Sample Report M Reference Range M Gender M F: Fill & Download for Free

Here’s your detailed answer:BMC Vet Res. 2016; 12: 162.Published online 2016 Aug 3. doi: 10.1186/s12917-016-0786-5PMCID: PMC4973095A mixed grape and blueberry extract is safe for dogs to consumeAnne-Sophie Martineau,1 Véronique Leray,1 Anne Lepoudere,2 Géraldine Blanchard,3 Julien Bensalem,4 David Gaudout,4 Khadija Ouguerram,5 Patrick Nguyen,On behalf of Neurophenols ConsortiumAuthor information ► Article notes ► Copyright and License information ►This article has been cited by other articles in PMC.Go to:AbstractBackgroundGrape and blueberry extracts are known to protect against age-related cognitive decline. However, beneficial effects achieved by mixing grape and blueberry extracts have yet to be evaluated in dogs, or their bioavailability assessed. Of concern to us were cases of acute renal failure in dogs, after their ingestion of grapes or raisins. The European Pet Food Industry Federation (2013) considers only the grape or raisin itself to be potentially dangerous; grape-seed extracts per-se, are not considered to be a threat. Our aim was therefore to evaluate the renal and hepatic safety, and measure plasma derivatives of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) in dogs. Polyphenol expression was analyzed by UHPLC-MS/MS over 8 hours, for dogs given PEGB at 4 mg/kg. Safety was evaluated using four groups of 6 dogs. These groups received capsules containing no PEGB (control), or PEGB at 4, 20, or 40 mg/kg BW/d, for 24 weeks. Blood and urine samples were taken the week prior to study commencement, then at the end of the 24-wk study period. Routine markers of renal and liver damage, including creatinine (Creat), blood urea nitrogen, albumin, minerals, alkaline phosphatase (ALP), and alanine transaminase (ALT) were measured. Biomarkers for early renal damage were also evaluated in plasma (cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL)), and urine (CysC, clusterin (Clu), and NGAL). Ratios of urinary biomarkers to Creat were calculated, and compared with acceptable maximal values obtained for healthy dogs, as reported in the literature.ResultsWhile several PEGB-specific polyphenols and metabolites were detected in dog plasma, at the end of the PEGB consumption period, our biomarker analyses presented no evidence of either renal or liver damage (Creat, BUN, ionogram, albumin and ALT, ALP). Similarly, no indication of early renal damage could be detected. Plasma CysC, urinary CysC/Creat, Clu/Creat, and NGAL/Creat ratios were all beneath reported benchmarked maximums, with no evidence of PEGB toxicity.ConclusionsLong-term consumption of a pet specific blend of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium), was not associated with renal or hepatic injury, and can therefore be considered safe.Keywords: Dog, Neurophenols, Grape, Blueberry, Kidney, Cystatin C, Clusterin, NGAL, FlavonoidsGo to:BackgroundThis work comprises part of a project dedicated to the study of age-related cognitive decline in humans and dogs (the Neurophenols Consortium). We aim to complete a novel study into the efficacy of mixed extracts of grape and blueberry in counteracting age-related deterioration of function. In aged dogs, polyphenol ingestion (including grape pomace), and vitamin use, are both thought to ameliorate the effects of aging on learning ability [1]; similarly beneficial effects have been reported for humans [2]. A popular hypothesis is that protection against oxidative stress explains these effects. In aged mice, the consumption of a mixed grape and blueberry extract, has also been shown to improve spatial navigation; one of the skills that declines with age. In this scenario, increased expression for hippocampal nerve growth factor mRNA [3] may play a causal role [4].Studies reporting grape extract consumption in dogs (using grape seed and skin extracts, or grape seed proanthocyanidins), or grape pomace in aged dogs have, to date, not reported any side effects [1, 5, 6]. However, acute renal failure has been reported in dogs after their consumption of grapes, with kidney histopathology revealing tubular degeneration leading to necrosis, particularly in the proximal tubule [7–9]. In a retrospective study involving a cohort of 43 dogs, all of whom had eaten grapes, raisins, or both, animals presented with clinical signs consistent with kidney deterioration during a window extending from 24 h, until 5 days, after consumption. Vomiting, diarrhea, lethargy, and either olig- or, anuria, were the common clinical signs. A diagnosis of renal damage was supported by biochemical abnormalities showing higher plasma creatinine (Creat), blood urea nitrogen (BUN), an altered ionogram, glycosuria, and proteinuria. Again, histopathology revealed severe diffuse renal tubular degeneration, especially in proximal cells, with glomerular deterioration. Half of the dogs died [9]. In these cases, the precise amount of fruit eaten varied greatly (from 3 g/kg BW of raisin, to 150 g/kg BW of grape), as did the type of fruit (grape, raisin, seedless grape), and the affected breed [7–9]. Hepatic toxicity has also been associated with the consumption of plants such as greater celandine, green tea, valerian, or ayurvedic products. In these cases, higher concentrations of alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and bilirubin, were all demonstrated (reviewed in [10, 11]). Abnormal values of ALT and ALP provoked by grape consumption also point to the liver being a target of grape toxicity [9], although the factors responsible for hepatic damage, as well as the acute renal failure, have yet to be identified.The Neurophenols Consortium is a Europe-North America research collaboration dedicated to the research, and development of natural ingredients and products to prevent age-related cognitive decline in humans and pets. The Consortium brings together scientists in the fields of phytochemistry, neuroscience, psychology and nutrition with companies specialized in the development of active ingredients and food supplements. The specific aims of the program are to characterize and formulate fruit extracts from blueberry and grape, to evaluate their safety and efficacy in pre-clinical and clinical trials.The aim of this study was to assess the safety of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium). We studied the safety of this extract following chronic use in dogs, by monitoring renal and hepatic health, using early biomarkers of renal damage as well as a biochemical approach.Go to:MethodsAnimalsTwenty-four experimental Beagle dogs (4 groups of 5 males, and a single female, body condition score (BCS) 5/9, mean age 31 ± 3 months, mean body weight (BW) 11.4 ± 0.2 kg), originally from CEDS (Centre d’élevage du Domaine des Souches, Mézilles, France), were used. They were fed with a dry maintenance diet (Medium Adult Royal Canin), according to the National Research Council (NRC 2006) [12] recommendation (130 kcal metabolizable energy per kg metabolic body weight).Study designFour groups of 6 dogs (each comprising 5 males and a female) were given a polyphenol-rich extract from grape and blueberry (PEGB) for 24 weeks. The constituents of this PEGB extract were devised by the Neurophenols Consortium; these were grape (Vitis vinifera L.), and blueberry (Vaccinium angustifolium) extracts, containing specific polyphenols with low molecular weight monomers, including catechin (6 % dry matter), oligomers, flavonols (for a total of 0.15 % dry matter), anthocyanins, phenolic acids, and resveratrol formulated in a unique ratio of molecules. The intended dosage was 4 mg/kg BW/d. One group also received a control dose of 0 mg/kg BW/d (control), with two other groups receiving higher doses of the extract; 20 mg/kg BW/d, and 40 mg/kg BW/d. Each dose was given in the daily meal as a gelatin capsule (Cooper, Melun Cedex, France) containing the formulation and maltodextrin. All extracts were prepared in accordance with good laboratory practices.Plasma and urine samplesBlood and urine samples were collected in the week prior to the study commencing, and then at the end of the 24-wk period. Blood samples were obtained by jugular venipuncture into heparin tubes in 24-hour unfed animals. Each blood draw was immediately centrifuged (2124 g for 10 min at 4 °C), and the plasma fraction aliquoted and frozen at -80 °C. Twenty-four-hour urine samples were collected by voiding, following the consumption of the daily meal and the capsules. The expression of specific polyphenols, derived from the PEGB extract, were measured, on the 8th days of exposure, in plasma samples from dogs that were given PEGB at 4 mg/kg/d. For this purpose, plasma samples were taken for 8 h, with polyphenol analyses performed by UHPLC-MS/MS.Chemical analysesConcentrations of Creat, BUN, minerals (sodium (Na+), potassium (K+), calcium (Ca2+), and phosphate (PO43-), albumin, ALT, and ALP, were determined using a VetScan reagent rotor (Comprehensive Diagnostic Profile, VetScan VS2, Abaxis, Ca, USA). Biomarkers of renal damage, including CysC (cystatin C), Clu (clusterin), and NGAL (neutrophil gelatinase-associated lipocalin), were measured by species-specific ELISA (canine cystatin C, Biovendor, Czech Republic; canine clusterin, Biovendor, Czech