B. subtilis), Gram-negative bacteria (E. coli), and fungi  by disc-diffusion method. All extracts, even the aqueous ones, demonstrated antibacterial and antifungal activity. Chemometric methods  and a saturation-type model were used to evaluate the contribution of various bioactive principles in building the antioxidant capacity of extracts. Both experimental and modelling results show that 50% ethanolic extracts provide a rich polyphenolics profile and ensure a good antioxidant capacity.Propolis, a complex bee product, is a source of numerous bioactive principles, beneficial for human health, therefore it is intensively studied. In the present work, extracts of propolis from Bihor Romanian County were studied to identify the relationship between the polyphenolic derivatives profile and their antioxidant and antimicrobial activity. Extracts were obtained using water and 25%, 50%, and 70% ethanolic solutions (w/w), at 2:1, 4:1, and 6:1 liquid: solid ratios (w/w). 21 polyphenolic derivatives were quantified by UHPLC-MS, proving that the extracts composition strongly depends on the solvent. The sum of quantified polyphenolics extracted varied between 1.5 and 91.2 mg/g propolis. The antioxidant capacity was evaluated using the free radicals 2,2’-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid) diammonium salt (ABTS) and 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging methods. Antimicrobial efficiency was tested against Gram-positive ( The most representative biologically active compounds in temperate zones propolis are phenolic acids and flavonoids6.The social and economic value of propolis has been demonstrated by addressing health, nutrition, and product-development issues. Access to its potentially useful bioactive principles is restricted by different other plant debris, present in diverse proportions, depending on the collection time and geographical origin10. Chloroform, acetone2, methanol11, hexane12, glycerol with 5% (v/v) ethanol or propylene glycol with 2% (v/v) ethanol13, olive oil, β-cyclodextrin, 1.5% (w/v) aqueous solution, or 1:1 combination of these14 were also employed in macerations at ambient temperature.Due to the large extent of wax and debris present in propolis, ethanol or its aqueous mixtures represented the preferred extraction media over the time, given its selectivity the active principles of interest17 or pressurized liquid extraction systems18 led to better recovery yields. The liquid: solid ratio (w/w) was a widely varied experimental parameter, ranging from 2500:1 to 3:125. The process was mainly centred on increasing the global yield, without much attention towards the extracted profiles of phenolic acids and flavonoids.Several research groups have studied the effect of experimental factors upon the extraction yield and extracts properties. Temperature raising in stirred vessels20, microwave fields20, or supercritical CO2 extraction16. Process time decrease was remarkable, the 24 h cycles for ambient conditions extraction with 70% ethanol dropping between 10 s and 45 min cycles26, with intermittent cooling, to avoid excessive heating and polyphenolics degradation.Process intensification, aiming to increase the active principles concentration and to decrease the process time, has been sought by applying ultrasoundsDespite duration, maceration is still the method of choice for many propolis-selling beekeepers, due to its simplicity in terms of operations and equipment. Intensification techniques like sonication, microwaves, pressurized liquid extraction are not considered by most beekeepers, as they come with high initial investment costs and safety operation requirements. Therefore, mastering the traditional maceration to obtain dedicated usage extracts remains imperative in the artisanal apiculture.p-coumaric acid, 3-acetylpinobanksin, pinobanksin-3-acetate, and caffeic acid were demonstrated to have anti-fungal activity against C. albicans27. Propolis antimicrobial activities begin to be documented against different bacteria28, yeasts29, viruses30, and parasites31. In vitro, propolis may act directly on microorganisms, and in vivo it may stimulate the immune system, activating the mechanisms involved in the microorganisms killing.The targeted compounds in antibacterial, antifungal, antioxidant and antitumoral effects are present in small and variable amounts in the natural propolis. Pinocembrin, 32. Compounds like caffeic acid and caffeic acid phenethyl ester (CAPE), quercetin, kaempferol, p-coumaric acid, galangin, chrysin, with anti-inflammatory and immuno-regulatory effects, make propolis a possible significant component in treating various viral diseases, including Covid-1933. Kowacz and Pollack34 have shown that propolis could also be used as a spray to physically prevent viruses from entering the body by forming an extensive layer of water exclusion zone.Studies on the propolis antiviral activity showed that it can be used as an adjunct in fighting against respiratory infections caused mainly by coronaviruses2. It had been accepted that phenolic compounds in propolis donate hydrogen ions to free radicals, thus hindering lipids, proteins, and nucleic