects contained only a single sampled tree, while most contained three or fewer trees. Excluding the transect also allowed us to include opportunistic samples that were not on any transect in the model. Stepwise regression using backwards elimination was used to sequentially remove fixed effects from the full model. Small sample corrected Akaike Information Criterion (AICc) values were generated for each model produced, and the models were ranked from the lowest to the highest AICc value. The difference in AICc value (∆AICc) between each model and the model with the lowest/best AICc was also calculated. Models with a ∆AICc lower than 2 were considered equivalent to the model with the lowest AICc [The sun index, along with the obtained measurements of the tree height, tree diameter, hollow height from the ground level, hollow size, and tree species were used as fixed effects to generate a binomial generalized linear mixed-effects model (GLMM) with logit link function using the ‘lme4′ package to determine predictors of est AICc . In the C. gattii on each transect (Variable A) and the number of hollows which were positive for C. gattii on each transect (Variable B) were standardised to a length of 100 m (or per 100 m) prior to analysis. A ‘load index’ (Variable C) was also developed to represent the ‘environmental load’ of C. gattii on the transect, which was derived from the total score of colony counts for the hollows sampled on the transect  and then subsequently standardised to a transect length of 100 m.In order to account for varying transect lengths, the measurements of the number of trees positive for C. gattii. In the case that a transect overlapped the home range of more than one koala, the information from the transect was used for each koala  with a logit link function was produced for each of the standardised variables above in relation to whether a koala on the transect was colonised by ch koala . VariablEucalyptus trees were obtained from opportunistic sampling, transects within known koala home ranges  and opportunistic transects (Transects 16 and 17)  of all of the trees sampling positive. Forty (31%) environmental samples from tree hollows were positive for C. gattii and/or C. neoformans. Sixteen (40%) of these positive tree hollow samples were isolated from transects of koalas without colonisation, while 13/40 (33%) were from transects of colonised koalas. The remaining 11/40 (27%) positive tree hollow samples were from opportunistic sampling  . No sampsampling . A totalC. gattii  koalas were culture positive for . gattii . Of thes. gattii .C. gattii VGI (n = 237) and C. neoformans VNI (n = 23). Of the positive samples, 37/40 were only positive for C. gattii VGI, one out of 40 was only positive for C. neoformans VNI, and two out of 40 were positive for both molecular types: VGI and VNI. All nine koala isolates were positive with C. gattii VGI for all cultures , followed by ST51 (n = 6). ST257 (n = 2) was detected only at the Watermark site. Two new STs were also identified: ST462 (n = 1) was only found at the Dimberoy sampling site, whereas ST463 (n = 2) was isolated from two individual trees from the Watermark sampling site.Twenty-eight of the environmental isolates were sequence typed and five STs were identified in the environment . The mosn = 2) and ST51 (n = 3)—which were found in the environment—were also identified in samples from the nasal colonisation of koalas , and only seven SNPs were identified among all of the alleles in the most variable locus, SOD1  (C. gattii VGI in a hollow (AUC = 0.747). Probability plots for the likelihood of hollow colonisation as a function of hollow size showed that there was an approximately 20% increase in the likelihood of a hollow being colonised between a small (0–15 cm) and a large hollow (>30 cm).The step-by-step removal of the least informative fixed effects in subsequent models from the original model containing all fixed effects showed that the most predictive fixed effect was hollow size ( 0.0528) . A modelC. gattii in a tree hollow and no random effects, hollow size was significant (p = 0.04851) (p = 0.2888), the random effect of individual trees was significant (p = 0.0394). The significance of this random effect suggests that other variables of trees which went uninvestigated in this study exist, and that these may better predict the colonisation of hollows by C. gattii VGI, in addition to the variable of the hollow size.GLM was used to test the effect of hollow size on the likelihood of hollow colonisation alone. In this particular model with only hollow size as a predictor for the presence of 0.04851) , but wheC. gattii were found to be significant