Republic; dog NGAL, Bioporto, Denmark). Urinary Creat was assayed using an enzymatic colorimetric kit (Creatinine, Randox Laboratories, UK).Data analysesResults are reported as means ± standard error of the mean (SEM). For each early biomarker of renal damage, we compared their maximal values after PEGB consumption with previous maximal values reported for healthy dogs. As replicate datasets were collected, linear mixed-effects model analyses could be undertaken to investigate any interaction between PEGB dose, and time. Moreover, an inter-group analysis was performed using a linear model to compare data for each of the experimental groups (4, 20, and 40 mg/kg/d), with the control group, at the beginning and end of the study. Finally, an intra-group analysis was completed using a linear mixed effects model to compare data from the initiation and end of the study. These analyses were completed using the R software (R Core Team (2013)). The alpha level for determination of significance was 0.05.Go to:ResultsSpecific polyphenols in plasma following PEGB consumptionPolyphenols and their metabolites were detected in plasma samples, and their maximum concentrations (Cmax) determined. These metabolites comprised: hydroxy and dihydroxyphenyl-γ-valerolactone, both derived from flavan-3-ols; the resveratrol derivatives, reseveratrol glucuronide, dihydroresveratrol sulfate, and glucuronide; the flavonol and its metabolite, quercetin and isorhamnetine sulfate; and the anthocyanin metabolite, malvidin. A Cmax for the flavan-3-ol metabolites of 2028 nM was attained after 8 h. Flavonol metabolites reached a Cmax of 5nM, also after 8 h, with malvidin also peaking (7nM) at this timepoint. Peak concentrations for resveratrol metabolites were reached much earlier, after 30 min (Cmax 161 nM).Plasma and urine biomarkersMarkers of liver damagePlasma hepatic biomarker concentrations are shown in Table 1. All ALT and ALP concentrations were within the reference range.Table 1Plasma biomarkers of kidney damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; minima and ...Markers of renal damagePlasma creatinine, urea, sodium, potassium, calcium, phosphate, and albumin concentrations are shown in Table 2. All values were found to be in the reference range, i.e. the 95 % prediction interval (for a normal population).Table 2Plasma biomarkers of liver damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; minima and ...The concentration of early renal biomarkers, and their ratios, are presented in Table 3. For each biomarker, interaction analyses failed to identify any difference between the experimental and control groups, either at the beginning, or at the end of the study. No inter-group or intra-group variations could be noted between experimental groups, compared to controls, or between the initiation and the end of the study.Table 3Concentrations, and ratios, of early biomarkers of renal damage in dogs, at the initiation (Week -1) and the end (Week 24) of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d. (Data are means ± SEM, n = 6/group; ...In the experimental groups (PEGB given at 4, 20 or 40 mg/kg/d), the mean plasma CysC concentrations were found to be similar to control group. Mean concentrations ranged from 1.2 to 1.5 μg/mL (Fig. 1a). Intra-group analyses also showed no differences across the experimental groups. Collectively, their mean urinary CysC/Creat ratio varied from 4 to 27 μg/g. These ratios were not significantly different to those determined for the control group, for whom no intra-group difference was noted (Fig. 1b). Mean urinary Clu/Creat ratios varied between 44 and 94 ng/g in groups given PEGB at 4 to 40 mg/kg/d, again with no significant changes compared to control group nor intra-group differences between the initiation and the end of the study (Fig. 2). The mean urinary NGAL/Creat ratios were similar to control group in the experimental groups, varying between 4 and 8 ng/g. Intra-group analyses also failed to determine any significant differences between the initiation and the end of the study (Fig. 3).Fig. 1a Plasma Cystatin C concentration (μg/mL) in dogs at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value ...Fig. 2Urinary Clusterin/Creatinine ratio (ng/g) in dogs, at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value in normal ...Fig. 3Urinary NGAL/Creatinine ratio in dogs (ng/g) before at the initiation () and the end () of a 24-wk period of consumption of PEGB at 4, 20 or 40 mg/kg/d (n = 6 dogs per group). The line indicates the reported maximal value in normal ...Go to:DiscussionOur aim was to assess the safety of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) for dogs, by monitoring early biomarkers of renal damage over a 24-week period. This work considerably extends the previous study periods reported, where platelet effects, and gene expression profiles, were interrogated after 7 days, or 3 months of supplement use [5, 6].After PEGB consumption, biomarker values exceeded the reported maximal limits in no dog, with no differences observed at the end of the 24-week period, compared to beginning, for plasma CysC, and urinary CysC/Creat, Clu/Creat, or NGAL/Creat ratios. When considering these data, we conclude that the dogs neither presented with renal, nor hepatic injury, at the end of the study.While bioavailability of the Neurophenols Consortium PEGB had never been evaluated in dogs, our evaluation of the safety of this supplement necessitated measurement of PEGB derivatives in plasma. The main polyphenols in the extract were flavan-3-ols, resveratrol, anthocyanins (malvidin, petunidin, peonidin, petunidin, cyanidin), and flavonol (quercetin). Some polyphenols and polyphenol metabolites were found in plasma. Malvidin, which is present in blueberry but not in grape, has been the only anthocyan detected, but it is known that anthocyanins are less absorbed than other flavonoids. The finding of resveratrol derivatives (which are grape specific) is in accordance with a study that also showed appearance of resveratrol conjugates (sulfate & glucuronide) in the plasma of dogs after resveratrol administration [13]. The valerolactones detected resulted from the metabolization of flavan-3-ols by gut microflora. Quercetin and isorhamnetin sulfate, which are present in both fruits, were also found. Other compounds may have been absorbed, but either they have not been identified, or their concentration was under the detection threshold, or they were rapidly metabolized and excreted. Very few data on polyphenols pharmacokinetics in dogs are available. Regarding resveratrol, Cmax could not be compared since in previous report [13] it was given to dogs at much higher doses than the intended dose in the present study (200–1200 mg/kg/d, compared to 4 mg/kg/d). When anthocyanins were given to pigs at 1 to 4 % of the diet (w/w), several metabolites were measured in liver, eye and brain while there were not detected in plasma [14], and again the doses were far higher than in the present study. Catechin and epicatechin glucuronides from a grape extract given to mice were measured in plasma [15], which was not the case in our study, but the dose used was still much higher (grape-derived polyphenols: 80 mg/kg/d). When green tea catechins (13 mg/kg/d, [16], 170 mg/kg/d [17]) and epigallocatechin gallate (EGCG; 250 mg/kg/d [18]) were given to dogs, respective metabolites were found in plasma, which was not the case after PEGB consumption where only valerolactones were detected. The difference could be explained either by the catechin sources or higher doses or both. Another possible explanation is that dogs were given the PEGB at the same time of their daily meal, and the plasma measurements were done after a relatively short period of exposure. Indeed in dogs given EGCG at 300 mg/kg/d, plasma area under the curve (AUC) for EGCG was higher in unfed than fed dogs [19]. When EGCG was given at 500 mg/kg/d, authors reported, although the difference did not reach the significance level, that the AUC for EGCG was 1.6 time higher after 28 days of dosing than after 14 days [19]. The data of the present study demonstrated that the polyphenols of the PEGB extract were, at least in part, bioavailable, and this is the first report on the appearance of valerolactones as well as quercetin, isorhamnetin sulfate and malvidin in the plasma of dogs after consumption of a mixture of polyphenols.The origin of the grape toxicity described in the literature for dogs is still obscure, but numerous hypotheses have emerged. Among them, it was reported that exogenous compounds on grapes, such as mycotoxin, pesticides, or herbicide residues, could be responsible for the kidney toxicity, with histopathology indicating that the proximal cells are the primary target [8]. These findings provoked further hypotheses, such as the toxic accumulation of a foreign chemical (a xenobiotic), with a particular affinity for tubular specific transporters. Additionally, the expression of a perinuclear golden brown pigment [8], could imply its cytotoxic accumulation, with failed cellular clearance. Hypercalcemia and renal mineralization induced by the high sugar content of grapes are also current hypotheses.The resveratrol concentration in grapes could also be responsible for renal damage. A