acids oxidation35.Most of the propolis antioxidant properties were attributed to galangin and pinocembrin36 reported that an aqueous propolis extract presented higher antioxidant effects than ethanolic extracts, with galangin showing more efficiency than pinocembrin, regardless the extraction solvent. Kubiliene et al37 compared composition and biological activities of propolis extracts prepared with non-alcoholic solvent mixtures and found no significant differences in the total content of phenolic compounds compared to ethanolic extracts.WaghDespite the reduced phenolic acids’ and flavonoids’ aqueous solubility, water should not be overlooked as extractant. Fine-tuning the water–ethanol ratio, contact time, and/or solid: liquid ratio could open way to operational parameters customized for yielding extracts with high content of targeted compounds, displaying high antioxidant capacity, and equally high antimicrobial activity.23. Variability of debris or access to certain processing practices endorse reporting results as mass of extracted matter per weight of ‘balsam’22, ethanolic extract of propolis38, or crude propolis20.Composition reports on propolis are extremely diverse, given the specificity of flora, climate, geographical and hydrological conditions at the site of collection. It is an established fact that propolis from temperate zone, called poplar propolis contains mainly phenolic acid, flavonoids, aromatic acids, and their esters1, with a large variability according to the provenance region, harvest time, and beekeeping practices. Caffeic acid, chrysin, CAPE, and pinocembrin are most commonly found40, accompanied by ferulic and p-coumaric acids, galangin, kaempferol and quercetin. Apigenin, rutin, luteolin, narigenin, and pinostrobin are less often quantified39.Romanian propolis, as a poplar type propolis, is reported to contain around 300 mg/g polyphenolic compoundsExperimentally, there should be a quest for better extraction conditions, using environmentally friendly solvents or solvent mixtures. Within this frame, the present work carries on a large experiment involving solvent composition, solid: liquid ratio, and process duration. The detailed composition of the polyphenolic derivatives in propolis, obtained by UHPLC-MS, gives the chance to evaluate the importance of the operating conditions for the extraction of the main bioactive principles which contribute to the antioxidant and antimicrobial activity. Chemometric methods  were used to emphasize a relationship between the chemical compounds’ nature and extracts antioxidant/antimicrobial activity. A saturation-type model was used to obtain a general insight of the contribution of the polyphenolics profile to the variation of antioxidant capacity.Apis Meliffera Carpatica, was donated by dr. Roxana Spulber, Institute for Research and Development for Beekeeping, Bucharest, Romania; it originated from an apiary registered with the Romanian Beekeepers’ Association in Bihor County, Romania, being harvested by the beekeepers in March-November 2016 season. It was stored in laboratory at − 20 °C until processing and analysis. Prior to extractions, propolis was grounded to fine powder with a Retsch 200 mill .Propolis, produced by 2S2O8 (99%), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), quercetin (95%), ethanol  were purchased from Sigma-Aldrich  and used without further purification.Trolox (95%), 2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)–ABTS–(98%), Kp‐coumaric acid, syringic acid, caffeic acid, caffeic acid phenyl ester—CAPE, chlorogenic acid, ferulic acid, ellagic acid, vanillic acid, p-hydroxybenzoic acid, 3,4‐dihydroxybenzoic acid, t‐cinnamic acid, (+)–catechin, and (−)–epicatechin), from Sigma-Aldrich , were used to prepare individual 500 mg/L methanolic stocks. A 10 mg/L mixed working standard in methanol was obtained by appropriate dilution of individual stocks. Automatic pipettes and class A volumetric glass flasks were used.Analytical standards , and formic acid (98–100%) were purchased from Merck . Aqueous solutions were prepared with deionized water produced by a Milli-Q Millipore system .All chemicals for antimicrobial activity were microbiologically pure. Nutrient agar (NA) was purchased from Sigma–Aldrich , and yeast extract peptone dextrose (YPD) from Carl Roth .Water and ethanolic solutions  were used for extraction. The liquid:solid ratios (w/w) were 2:1, 4:1, and 6:1. Phases were contacted at room temperature, 150 rpm for 1, 3, and 5 days, in an ES 80-Grant Instruments orbital shaker (UK). Extracts were separated from waxes with Filtrak No 389, Ø 12.5 cm filter paper, and stored at − 20 °C until analysis.2:1, 4:1 and 6:1), while the second digit stands for the contact duration .The extracts coding system is an alpha-numerical combination with the letter representing the solvent , the first digit, the liquid:solid ratio (The phenolic acids and flavonoids were quantified with an UltiMate 3000 UHPLC System (Thermo Fisher Scientific), coupled with a Q Exactive Focus Hybrid Quadrupole‐Orbitrap mass spectrometer equipped with Heated Electrospray Ionisation (HESI) probe (Thermo Fisher Scientific).The Kinetex C18 