in GLMs containing only the individual predictors, i.e., the number of trees which were positive for C. gattii VGI on each transect (Variable A) (p = 0.3976), the number of hollows which were positive for C. gattii VGI on each transect (Variable B) (p = 0.2294), and the Load Sum of each transect (Variable C) (p = 0.1616). Similarly, none of the models containing two variables were found to be informative, i.e., Variable A and Variable B (p ≥ 0.3765), and Variable A and Variable C (p ≥ 0.2546).None of the three predictors of likelihood of koala nasal colonisation with C. gattii within known home ranges of wild koalas with identified colonisation/subclinical disease status. It allowed an evaluation of the molecular links between environmental and koala isolates of C. gattii, and the assessment of the role of possible environmental predictors for tree hollow and koala colonisation. From the 260 environmental isolates from 69 Eucalyptus trees, we established C. gattii VGI  to be the dominant molecular type in the Liverpool Plains region. C. neoformans VNI  was also isolated from Eucalyptus tree hollows, but infrequently  [C. gattii VGI within the region.Relative to other countries ,48, the  (64.3%)  in the sSOD1, only containing a maximum of seven SNP differences between two alleles , followed by ST51 (21%), although worldwide ST51 is one of the most commonly isolated STs ,50. Whil alleles . The varisolates .GPD1 locus. In Cluster 2, ST462 may have originated from ST51, as there is only one SNP difference in the SOD1 locus, with all of the other alleles being the same.Phylogenetic analysis confirmed the close association between the STs isolated, showing strong bootstrap support for the formation of two clusters—one containing ST154 and six other STs, and another containing ST51 and ST462 —suggestiC. gattii VGI, three were identified as ST51 and two were ST154, with both STs being the most commonly isolated from the environment for both the Watermark and Dimberoy sites. The remainder of the koala isolates were identified as novel STs, but all were closely related to either ST154 or ST51. Of these koalas, three had genotyped environmental isolates from within their home range.Of the nine koalas which were positive for CAP59 locus, and the SOD1 locus  in our phylogenetic analysis , they arEucalyptus trees as the primary habitat for koalas, and the close association of both to C. gattii [C. gattii VGI in Eucalyptus tree hollows were significant. It is important to note that we only had data on a small number of colonised koalas (5/19) compared to naïve koalas, and of these, no samples were obtained from the transect of one due to a lack of hollows (Transect 4), reducing the power of this statistical analysis. We are therefore unable to conclude whether there is an association between nasal colonisation in koalas and the environmental load of C. gattii VGI in Eucalyptus tree hollows across their home range.The role of . gattii ,21,22,61C. gattii VGI is present within their home range, and in some cases these overlap with the home ranges of colonised animals  has been shown to be linked to the increased susceptibility of chlamydial infection [Cryptococcus species colonisation/infection is only postulated, but cannot be excluded at this point in time [C. gattii colonisation/disease in seemingly healthy or KoRV-free koalas. Although we cannot be certain due to the low power of our statistical analysis, the observed abundance of C. gattii VGI in the environment studied, in combined with the results of our previous study, in which only 12/181 (6%) of the same koala population were colonised [C. gattii is therefore warranted.Conversely, the lack of a statistical association between the environmental load of nfection , with Konfection . An asso in time ,64. A re in time . Alterna in time , could bolonised , seem toC. gattii VGI in the home ranges of wild koalas has an attenuated effect compared to captive koala populations. However, the presence of C. gattii in the environment is still an obvious prerequisite for the colonisation of koalas. Therefore, understanding the variables that affect the presence of C. gattii VGI is still essential in order to provide insights into the host-pathogen-environment interaction for this organism. Our statistical analysis of predictors of hollow colonisation by C. gattii determined the best fitting model to contain only hollow size as a fixed effect. All of the other features of the hollow and tree from which isolates were obtained, such as the tree species, height and diameter, hollow height from ground level, and hollow directional aspect (in the form of a sunlight exposure index) were found to