previous study described that the no-observed-adverse-effect level of resveratrol consumption was 600 mg/kg BW/d in dogs. Consumption of twice this dose (1200 mg/kg BW/d) induced a loss of appetite, and weight [20]. Given that grapes contain 1.5 to 7.8 μg of total resveratrol per gram of fresh weight [21], it is highly unlikely that resveratrol is responsible for the acute kidney injury observed in clinical cases in dogs.Plasma creatinine and urea are the most frequently measured parameters used to evaluate renal damage. High creatinine concentrations are seen when at least 75 % of renal function has already been lost [22]. In previous studies describing acute renal failure after grape consumption, symptoms appeared rapidly [9]. Therefore, we reasoned that to monitor kidney health, earlier biomarkers of renal damage would be required. In 2010, the Nephrotoxicity Working Group established a consortium between the European Medicines Agency, and the Food and Drug Administration. They listed seven biomarkers needed to detect the early development of renal injury [23]. Among these, we chose to assess CysC and Clu, because of their ease of use in dogs. In addition, NGAL was measured, as a promising early biomarker of drug-induced kidney injury. Collectively, these early biomarkers of renal damage are ideal for monitoring renal health, before irreversible damage, as they survey different renal functions, and compartments of the kidney.Ordinarily, cystatin C, which is a low molecular weight protein produced at a constant rate by all cells, is completely reabsorbed and catabolized in proximal tubular epithelial cells [24]. Following renal injury, CysC concentration increases in the plasma, as the glomerular filtration rate declines [25]; an increased concentration in urine reflects tubular impairment [26]. Plasma CysC has previously been measured in healthy dogs (urea and creatinine concentrations within reference intervals), with the highest reported values of 2 μg/ml [27]. For all dogs that had received PEGB, at any dose, plasma CysC concentrations were beneath this upper limit. To the best of our knowledge, the referenced study [27] is the only one in which plasma CysC concentrations have been measured in healthy dogs by canine ELISA. We therefore conducted the same tests, in our study. In other studies, CysC was measured in serum and/or with a different ELISA kit or technique (i.e. Particle-Enhanced Turbidimetric Immunoassay), which may explain the slightly different reference ranges reported [28–30]. In our study, the maximum urinary CysC/Creat ratio that we measured in dogs following PEGB consumption (regardless of dose) was 79 μg/g, whereas reported urinary CysC/Creat ratios have been as higher as 0.11 ± 0.02 mg/g [31]. Therefore, we conclude that our CysC results revealed no glomerular or tubular impairments.Clusterin is a high molecular weight glycoprotein expressed in epithelial cells (reviewed in [32]); in cases of acute renal failure, clusterin is found at high concentrations in the urine, indicating glomerular damage [33]. The highest urinary Clu/Creat ratio previously reported in healthy dogs was 4.87 μg/g [33]. In this study, the urinary Clu/Creat ratio measured in dogs after PEGB consumption (4 to 40 mg/kg/d) was far lower, ranging from 10 to 437 ng/g. Therefore, clusterin analyses also revealed no evidence of glomerular damage after PEGB consumption.NGAL is a protein that has raised some interest since its mRNA and protein were detected in urine after induction of acute kidney injury in rodents [34]. NGAL mRNA has been found in the ascending limb of Henle, and in collecting duct cells after ischemia-reperfusion [35]. NGAL is ordinarily reabsorbed by the proximal tubule [35, 36]. However, in case of renal injury, reabsorption may decrease, which results in higher urinary concentrations. Tubular damage and reduced filtration may also cause the accumulation of plasma NGAL [37]. The reported ranges of urinary NGAL/Creat ratio have varied greatly in healthy dogs from 10 to 460 ng/g, or from 40 to 3660 ng/g [38, 39]. These variations could reflect reporting from client-owned dogs of various breeds, age, and gender, fed with various diets. In our study, the urinary NGAL/Creat ratios after PEGB consumption (at any dose), ranged from 0.9 to 10 ng/g, leading us to conclude that there was no evidence of tubular damage. Recently, it was found that plasma NGAL was not an absolute criterion with which to discriminate between a healthy dog, versus a dog with either chronic, or acute kidney disease [38] contrary to urinary NGAL [39] and this shows how we must be cautious when interpreting these values. Moreover, increasing plasma NGAL would reflect tubular and filtration dysfunction, data already provided by other early biomarkers of renal damage (Plasma CysC, and urinary CysC/Creat, NGAL/Creat, and Clu/Creat ratios). Therefore, we suggest that plasma NGAL measurements represent redundant data and can be omitted.Intermediate measurements were also taken during the 24-week study period for all biomarkers; these did not reveal any significant differences.The PEGB doses ranged from 4 to 40 mg/kg/d, the intentional dose for dogs facing cognitive decline being 4 mg/kg/d [as recommended by the Neurophenols Consortium]. In studies where dogs were fed supplements with grape seed/skin extract at 20 mg/kg/d [5], or grape seed proanthocyanidins at 5 mg/kg/d [6], symptoms related to acute renal failure were not reported. In the group given the PEGB at 4 mg/kg/d, the dose of grape extract was beneath these previously reported doses. In addition, dogs consuming five or even ten times the intentional PEGB dose, showed no alteration of kidney or hepatic damage at 24 weeks. These data corroborated the 2013 European Pet Food Industry Federation (FEDIAF) advice that dogs could safely consume grape extract.We have considered why our extract, consumed long-term, as described in this study, appears to be entirely safe for consumption by dogs, in stark contrast to reports of acute renal failure in pets following their consumption of whole grapes or raisins. We can envisage some possibilities. The extract developed by our consortium is actually a complex mix of different extracts. How these extracts are derived (i.e. extracted from the grape), may have reduced, denatured, or eliminated, potentially toxic compounds. These factors may underlie the lack of any discernable toxicity when dogs consume the Neurophenols Consortium extract, even at high doses.Go to:ConclusionsFollowing consumption of the PEGB at all doses, conventional biomarkers of renal and liver damage were within the reference range throughout the study, with values of early biomarkers of renal damage CysC, Clu, NGAL unremarkable. To our knowledge, this is the first study demonstrating that chronic consumption of the PEGB extract can be achieved with neither renal, nor hepatic damage, at least based on plasma and urine analyses. Of note, renal health was monitored using a panel of parameters encompassing both early biomarkers of renal damage, as well as conventional biochemistry; this complementary approach is recommended in future studies. To conclude, dogs can safely consume a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium).Go to:AbbreviationsALP, Alkaline phosphatase; ALT, Alanine transaminase; AST, Aspartate aminotransferase; AUC, Area under the curve; BCS, Body condition score; BUN, Blood urea nitrogen; BW, Body weight; Clu, Clusterin; Cmax, maximum concentrations; Creat, Creatinine; CysC, Cystatin C; EGCG, Epigallocatechin gallate; NGAL, Neutrophil gelatinase-associated lipocalin; PEGB, Polyphenol-rich extract from grape and blueberry; UHPLC-MS/MS, Ultra high-performance liquid chromatography coupled to tandem mass spectrometryGo to:AcknowledgementsResearchers also thank Dr Chantal Thorin for assistance with statistical analysis and R software, to Samuel Ninet and the kennel staff as well as Philippe Bleis and Nutrition & Endocrinology Unit for taking good care of the animals and/or for technical assistance. The manuscript has been edited by San Francisco Edit.FundingThis work is part of the Neurophenols project. This project has been selected within the framework of the 12thcall for research projects launched by the French Governmental FUI (Fonds Unique Interministériel), and the present study was then supported by Bpifrance and the Conseil Régional des Pays-de-la-Loire.Availability of data and materialData supporting our conclusions are presented in the Results section of the manuscript.Authors’ contributionsVL, PN and GB conceived and designed the experiments; ASM performed the experiments; ASM, VL, PN, KO analyzed the data; ASM, VL, KO, PN, AL, JB, DG, GB wrote and/or revised the manuscript. All authors read and approved the final manuscript.Competing interestsAuthors’ institutions were affiliated with the Neurophenols Consortium, which produced the combined grape and blueberry extract, which safety is investigated in this study.Consent for publicationNot applicable.Ethics approval and consent to participateExperimental dogs were housed at Oniris (Nantes, France), according to animal welfare regulations of the French Ministry of Agriculture and Fisheries. Our experimental protocols complied fully with European Union guidelines (directive 2010/63 on the protection of animals used for scientific purpose), and our study was approved by the Animal Use and Care Advisory Committee of Pays-de-la-Loire (France), reference CEEA.2012.151.Adherence to ARRIVE guidelinesOur manuscript reporting adheres to the ARRIVE guidelines.Go to:Contributor InformationAnne-Sophie Martineau, Email: [email protected]éronique Leray, Email: [email protected] Lepoudere, Email: [email protected]éraldine Blanchard, Email: [email protected] Bensalem, Email: [email protected] Gaudout, Email: [email protected] Ouguerram, Email: [email protected] Nguyen, Email: [email protected] to:References1. Milgram NW, Head E, Zicker SC, Ikeda-Douglas CJ, Murphey H, Muggenburg B, et al. Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study. Neurobiol Aging. 2005;26:77–90. doi: 10.1016/j.neurobiolaging.2004.02.014. [PubMed] [Cross Ref]2. Krikorian R, Shidler MD, Nash TA, Kalt W, Vinqvist-Tymchuk MR, Shukitt-Hale B, et al. Blueberry supplementation improves memory in older adults. J Agric Food Chem. 2010;58:3996–4000. doi: 10.1021/jf9029332. [PMC free article] [PubMed] [Cross Ref]3. Bensalem J, Servant L, Alfos S, Gaudout D, Layé S, Lafenetre P, et al. Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice. Front Behav Neurosci. 2016;10:9. doi: 10.3389/fnbeh.2016.00009. [PMC free article] [PubMed] [Cross Ref]4. Henriksson BG, Söderström S, Gower AJ, Ebendal T, Winblad B, Mohammed AH. Hippocampal nerve growth factor levels are related to spatial learning ability in aged rats. Behav Brain Res. 1992;48:15–20. doi: 10.1016/S0166-4328(05)80134-2. [PubMed] [Cross Ref]5. Shanmuganayagam D, Beahm MR, Osman HE, Krueger CG, Reed JD, Folts JD. Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans. J Nutr. 2002;132:3592–3598. [PubMed]6. Salas A, Subirada F, Pérez-Encisco M, Blanch F, Jeussette I, Romano V, et al. Plant polyphenol intake alters gene expression in canine leukocytes. J Nutrigenet Nutrigenomics. 2009;2:43–52. doi: 10.1159/000200018. [PubMed] [Cross Ref]7. Mazzaferro EM, Eubig PA, Hackett TB, Legare M, Miller C, Wingfield WE, et al. Acute renal failure associated with raisin or grape ingestion in 4 dogs. J Vet Emerg Crit Care. 2004;14:203–212. doi: 10.1111/j.1534-6935.2004.00114.x. [Cross Ref]8. Morrow CMK, Valli VE, Volmer PA, Eubig PA. Canine renal pathology associated with grape or raisins ingestion: 10 cases. J Vet Diagn Invest. 2005;17:223–231. doi: 10.1177/104063870501700302. [PubMed] [Cross Ref]9. Eubig PA, Brady MS, Gwaltney-Brant SM, Khan SA, Mazzaferro EM, Morrow CMK. Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992–2002) J Vet Intern Med. 2005;19:663–674. [PubMed]10. Teschke R, Wolff A, Frenzel C, Schulze J, Eickhoff A. Herbal hepatotoxicity: a tabular compilation of reported cases. Liver Int. 2012;32:1543–1556. doi: 10.1111/j.1478-3231.2012.02864.x. [PubMed] [Cross Ref]11. Teschke R, Bahre R. Severe hepatotoxicity by Indian ayurvedic herbal products: a structured causality assessment. Ann Hepatol. 2009;8:258–266. [PubMed]12. National Research Council NRC. Nutrient requirements of dogs and cats. Washington, DC: National Academy Press; 2006.13. Muzzio M, Huang Z, Hu SC, Johnson WD, McCormick DL, Kapetanovic IM. Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharma-cokinetics in dogs. J Pharm Biomed Anal. 2012;59:201–208. doi: 10.1016/j.jpba.2011.10.023.[PMC free article] [PubMed] [Cross Ref]14. Kalt W, Blumberg JB, McDonald JE, Vinqvist-Tymchuk MR, Fillmore SA, Graf BA, et al. Identifica-tion of anthocyanins in the liver, eye, and brain of blueberry-fed pigs. J Agric Food Chem. 2008;56:705–712. doi: 10.1021/jf071998l. [PubMed] [Cross Ref]15. Wang J, Ferruzzi MG, Ho L, Blount J, Janle EM, Gong B, et al. Brain-targeted proanthocyanidin metabolites for Alzheimer’s disease treatment. J Neurosci. 2012;32:5144–5150. doi: 10.1523/JNEUROSCI.6437-11.2012. [PMC free article] [PubMed] [Cross Ref]16. Mata-Bilbao M, Andrés-Lacueva C, Roura E, Jáuregui O, Escribano E, Torre C, et al. Absorption and pharmacokinetics of green tea catechins in beagles. B J Nutr. 2008;100:496–502. doi: 10.1017/S0007114507898692. [PubMed] [Cross Ref]17. Kapetanovic IM, Crowell JA, Krishnaraj R, Zakharov A, Lindeblad M, Lyubimov A. Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs. Toxicology. 2009;260:28–36. doi: 10.1016/j.tox.2009.03.007. [PMC free article] [PubMed] [Cross Ref]18. Swezey RR, Aldridge DE, LeValley SE, Crowell JA, Hara Y, Green CE. Absorption, tissue distribution and elimination of 4-[3H]-epigallocatechin gallate in beagle dogs. Int J Toxicol. 2003;22:187–193. doi: 10.1080/10915810305101. [PubMed] [Cross Ref]19. Isbrucker RA, Edwards JA, Wolz E, Davidovich A, Bausch J. Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies. Food Chem Toxicol. 2006;44:636–650. doi: 10.1016/j.fct.2005.11.003. [PubMed] [Cross Ref]20. Johnson WD, Morrissey RL, Usborne AL, Kapetanovic I, Crowell JA, Muzzio M, et al. Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity. Food Chem Toxicol. 2011;49:3319–3327. doi: 10.1016/j.fct.2011.08.023. [PMC free article] [PubMed] [Cross Ref]21. Burns J, Yokota T, Ashihara H, Lean MEJ, Crozier A. Plant foods and herbal sources of resveratrol. J Agric Food Chem. 2002;50:337–340. [PubMed]22. Braun JP, Lefèbvre HP, Watson ADJ. Creatinine in the dog: a review. Vet Clin Path. 2003;32:162–179. doi: 10.1111/j.1939-165X.2003.tb00332.x. [PubMed] [Cross Ref]23. Dieterle F, Sistare F, Goodsaid F, Papaluca M, Ozer JS, Webb CP, et al. Renal biomarker qualification submission: a dialog between the FDA-EMEA and predictive safety testing consortium. Nature Biotechnol. 2010;28:455–462. doi: 10.1038/nbt.1625. [PubMed] [Cross Ref]24. Tenstad O, Roald AB, Grubb A, Aukland K. Renal handling of radiolabelled human cystatin C in the rat. Scand J Clin Lab Invest. 1996;56:409–414. doi: 10.3109/00365519609088795. [PubMed][Cross Ref]25. Herget-Rosenthal S, Marggraf G, Hüsing J, Göring F, Pietruck F, Janssen O, et al. Early detection of acute renal failure by serum cystatin C. Kidney Int. 2004;66:1115–1122. doi: 10.1111/j.1523-1755.2004.00861.x. [PubMed] [Cross Ref]26. Conti M, Moutereau S, Zater M, Lallali K, Durrbach A, Manivet P, et al. Urinary cystatin C as a specific marker of tubular dysfunction. Clin Chem Lab Med. 2006;44:288–291. doi: 10.1515/CCLM.2006.050. [PubMed] [Cross Ref]27. Tvarijonaviciute A, Ceron JJ, Holden SL, Biourge V, Morris PJ, German AJ. Effect of weight loss in obese dogs on indicators of renal function or disease. J Vet Intern Med. 2013;27:31–38. doi: 10.1111/jvim.12029. [PubMed] [Cross Ref]28. Miyagawa Y, Takemura N, Hirose H. Evaluation of the measurement of serum cystatin C by an enzyme-linked immunosorbent assay for humans as a marker of the glomerular filtration rate in dogs. J Vet Med Sci. 2009;71:1169–1176. doi: 10.1292/jvms.71.1169. [PubMed] [Cross Ref]29. Wehner A, Hartmann K, Hirschberger J. Utility of serum cystatin C as a clinical measure of renal function in dogs. J Am Anim Hosp Assoc. 2008;44:131–138. doi: 10.5326/0440131.[PubMed] [Cross Ref]30. Almy FS, Christopher MM, King DP, Brown SA. Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs. J Vet Intern Med. 2002;16:45–51. doi: 10.1111/j.1939-1676.2002.tb01605.x. [PubMed] [Cross Ref]31. Sasaki A, Sasaki Y, Iwama R, Shimamura S, Yabe K, Takasuna K, et al. Comparison of renal biomarkers with glomerular filtration rate in susceptibility to the detection of gentamicin-induced acute kidney injury in dogs. J Comp Path. 2014;151:264–270. doi: 10.1016/j.jcpa.2014.06.001.[PubMed] [Cross Ref]32. Jones SE, Jomary C. Clusterin. Int J Biochem Cell Biol. 2002;34:427–431. doi: 10.1016/S1357-2725(01)00155-8. [PubMed] [Cross Ref]33. Garcia-Martinez JD, Tvarijonaviciute A, Ceron JJ, Caldin M, Martinez-Subiela S. Urinary clusterin as a renal marker in dogs. J Vet Diagn Invest. 2012;24:301–306. doi: 10.1177/1040638711435112. [PubMed] [Cross Ref]34. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14:2534–2543. doi: 10.1097/01.ASN.0000088027.54400.C6. [PubMed][Cross Ref]35. Schmidt-Ott KM, Mori K, Kalandadze A, Li JY, Paragas N, Nicholas T, et al. Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia. Curr Opin Nephrol Hypertens. 2006;15:442–449. doi: 10.1097/01.mnh.0000232886.81142.58. [PubMed] [Cross Ref]36. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005;115:610–621. doi: 10.1172/JCI23056. [PMC free article] [PubMed] [Cross Ref]37. Pickering JW, Endre ZH. The clinical utility of plasma neutrophil gelatinase-associated lipocalin in acute kidney injury. Blood Purif. 2013;35:295–302. doi: 10.1159/000351542. [PubMed][Cross Ref]38. Steinbach S, Weis J, Schweighauser A, Francey T, Neiger R. Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney injury or chronic kidney disease. J Vet Intern Med. 2014;28:264–269. doi: 10.1111/jvim.12282. [PMC free article] [PubMed][Cross Ref]39. Segev G, Palm C, LeRoy B, Cowgill LD, Westropp JL. Evaluation of neutrophil gelatinase-associated lipocalin as a marker of kidney injury in dogs. J Vet Intern Med. 2013;27:1362–1367. doi: 10.1111/jvim.12180. [PubMed] [Cross Ref]Articles from BMC Veterinary Research are provided here courtesy of BioMed CentralFormats:ArticlePubReaderePub (beta)PDF (1.2M)CitationShareFacebookTwitterGoogle+Save itemsAdd to FavoritesView more optionsSimilar articles in PubMedEffects of dietary supplementation with a mixed blueberry and grape extract on working memory in aged beagle dogs.