column  was operated at 30 °C. The injection volume was 10 µL, each injection being repeated three times. Mobile phase A contained formic acid, 0.1% aqueous solution, while solution B was a methanolic 0.1% formic acid solution. The gradient elution program started with 100% A: 0–2 min, from 100% A to 98% A, 2% B at 400 µL/min; 2–5 min, from 98% A, 2% B to 50% A, 50% B at 300 µL/min; 5–17 min from 50% A, 50% B to 2% A, 98% B at a 300 µL/min; 17–18 min, from 2% A, 98% B to 98% A, 2% B at 400 µL/min; 18–20 min to 100% A.Mass spectra were recorded in the negative ionization mode in the 100–800 m/z range, at 70,000 resolution. Nitrogen was used as collision, sheath, and auxiliary gas at 11–48 arbitrary units flow rates. The spray voltage was 2.5 kV and the capillary temperature 320 °C. Energy of the Collision-induced Dissociation cell was varied in the 30–60 eV range. Data were acquired and analysed with the Thermo Xcalibur software package (Version 4.1)., together with other measurements quality information.Calibrations were carried out in the 50–1750 μg/L concentration range, by serial dilution of the 10 mg/L methanolic standard mix. Calibration curves parameters are available in Supplementary Table Propolis extracts were filtered through a 0.45 μm polytetrafluoroethylene membrane and diluted before injection into the UHPLC-MS system. Typical chromatograms recorded are presented in Supplementary Figure + and DPPH, Trolox being model compound8. ABTS assay was carried out 734 nm, against a reagent blank in water, while DPPH assay was performed at 515 nm41.Extracts were tested for antioxidant capacity by scavenging the long-lived free radicals ABTS–3 μg/mL slope, 0.258 ± 0.009 μg/mL intercept, 0.9956 coefficient of determination, and 0.008 μg/mL standard error of response. Bias did not exceed 5.4%, with 94.9–103.5% recovery for the investigated concentration range.Calibration curve parameters for ABTS assay were: 5.76 ± 0.07 μg/mL slope, 3.76 ± 0.63 μg/mL intercept, 0.9959 coefficient of determination, and 2.07 μg/mL standard error of response. Bias did not exceed 3.8%, with 95.5–101.7% recovery for the investigated concentration range. Calibration curve parameters for DPPH assay were: (− 7.3 ± 0.5) × 10Propolis extracts were diluted prior reacting with the free radicals’ solution. The antioxidant capacity was calculated, irrespective of the method at hand, as equivalent concentration of Trolox from the calibration curves, namely Trolox Equivalent Antioxidant Capacity (TEAC), and reported as μg TEAC/mL.−), Escherichia coli (K12-MG1655) - E. coli, Gram-positive bacteria (G+), Bacillus subtilis spizizenii nakamura (ATCC 6633) - B. subtilis, and yeast, Candida albicans  - C. albicans. The bacterial strains were cultured on NA at 37 °C for 24 h. Bacterial inocula were prepared in sterile saline solution, 0.85% (w/v) NaCl, with the cell density corresponding to 0.5 McFarland (~ 108 CFU/mL). Yeast was cultured on YDP at 28 °C for 48 h. The yeast inoculum was prepared in a sterile solution, 0.85% (w/v) NaCl, to reach an approximately 106 CFU/mL population, using a hemacytometer.Strains used in the tests included Gram-negative bacteria ). The plates with bacteria were incubated at 37 °C for 24 h, and with yeast at 28 °C for 48 h. The diameter of inhibition zones  around the discs was measured after incubation. The inhibition zones were calculated as differences between the measured diameters of the clear zones and the disc diameter (standard), with the solvent inhibition zone subtraction. All tests were performed in triplicate. The three concentrations obtained for different liquid: solid ratios were used to determine the minimum inhibitory concentration  for the extractions performed at different time intervals. The inoculum suspensions of the used strains were distributed into a 96-well microtiter plate, containing two-fold serial dilution of samples. MIC value was established as the lowest extract concentration which inhibited bacterial or fungal growth after incubation at optimal temperaturecp, for which the antioxidant capacity, Qa, attains saturation.Qa is the antioxidant capacity , while cp is the polyphenolic derivatives concentration (μg/mL). The model parameters are Kmax, (μg Trolox /mL), the extract antioxidant potential at theoretically very high cp values (cp → ∝), and Kc (μg/mL), the critical concentration . The model parameters were identified minimizing the objective function (2):n being the number of samples considered.Statistical modelling was applied to investigate the correlation between the extracts’ polyphenolics content and the antioxidant capacity. A saturation type model Eq.  was assuThe saturation model regression against the experimental data was performed for correlating the concentration of chemical compounds identified in each extraction solvent with the corresponding antioxidant capacity of the extract.Statistical analysis also correlated individual polyphenolics concentration in the extracts with the corresponding antioxidant capacity. Therefore, the link between the antioxidant capacity and the polyphenolic composition identifies the chemical compounds synergistically involved in the