be uninformative. Although the p-value of hollow size in the best GLMM model is only 0.0528, subsequent GLM modelling and AUC analysis reinforce the significance of the hollow size on the likelihood of hollow colonisation by C. gattii VGI, with an approximately 20% increase in the likelihood of hollow colonisation between small (0–15 cm diameter) and large (>30 cm diameter) hollows. The random effect of trees was also found to be informative in the best GLMM model, and significant in a GLM model predicting the presence of C. gattii VGI in a tree hollow (p = 0.0394). This suggests that other variables of trees uninvestigated in this study may also be informative in the prediction of the presence of C. gattii VGI in tree hollows, in conjunction with hollow size.Our evidence suggests that the environmental load of C. gattii VGI from six Eucalyptus species, from three of which C. gattii had previously not been documented to the best of our knowledge: E. conica, E. melliodora, and E. microcarpa. Ellis and Pfeiffer have previously postulated a link between E. camaldulensis [E. tereticornis with C. gattii [C. gattii has been isolated from over 50 tree species, with most being outside of the genus Eucalyptus [C. gattii from Eucalyptus species other than in Australia is rare [Eucalyptus species were found to be positive for C. gattii in India [Eucalyptus trees sampled in an extensive collaborative study were found to be positive for C. gattii [In this study, we documented the isolation of dulensis  and E. t. gattii , but sincalyptus . In fact is rare . Only fiin India , whereas. gattii . TherefoC. gattii species complex have been isolated [C. gattii by wildlife—particularly by psittacine birds, which are dependent on tree hollows and are susceptible to cryptococcosis [Considering the wide spectrum of wild animals from which members of the isolated , and itsisolated , the discoccosis —is anothC. gattii/C. neoformans species complexes was not investigated as a variable for tree hollow colonisation in this study, as it does not seem to have an effect in Australia [C. gattii in the environment [C. gattii survival throughout the year.Seasonal variation in the prevalence of ustralia ,42. In cironment ,69,70,71Eucalyptus tree hollows was examined previously, and no correlation between the presence of other fungi and either C. gattii or C. neoformans was found [Cryptococcus species—such as Staphylococcus aureus [Pseudomonas aeruginosa [Acanthamoeba polyphaga [Cryptococcus species.The effect of other fungi in the mycobiome of as found . The rols aureus  and Pseuruginosa —and someolyphaga  is yet tC. gattii species in the environment and tree hollows. Further exploration of these factors is needed in order to evaluate their impact and potential to predict the environmental presence of the organism.All-in-all, a wide range of environmental variables may influence the presence of the C. gattii within a free-ranging koala population and their environment in the Liverpool Plains identified a genetic link between nose-colonising isolates in koalas and those isolated from tree hollows of Eucalyptus species in their home ranges. This provides further evidence for the role of tree hollows as reservoirs of C. gattii that cause nasal colonisation and, potentially, clinical disease in wild koala populations. Not only does this impact the future management of captive koalas, it also implies the possibility of tree hollows as a source for human infection, particularly for those that work in close proximity to or with materials from Eucalyptus species. Furthermore, the role of koalas as sentinels for this pathogen is emphasised. Interestingly, we could not determine an association between the environmental load of C. gattii VGI in tree hollows with the likelihood of nasal colonisation. However, the isolation of C. gattii VGI from the home ranges of naïve koalas, and in some cases, where the home ranges of naïve and colonised koalas overlap indicate that host behaviour or immunity may play a greater role in the nasal colonisation of wild koalas compared to environmental load. This is not to say that variables that affect the presence of the C. gattii VGI in the environment become irrelevant, as the pathogen must still exist in the environment. Our analysis of the predictors of hollow colonisation by C. gattii VGI determined hollow size to be a significant variable, with an opening larger than 30 cm in diameter having up to a 20% greater likelihood of being colonised by C. gattii VGI. This finding may have implications for the management of captive koalas, for example, avoiding the use of natural environmental enrichment with large hollows.Our study of