[J Nutr Sci. 2017]Potentiation of the bioavailability of blueberry phenolic compounds by co-ingested grape phenolic compounds in mice, revealed by targeted metabolomic profiling in plasma and feces.[Food Funct. 2016]Uptake and bioavailability of anthocyanins and phenolic acids from grape/blueberry juice and smoothie in vitro and in vivo.[Br J Nutr. 2015]Recent advances and uses of grape flavonoids as nutraceuticals.[Nutrients. 2014]Final report on the safety assessment of capsicum annuum extract, capsicum annuum fruit extract, capsicum annuum resin, capsicum annuum fruit powder, capsicum frutescens fruit, capsicum frutescens fruit extract, capsicum frutescens resin, and capsaicin.[Int J Toxicol. 2007]See reviews...See all...Cited by other articles in PMCEffects of dietary supplementation with a mixed blueberry and grape extract on working memory in aged beagle dogs[Journal of Nutritional Science...]See all...LinksPubMedTaxonomyRecent ActivityClearTurn OffA mixed grape and blueberry extract is safe for dogs to consumeA mixed grape and blueberry extract is safe for dogs to consumeBMC Veterinary Research. 2016; 12()Discriminatory power of 3.5 MHz convex and 7.5 MHz linear ultrasound probes for ...Discriminatory power of 3.5 MHz convex and 7.5 MHz linear ultrasound probes for the imaging of traumatic splenic lesions: a feasibility study.J Trauma. 2001 Jul;51(1):37-43.PubMedSee more...Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study.[Neurobiol Aging. 2005]Blueberry supplementation improves memory in older adults.[J Agric Food Chem. 2010]Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice.[Front Behav Neurosci. 2016]Hippocampal nerve growth factor levels are related to spatial learning ability in aged rats.[Behav Brain Res. 1992]Learning ability in aged beagle dogs is preserved by behavioral enrichment and dietary fortification: a two-year longitudinal study.[Neurobiol Aging. 2005]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992-2002).[J Vet Intern Med. 2005]Review Herbal hepatotoxicity: a tabular compilation of reported cases.[Liver Int. 2012]Severe hepatotoxicity by Indian Ayurvedic herbal products: a structured causality assessment.[Ann Hepatol. 2009]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharmacokinetics in dogs.[J Pharm Biomed Anal. 2012]Identification of anthocyanins in the liver, eye, and brain of blueberry-fed pigs.[J Agric Food Chem. 2008]Brain-targeted proanthocyanidin metabolites for Alzheimer's disease treatment.[J Neurosci. 2012]Absorption and pharmacokinetics of green tea catechins in beagles.[Br J Nutr. 2008]Exposure and toxicity of green tea polyphenols in fasted and non-fasted dogs.[Toxicology. 2009]Absorption, tissue distribution and elimination of 4-[(3)h]-epigallocatechin gallate in beagle dogs.[Int J Toxicol. 2003]Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies.[Food Chem Toxicol. 2006]Canine renal pathology associated with grape or raisin ingestion: 10 cases.[J Vet Diagn Invest. 2005]Subchronic oral toxicity and cardiovascular safety pharmacology studies of resveratrol, a naturally occurring polyphenol with cancer preventive activity.[Food Chem Toxicol. 2011]Plant foods and herbal sources of resveratrol.[J Agric Food Chem. 2002]Review Creatinine in the dog: a review.[Vet Clin Pathol. 2003]Acute renal failure in dogs after the ingestion of grapes or raisins: a retrospective evaluation of 43 dogs (1992-2002).[J Vet Intern Med. 2005]Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.[Nat Biotechnol. 2010]Renal handling of radiolabelled human cystatin C in the rat.[Scand J Clin Lab Invest. 1996]Early detection of acute renal failure by serum cystatin C.[Kidney Int. 2004]Urinary cystatin C as a specific marker of tubular dysfunction.[Clin Chem Lab Med. 2006]Effect of weight loss in obese dogs on indicators of renal function or disease.[J Vet Intern Med. 2013]Evaluation of the measurement of serum cystatin C by an enzyme-linked immunosorbent assay for humans as a marker of the glomerular filtration rate in dogs.[J Vet Med Sci. 2009]Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs.[J Vet Intern Med. 2002]Comparison of renal biomarkers with glomerular filtration rate in susceptibility to the detection of gentamicin-induced acute kidney injury in dogs.[J Comp Pathol. 2014]Review Clusterin.[Int J Biochem Cell Biol. 2002]Urinary clusterin as a renal marker in dogs.[J Vet Diagn Invest. 2012]Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury.[J Am Soc Nephrol. 2003]Review Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia.[Curr Opin Nephrol Hypertens. 2006]Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury.[J Clin Invest. 2005]The clinical utility of plasma neutrophil gelatinase-associated lipocalin in acute kidney injury.[Blood Purif. 2013]Plasma and urine neutrophil gelatinase-associated lipocalin (NGAL) in dogs with acute kidney injury or chronic kidney disease.[J Vet Intern Med. 2014]Evaluation of neutrophil gelatinase-associated lipocalin as a marker of kidney injury in dogs.[J Vet Intern Med. 2013]Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.[J Nutr. 2002]Plant polyphenol intake alters gene expression in canine leukocytes.[J Nutrigenet Nutrigenomics. 2009]Support CenterSupport CenterExternal link. Please review our privacy policy.NLMNIHDHHSUSA.gov

They are not. Differing definitions and cut-off rates give differing estimates of prevalence (the percentage of population affected), with the highest being 20% and the lowest being aroundSee Seigal 2006:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2528651/Another way of looking at it: take the adult population with low reading skills. Of those, up to 80% will have some degree of dyslexia. The other fraction will have had poor instruction (or poverty, or missing school, or all three together).There MAY be variances in how dyslexic male brains function vs. dyslexic female brains. Guinevere Eden’s 2013 study did so find, but it hasn’t been replicated and used fairly small sample sizes (N <100).Evans T. M., Flowers D. L., Napoliello E. M., & Eden G. F. (2013). Sex-specific gray matter volume differences in females with developmental dyslexia. Bain Struct Funct. 2013 Apr 27. [Epub ahead of print] PMID: 23625146Another, another study earlier found that:“The ratio of males to females in samples of children with reading difficulties varies widely depending upon the method of ascertainment. In studies where subjects are ascertained employing clinical or referral methods, gender ratios range from 2:1 to 15:1 males to females (e.g., Finucci & Childs, 1981; Vogel, 1990); however, in research-identified samples, gender ratios are closer to 1:1 (e.g., Harlaar, Spinath, Dale, & Plomin, 2005; Hawke, Wadsworth, Olson, & DeFries, 2007; Shaywitz, Shaywitz, Fletcher, & Escobar, 1990; Stevenson, 1992). Nevertheless, in both referred and research-identified samples, greater numbers of males with reading problems have typically been reported. For example, in a recent review of sex differences in reading disability, Rutter et al. (2004) reported the gender ratios in four independent epidemiological studies in which the samples had been ascertained using research criteria. In all four of the studies, significantly more males than females with reading disabilities were reported. Moreover, gender ratios for reading difficulties are greater in more severely affected samples (Hawke et al., 2007; Olson, 2002).”https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739722/#R13

We are far from understanding function of fungi in the human microbiome. Our understanding is at the 1st rung, the most primitive stage, where we continue to examine mammalian-microbial interactions through the biased lens of pathogenesis, as in 'eeks! Microbes are bad, cause diseases'. For the picture to become clearer, this bias needs to go the way of the dodo and that'll take a few years yet.Fungal communities of the human body: the Human MycobiomeI've organized this answer into 4 sections:I. To identify and enumerate the human mycobiome (human body fungal communities) is no easy task. Why?II. Mycobiome-human relationship in health and disease.III. Location-wise human mycobiome in health and disease (skin-scalp, gut-oral cavity, lung, plasma, vagina).IV. Saccharaomyces boulardii, example of probiotic fungus.I. To identify and enumerate the human mycobiome is no easy task. Why?Need to identify to understand function. However, human microbiota fungi are so rare as to make identification a challenge.From 1Traditionally, fungi were identified by culture. Human mycobiome inhabitants are likely novel and we don't yet know how to grow them in culture, at least many of them.Taking a leaf out of bacterial studies, where efforts center on the bacterial 18S rRNA locus, now PCR (Polymerase Chain Reaction)-based methods focus on the fungal genetic locus encompassing 18S, 5.8S and 28S rDNA genes, and the internal transcribed spacer regions (ITS1 and ITS2) that encode nonfunctional RNA transcribed during rRNA synthesis.Which region of this fungal locus to amplify is still debated, and different studies make different choices. With as many as 6 choices, studies run the gamut in target choice. Problem? Yes, makes it difficult to generalize datasets across studies.Debate also rages on which is better choice, 18S rDNA or ITS, 28S rDNA having been side-lined recently.From 2As expected from a rapidly growing field, much chaos, redundancy and mis-interpretation reigns.While the Fungal Genome Initiative (FGI; Fungal Genomics | Broad Institute of MIT and Harvard) aims to establish genomes throughout the fungal kingdom, such databases prioritize human disease-associated fungi with as yet little or no information on human mycobiome, i.e. commensal, inhabitants.From 3.Fungal databases are puny and growing slowly compared to those for bacteria. For example, a major database on fungal ITS regions maintained by Henrik Nillsson at the University of Gothenburg, Sweden, was last updated in 2012 Page on emerencia.org.Genome sequences of over a hundred fungal species are publicly available (3) but few of them are human-associated.From 3.Among Next-Generation Sequencing (NGS) technologies, RNA deep sequencing or RNA-seq offers several advantages for human mycobiome analysis: not hybridization-based; provide insight into transcriptional mechanisms (boundaries, links between exons).RNA-seq studies that pyrosequence (Pyrosequencing) fungal ITS and rDNA genes to study the human mycobiome started appearing in the past 4 or 5 years.Different studies use hugely varying approaches, ranging from different DNA isolation kits, analysis of different genes, different qPCR primer pairs and reactions ranging from 25 to even 45 cycles. Let's remember PCR is not only exquisitely sensitive but also exponential so a 20 cycle difference runs the gamut from entirely missing low abundance species to detecting many artifacts.Molecular techniques such as qPCR are also so sensitive that environmental contaminants turn up frequently in the datasets. Lacking prior knowledge of what to look for, it's difficult to exclude them.Not only do such differences make meta-analyses near-impossible, they make it difficult to distinguish clinically relevant datasets from experimental artifacts, unwittingly generated by sub-optimal experimental design and decisions.Problem with molecular analyses in a field of sparse databases is that in each study, data that doesn't align to database sequences is discarded. Is such discarded data truly irrelevant or is it the missed iceberg?Fungal nomenclature is a major problem. Synonyms and different names for different sexual stages of the same species abound. For example, a recent study re-annotated 'set of marker reference sequences that represent each currently accepted order of Fungi' (4). Careful and methodical curation required? Certainly!From 5.II. Mycobiome-human relationship in health and diseaseCommensal and environmental fungi constantly interact with the human body. How do they cause disease? Typically, underlying body perturbations such as immuodeficiencies and dysregulated immune function promote opportunistic fungal growth.For e.g., environmental Aspergillus spores that normally get killed could instead develop hyphae and invade tissue.From 6or commensal Candida could switch from yeast to biofilms, which in turn provide rich nutritious milieu for variety of pathogenic bacteria as well as an effective barrier against antibiotics.III. Human mycobiome health-versus-disease comparison by locationHuman Skin MycobiomeMalassezia, a common human skin mycobiome inhabitantLipid-dependent fungus Malassezia are the most abundant fungi living on human skin (7, 8)Lack fatty acid synthase, and express lipases and hydrolases, helping them adapt to human skin (3).Almost all epidermal skin cells express aryl-hydrocarbon receptor (AhR) (9) and Malassezia synthesizes powerful AhR ligands, indirubin and indolo [3,2-b] carbazole (ICZ) (10). Thus, Malassezia influences skin metabolism and function by exploiting the AhR-AhR ligand pathway.Requiring lipids for its growth, Malassezia preferentially colonizes face, scalp and upper trunk rather than limbs , i.e. the sebaceous gland-rich areas of skin.Malassezia is dimorphic, i.e. has yeast and mycelial (hyphal) phases.Its lipid dependence makes it challenging to isolate and grow in culture. For example, Sabouraud's dextrose agar is a commonly used fungal culture medium. Yet it does not support growth of many Malassezia species (11). Rather all Malassezia yeasts identified thus far grow in the nutritionally complex (containing Ox bile and Tween 60, a fatty acid) and unconventional Leeming and Notham agar (LNA). Even so, such approaches can miss M. restricta. Slower-growing than others in such cultures, it's easily overgrown by related species.Mis-identification is another common problem with purely phenotypic approaches.Careful characterization of microbial species requires isolation in culture, freeze-down and subculture of frozen aliquots. Even this routine task is difficult in the case of Malassezia which, poorly viable in culture, only maintains viability when stored at -80oC, and not at 4 to 12oC, the norm for yeasts (12).Culture-independent, molecular approaches are thus more suitable. Tape or swab is used to take the skin sample, fungal DNA extracted and subjected to PCR. DNA extraction method, and sensitivity and specificity of the particular PCR method used greatly influence the outcome. The specific approach taken to accurately identify Malassezia needs to keep in mind that human skin is after all host to multitude of bacteria as well as other fungi such as Candida.Malassezia in human skinPresumed to colonize immediately post-birth (13).Found in skin swabs from 78 out of 245 British neonates (<28 days of age) on LNA culture, with 41 out of 42 still positive at follow-up (14).M. furfur and globosa found in 60.5% and 7.2%, respectively, cultures of 195 Iranian neonates (15). Melassezia species colonizing human skin are thus neither random nor inter-changeable.M. dermatis found in skin of 19 healthy Koreans aged 17 to 55 years by both culture-dependent and -independent (ITS-1 and 26 rDNA) (16). Geographic difference?Why do we have Malassezia in our skin? Are its lipid dependence and its human skin colonization pattern related to age-related human sebaceous gland activity? Data suggest so.Minimal in children, sebaceous gland activity increases during the teens in response to androgens, and then stabilizes from late teens until old age (17).Sebum, product of sebaceous glands, consists of ceramides, fatty acids, cholesterol, squalene (cholesterol precursor and also popular vaccine adjuvant but that's another story!), triglycerides and wax esters. Sebum fatty acid composition changes markedly with puberty (18).Healthy human skin from 245 Canadians ranked by age (0–3, 4–14, 15–25, 26–40, 41–60, and >60-years old), swabbed on LNA cultures, showed marked increase in Malassezia-positive cultures starting from age 15, with no noticeable difference in positivity between genders (19).DNA analysis of 770 healthy Japanese aged 0 to 82 years also showed marked increase in early teens but had major gender differences, being much more abundant in males. M. restricta predominated in males of all ages, while doing so only in females >23 years, with M. globosa dominating at 1o to 18 years (20).From 20.Similar dominance of M. restricta followed by M. globosa was also found by 5.8S/ ITS2 rDNA analysis in a small Brazilian study of scalp and forehead skin from healthy and seborrheic dermatitis subjects (21).Sampling trunk, arms, plantar heel, toenail and toeweb fungal communities using ITS 1 and 18S rDNA gene in 10 healthy adults, Findley et al found Malassezia dominance at all sites with much greater species diversity in the foot sites (22).Clearly Malassezia colonization of human skin closely mirrors sebaceous gland distribution and activity and sebum fatty acid composition.Malassezia-human skin interactions (adapted from 23) range all the way fromHealthy commensalismMild, non-clinical altered skin melanocyte pigmentation and plaques that mildly alter epidermal barrier function (Pityriasis Versicolor)Inflammation without adaptive immune function involvement (Seborrheic dermatitis; SD and dandruff)Inflammation with adaptive immune function involvement (Atopic dermatitis; AD)Hair follicle invasion and inflammation (Malassezia folliculitis).Much less is known about Malassezia's role in psoriasis (20).- Geographic differences in Malassezia distribution: Rare elsewhere, M. dermatis and M. japonica are more frequently found in East Asia, (20, 24, 25), and in India (26).Malassezia and PsoriasisPsoriasis is a chronic skin inflammation marked by hyperproliferation and hyperkeratinization of the epidermis. Malassezia's role in this disease is as yet undetermined. Data are all over the place.While an Indian study that examined ITS 2 in addition to 28S rDNA gene found no difference in Malassezia prevalence in skin from psoriatic and healthy subjects (n=50 each) (26), a Japanese analysis of 28S rDNA gene sequences from skin samples from healthy (n=12) and psoriasis (n=12) subjects found psoriatic skin contained more diverse fungi compared to healthy skin though Malassezia was less abundant (27).Itraconazole, ketoconazole, and posaconazole are the most effective drugs for treating Malassezia infections (28, 29).Malassezia and DandruffIn a French study of 49 volunteers examining fungal ITS 1-5.8S-ITS2 and part of the 28S rDNA genes, Malassezia restricta was found more frequently associated with dandruff (30).Similar dominance of M. restricta (and globosa) in dandruff scalps also seen in comparison of 62 and 57 dandruff and healthy scalp in Japanese volunteers (31).Human Gut MycobiomeHuman Gut Mycobiome is influenced by dietYeasts in human stool were first reported in 1917 (32) so human gut-fungal association is not a novel finding.Geotrichum candidum and Saccharomyces cerevisiae were found in gut mycobiome of people who ate cheese and drank sake among French and Japanese, respectively (33, 34).Reduced gut fungal diversity in US urban/suburban residents (Boulder, CO and Philadelphia, PA) eating typical western diets compared to rural Malawi residents eating diets 'rich in maize, legumes and other plants' (35) revives the old question of the hygiene hypothesis, namely, whether loss of our co-evolved microbial diversity triggered the greater autoimmune prevalence in Western populations. Differences between these 2 populations include diet, hygiene and contact with animals to mention just a few of the more obvious ones.ITS 1 pyrosequencing found fungal genome signals in every one of 96 stool samples from healthy American volunteers (36). Proportionally Saccharomyces (89%), Candida (57%) and Cladosporium (42%). Candida correlated positively with carbohydrates and negatively with total saturated fatty acids, while Aspergillus correlated negatively with SCFA (Short-chain fatty acids). Saccharomyces showed no particular dietary trend.The Wayampi people are an indigenous Amerindian tribe living in French Guiana and Brazil in South America. Fungal ITS1-ITS4 and NL1-NL4 PCR and pyrosequencing of stool samples from 151 healthy volunteers on two different occasions, 2006 and 2010, showed not Candida albicans but Candida krusei and Saccharomyces cerevisiae were the most abundant gut fungal species. In other words a very different gut mycobiome in an isolated rural human population from the one observed in humans living in industrialized environments.From 37Human Gut Mycobiome in health and diseaseA 2005 stool culture study of 80 pediatric bone marrow transplant or cancer patients and 61 healthy controls on Sabouraud's Dextrose agar found Candida albicans in 41.2% and 40.5%, respectively, and non-albicans Candida in 50% and 40.5%, respectively (38). In other words, similar Candida proportion in stool.Anti-Saccharomyces cerevisiae antibodies are found more frequently in Crohn's disease (CD) patients compared to Ulcerative colits patients and healthy controls (39, 40). No consensus yet on what this signifies.18S rDNA pyrosequencing of distal colon (rectal/sigmoid) biopsies from 25 children with IBD (Inflammatory Bowel Disease) compared to 12 age-matched controls, and 2 adults each either normal or with Ulcerative Colitis (UC) showed that Ascomycota and Basidiomycota were the dominant phyla. Fungal DNA was only found in few, not all, subjects (41). Antibiotics or immunosuppression weren't responsible for these differences since these newly diagnosed IBD patients hadn't been administered them yet. Why such poor recovery? Could it be site (colon biopsy versus stool) or choice of fungal gene (18S rDNA versus ITS 1 in other studies)?A Chinese ileal biopsy and stool sample study of 19 CD patients and 7 healthy volunteers (42) found CD patients had increased Candida prevalence and different gut mucosa- and stool-associated fungi species compared to controls. Red: CD; Green: Controls.From 42Human Oral MycobiomeIn a study of 20 healthy volunteers, ITS 1 pyrosequencing found Candida (75%), Cladosporium (65%), Aureobasidium and Saccharomycetales (50% each), Aspergillus (35%), Fusarium (30%) and Cryptococcus (20%) but no Malassezia (43).But a more recent ITS 1 pyrosequencing found Malassezia in saliva of 6 out of 6 healthy adult volunteers, identifying it for the 1st time as a commensal fungal inhabitant of the human oral cavity (44). This raises the question how previous studies missed such a basic finding? Malassezia culture is difficult, requiring specialized culture media, nomenclature is muddled, fungal databases are incomplete and confusing, Malassezia is dimorphic, all possible reasons.Summary of Human Gut-Oral Mycobiome studiesThe table to the right is my original summation of the differences between the major human gut and oral mycobiome studies published thus far.Differences include different diseases, tissue samples and methods, and absence of controls.Boy, are the methods different!Any generalizable observations? Yes, fungal diversity increases in GI tract-associated diseases such as CD, HBV (Hepatitis B).From 45.Human Lung Mycobiome changes with lung disease: cause or effect? Not clear- Mouthwash/gargle (oropharyngeal wash, OW) and BAL (bronchoalveolar lavage) ITS PCR and pyrosequencing comparison of lung transplant patients and controls indicated fungi had colonized deeper lung tissues in lung transplant patients.From 46Same group showed clinically relevant fungi like Candida and Aspergillus were enriched in BAL of HIV-infected and lung transplant patients, and more frequently present, i.e. more samples positive compared to healthy controls (47).Comparison of mouthwash, induced sputum and BAL (bronchoalveolar lavage) of HIV-infected, COPD (Chronic Obstructive Pulmonary Disease) and normal people (48) using 18S rDNA and ITS PCR and pyrosequencing. Showed two things, one, the three sites had overlapping as well as distinct fungi with Candida dominating in mouthwash and sputum, two, Pneumocystis jirovecii was enriched in HIV-infected and COPD samples.Common theme emerging as in human lung mycobiome changes with diseases as it does in gut. Cause-effect distinction not yet clear.Human Lung Mycobiome SummaryFrom 49.Human Plasma and Vagina MycobiomeReports of mycobiome in human plasma, milk, vagina are currently restricted to only one or few peer-reviewed studies.One human plasma study found a surprise. Ascomycota, in particular the order Hypocreales, was dominant fungal signature in 5 of 6 subjects (50). Could source be gut since Ascomycota are prevalent there?A large study of 494 pre-menopausal Estonian women examined fungal mycobiome using ITS1 and 2 pyrosequencing in vaginal fornix and cervix brush samples. They found great diversity consisting of 196 OTUs including 16 for Candida alone (51). As with other mycobiome studies, results were plagued with the issue of air-borne contamination.IV. Saccharaomyces boulardii, example of probiotic fungusEffective against diarrhea in human clinical trial (52) in a study of 35 children each given either S. boulardii (250mg orally twice a day) or not. Children given S. boulardii recovered faster from both diarrhea (3.4 versus 5.5 days) and vomiting (2.5 versus 3.3 days) (statistically significant).Could such differences be biologically relevant? Absolutely and this table explains why.From 53.Other S. boulardii studies: 11 randomised clinical trials for acute infectious diarrhea (AID), 9 for antibiotic-associated diarrhea (ADD), 4 each for Helicobater pylori infection, and Crohn's disease (CD), 1 for Ulcerative colitis (UC), 5 newborn studies, and 3 for IBS (Irritable Bowel Syndrome).S. boulardii is very effective in disease treatment either alone (for AID and ADD) or as an adjunct (for H. pylori, CD, UC).Extensive and larger trials of S. boulardii for CD, UC and IBS are very much warranted.A good place to learn more about S. boulardii, especially its history is here: Saccharomyces boulardiiHow does S.boulardii work against so many diseases? Many hypotheses (adapted from 53):Secretes polyaminesRestores normal levels of colonic Short-chain fatty acids (SCFA)Stabilizes gut epithelium barrier functionRestores fluid transport pathwaysInduces enhanced gut mucosal secretory IgA productionNeutralizes bacterial toxins, specifically those of Clostridium difficileIts metabolic functions, such as polyamines, accelerate re-establishment of normal gut microbiotaMycobiome BibliographyHuffnagle, Gary B., and Mairi C. Noverr. "The emerging world of the fungal microbiome." Trends in microbiology 21.7 (2013): 334-341. Page on europepmc.orgCui, Lijia, Alison Morris, and Elodie Ghedin. "The human mycobiome in health and disease." Genome Med 5 (2013): 63. Page on biomedcentral.comSharma, Krishna Kant. "Fungal genome sequencing: basic biology to biotechnology." Critical reviews in biotechnology 0 (2015): 1-17.Schoch, Conrad L., et al. "Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi." Database 2014 (2014): bau061. Finding needles in haystacks: linking scientific names, reference specimens and molecular data for FungiUnderhill, David M., and Iliyan D. Iliev. "The mycobiota: interactions between commensal fungi and the host immune system." Nature Reviews Immunology 14.6 (2014): 405-416.Iliev, Iliyan D., and David M. Underhill. "Striking a balance: fungal commensalism versus pathogenesis." Current opinion in microbiology 16.3 (2013): 366-373.Page on nih.govFindley K, Oh J, Yang J, Conlan S, Deming C, et al. (2013) Topographic diversity of fungal and bacterial communities in human skin. Nature 498: 367–370.Grice EA, Segre JA (2011) The skin microbiome. Nat Rev Microbiol 9: 244–253).Gaitanis G, Magiatis P, Stathopoulou K, Bassukas ID, Alexopoulos EC, et al. (2008) AhR ligands, malassezin, and indolo [3,2-b] carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis. J Invest Dermatol 128: 1620–1625.Magiatis P, Pappas P, Gaitanis G, Mexia N, Melliou E, et al. (2013) Malassezia yeasts produce a collection of exceptionally potent activators of the Ah (dioxin) receptor detected in diseased human skin. J Invest Dermatol 133: 2023–2030.Gue ́ho-Kellermann E, Boekhout T, Begerow D. (2010) Biodiversity phylogeny and ultrastructure In: Malassezia and the Skin: Science and Clinical Practice. Berlin: Springer. Boekhout T, Gue ́ho E, Mayser P, Velegraki A (Editors), pp 17– 63.Crespo MJ, Abarca ML, Caban ̃es FJ (2000) Evaluation of different preservation and storage methods for Malassezia spp. J Clin Microbiol 38: 3872–3875.Nagata, Rie, et al. "Transmission of the major skin microbiota, Malassezia, from mother to neonate." Pediatrics International 54.3 (2012): 350-355.Ashbee HR, Evans EG (2002) Immunology of diseases associated with Malassezia species. Clin Microbiol Rev 15:21–57.Zomorodian K, Mirhendi H, Tarazooie B et al (2008) Molecular analysis of Malassezia species isolated from hospitalized neonates. Pediatr Dermatol 25:312–316.Lee YW, Kim SM, Oh BH et al (2008) Isolation of 19 strains of Malassezia dermatis from healthy human skin in Korea. J Dermatol 35:772–777.Pochi PE, Strauss JS, Downing DT (1979) Age-related changes in sebaceous gland activity. J Invest Dermatol 73:108–111.Yamamoto A, Serizawa S, Ito M, Sato Y (1987) Effect of aging on sebaceous gland activity and on the fatty acid composition of wax esters. J Invest Dermat 89:507–512.Gupta AK, Kohli Y (2004) Prevalence of Malassezia species on various body sites in clinically healthy subjects representing different age groups. Med Mycol 42:35–42.Epidemiology of Malassezia-related Skin Diseases. Takashi Sugita, Teun Boekhout, Aristea Velegraki, Jacques Guillot, Suzana Hadina, and F. Javier Cabanes. In Malassezia and the Skin: Science and Clinical Practice. Berlin: Springer. Boekhout T, Gue ́ho E, Mayser P, Velegraki A (Editors). 2010.Soares, Renan Cardoso, et al. "Malassezia intra-specific diversity and potentially new species in the skin microbiota from brazilian healthy subjects and seborrheic dermatitis patients." PloS one 10.2 (2015): e0117921. Malassezia Intra-Specific Diversity and Potentially New Species in the Skin Microbiota from Brazilian Healthy Subjects and Seborrheic Dermatitis Patients.Findley, Keisha, et al. "Human Skin Fungal Diversity." Nature 498.7454 (2013): 367. Human Skin Fungal Diversity.Velegraki, Aristea, et al. "Malassezia Infections in Humans and Animals: Pathophysiology, Detection, and Treatment." PLoS pathogens 11.1 (2015): e1004523. Malassezia Infections in Humans and Animals: Pathophysiology, Detection, and TreatmentGaitanis G, Velegraki A, Alexopoulos EC, Kapsanaki-Gotsi E, Zisova L, et al. (2009b) Malassezia furfur fingerprints as possible markers for human phylogeography. ISME J 3: 498–502.Giusiano G, Sosa Mde L, Rojas F, Vanacore ST, Mangiaterra M (2010) Prevalence of Malassezia species in pityriasis versicolor lesions in northeast Argentina. Rev Iberoam Micol 27: 71–74.Rudramurthy SM, Honnavar P, Chakrabarti A, Dogra S, Pankaj S et al. (2014) Association of Malassezia species with psoriatic lesions. Mycoses 57: 483–488.Takemoto, Akemi, et al. "Molecular characterization of the skin fungal microbiome in patients with psoriasis." The Journal of dermatology (2014)) as did another Japanese study of 24 patients with SD (Tanaka, A., et al. "Molecular Characterization of the Skin Fungal Microbiota in Patients with Seborrheic Dermatitis." J Clin Exp Dermatol Res 5 (2014): 239.Cafarchia C, Figueredo LA, Iatta R, Colao V, Montagna MT, Otranto D (2012) In vitro evaluation of Malassezia pachydermatis susceptibility to azole compounds using E-test and CLSI microdilution methods. Med Mycol 50: 795–801.Velegraki A, Alexopoulos EC, Kritikou S, Gaitanis G (2004) Use of fatty acid RPMI 1640 media for testing susceptibilities of eight Malassezia species to the new triazole posaconazole and to six established antifungal agents by a modified NCCLS M27-A2 microdilution method and Etest. J Clin Microbiol 42: 3589– 3593.Clavaud, Cécile, et al. "Dandruff is associated with disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp." PloS one 8.3 (2013): e58203. Dandruff Is Associated with Disequilibrium in the Proportion of the Major Bacterial and Fungal Populations Colonizing the ScalpHiruma, Midori, et al. "Genotype Analyses of Human Commensal Scalp Fungi, Malassezia globosa, and Malassezia restricta on the Scalps of Patients with Dandruff and Healthy Subjects." Mycopathologia 177.5-6 (2014): 263-269.Anderson, Harry Warren. "Yeast-like fungi of the human intestinal tract." The Journal of Infectious Diseases (1917): 341-386. An Error Occurred Setting Your User CookieFirmesse, Olivier, et al. "Fate and effects of Camembert cheese micro-organisms in the human colonic microbiota of healthy volunteers after regular Camembert consumption." International journal of food microbiology 125.2 (2008): 176-181. Page on researchgate.netKitagaki, Hiroshi, and Katsuhiko Kitamoto. "Breeding research on sake yeasts in Japan: history, recent technological advances, and future perspectives." Annual review of food science and technology 4 (2013): 215-235.Parfrey, Laura Wegener, et al. "Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity." Frontiers in microbiology 5 (2014). Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversityHoffmann, Christian, et al. "Archaea and fungi of the human gut microbiome: correlations with diet and bacterial residents." PLoS One 8.6 (2013): e66019. Archaea and Fungi of the Human Gut Microbiome: Correlations with Diet and Bacterial ResidentsAngebault, Cécile, et al. "Candida albicans is not always the preferential yeast colonising humans: a study amongst Wayampi Amerindians." Journal of Infectious Diseases (2013): jit389. A Study in Wayampi AmerindiansAgırbaslı, H., SA Keceli Özcan, and Gündüz Gedikoğlu. "Fecal fungal flora of pediatric healthy volunteers and immunosuppressed patients." Mycopathologia 159.4 (2005): 515-520.Kaul, Amit, et al. "Serum anti‐glycan antibody biomarkers for inflammatory bowel disease diagnosis and progression: A systematic review and meta‐analysis." Inflammatory bowel diseases 18.10 (2012): 1872-1884.Russell, R. K., et al. "Anti-Saccharomyces cerevisiae antibodies status is associated with oral involvement and disease severity in Crohn disease." Journal of pediatric gastroenterology and nutrition 48.2 (2009): 161-167.Mukhopadhya, I., et al. "The fungal microbiota of de-novo paediatric inflammatory bowel disease." Microbes and Infection (2014). The fungal microbiota of de-novo paediatric inflammatory bowel diseaseLi, Qiurong, et al. "Dysbiosis of gut fungal microbiota is associated with mucosal inflammation in Crohn’s disease." Journal of clinical gastroenterology 48.6 (2014): 513. Dysbiosis of Gut Fungal Microbiota is Associated With Mucosal Inflammation in Crohn’s DiseaseGhannoum, Mahmoud A., et al. "Characterization of the oral fungal microbiome (mycobiome) in healthy individuals." PLoS pathogens 6.1 (2010): e1000713. Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy IndividualsDupuy, Amanda K., et al. "Redefining the human oral mycobiome with improved practices in amplicon-based taxonomy: discovery of malassezia as a prominent commensal." PloS one 9.3 (2014): e90899. Redefining the Human Oral Mycobiome with Improved Practices in Amplicon-based Taxonomy: Discovery of Malassezia as a Prominent CommensalMukherjee, Pranab K., et al. "Mycobiota in gastrointestinal diseases." Nature Reviews Gastroenterology & Hepatology (2014).Charlson, Emily S., et al. "Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after lung transplant." American journal of respiratory and critical care medicine 186.6 (2012): 536-545. Page on bushmanlab.orgBittinger, Kyle, et al. "Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing." Genome biology 15.10 (2014): 1-14. Page on biomedcentral.comCui, Lijia, et al. "Topographical Diversity of the Respiratory Tract Mycobiome and Alteration in HIV and Lung Disease." American journal of respiratory and critical care medicine ja (2015).Nguyen, Do Ngoc Linh, Eric Viscogliosi, and Laurence Delhaes. "The lung mycobiome: an emerging field of the human respiratory microbiome." Frontiers in Microbiology 6 (2015): 89.Beatty, Meabh, et al. "Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma." BMC genomics 15.1 (2014): 933. Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasmaDrell, Tiina, et al. "Characterization of the vaginal micro-and mycobiome in asymptomatic reproductive-age Estonian women." PLoS One 8.1 (2013): e54379. Characterization of the Vaginal Micro- and Mycobiome in Asymptomatic Reproductive-Age Estonian WomenBurande, Meeta Amit. "Comparison of efficacy of Saccharomyces boulardii strain in the treatment of acute diarrhea in children: A prospective, single-blind, randomized controlled clinical trial." Journal of pharmacology & pharmacotherapeutics 4.3 (2013): 205.Dinleyici, Ener Cagri, et al. "Saccharomyces boulardii CNCM I-745 in different clinical conditions." Expert opinion on biological therapy 14.11 (2014): 1593-1609.Thanks for the A2A, Matt Chanoff.