antioxidant capacity.43. PLS is used when the predictors number is larger than the samples number, as in spectral data correlation44. These components included, in decreasing order, the variability between samples in terms of polyphenolics composition and antioxidant capacity. The “variable influence on projection” known as “VIP” was used to identify the bioactive principles significantly influencing the antioxidant capacity variation. A VIP score summarizes the contribution of each variable to the regression model. Variables with VIP values larger than 1 are generally considered relevant. The VIP score, defined for each variable (extracted bioactive principle), was calculated as a sum over its PLS components’ loadings weighted by the percentage of explained Y variance by each PLS component45.Data correlation was performed by partial least squares (PLS), a combination of Principal Component Analysis (PCA) and regressionThe extraction procedure involved small liquid: solid (w/w) ratios as to yield extracts more concentrated in bioactive compounds, eliminating the necessity of concentrating more dilute hydroethanolic solutions. The largest ratio employed was 6:1, significantly lower than the usual values reported in literature.46. Data collected, with relative standard deviations below 5%, identified bioactive principles with average content lower than 100 μg/g, below 1 mg/g, and a major group exceeding 1 mg/g. Total extracted phenolic acids and flavonoids quantified by the UHPLC-MS method are presented in Fig. 21 polyphenolics derivatives were quantified in the 36 extracts prepared and analysed, most also present in other papers on Romanian propolisWater and 25% ethanol solubilize on average 2.78 and 3.29 mg/g polyphenolics respectively, since the saturation concentrations are attained faster, and, thus, the mass driving force ceases to exist. The concentration of aqueous and ethanolic solutions increased linearly with the extraction time, for the 2:1 ratio, the ethanol influence being negligible. When the liquid phase volume increased, the concentrations of the extracts slightly decreased, almost always having a maximum after three days contact time. This could be an effect of the dichotomy between the availability of the active compounds from the solid matrix and the extracts’ degradation. In these cases, the presence of ethanol improved extraction, but not significantly for phenolic acids , while in 25% ethanol is ferulic acid (1.5 mg/g). The trend is less clear cut for 50% ethanol, since ferulic and p-coumaric acids part the supremacy with chrysin. 70% ethanol extracts chrysin most in all tests.The best extracted compound in water is 6. A later focus on Transylvanian propolis reveals levels in the 0.55–3.91 mg/g range39, still lower than the content extracted from the Bihor county-originating product investigated here.Chrysin, the reference flavonoid in poplar propolis, amounts on average to 12.4 mg/g in 50 -70% ethanol, a value larger than 1.6 mg/g propolis reported for the Romanian propolisFor a given solvent composition, the ratio between the major and minor extracted components was not significantly affected by the liquid: solid ratio, as shown in Fig. 48, was absent in water, present in traces in 25% ethanol, raising to 4.2 mg/g in 50% ethanol, and 4.9 mg/g in 70% ethanol. Some propolis specimens subjected to 100:1 (solid: liquid) extraction in 96% ethanol revealed levels in the 0.86–3.87 mg/g range39; local and seasonal variation in propolis composition may explain these differences.CAPE, an established anti-tumoral agent35, represent 27.8% and 29.8% of extracted compounds in the 50% and 70% ethanol solution and in negligible amounts in the higher water-containing solvents. Supplementary Table Galangin and chrysin, also known for anti-tumoral effects46 also noticed. They extracted 5.6 mg/g in cold 20% ethanol at 20:1 liquid: solid ratio. The present study reports only 0.7 mg/g in 25% ethanol at 6:1 liquid: solid ratio and 4.0 mg/g in 50% ethanol, a value closer to the published 6.34 mg/g in 60% ethanol46. It decreases in 96% ethanol to 2.45 mg/g46, a trend spotted for the 70% ethanol extracts in the present study (3.58 mg/g). This is in line with the 3.4 mg/g caffeic acid content found in some Italian propolis5.Caffeic acid levels depend on the extraction conditions, as Coneac et al38 found higher levels of pinocembrin, CAPE, galangin, and chrysin in propolis from the neighbouring Arad County; only the p-coumaric acids levels are somewhat similar, 12.68 mg/g (Arad) to 9.6 mg/g (Bihor-present study). p-Coumaric acid was found in higher amounts in Suceava propolis10, accompanied by levels of galangin, pinocembrin, and quercetin comparable to the present study.Gatea et al.The antioxidant effects are imparted by all analytes quantified in this study. The registered antioxidant effects in aqueous type solvents, determined by ABTS quenching, are induced by acids and their esters, as flavonoids are present in negligible amounts Fig. . Larger 38.After unit conversion, the antioxidant capacity average (11.47 mM TEAC/g in 70% ethanol) is in line with the 10.3 mM TEAC/g reported for samples collected in a neighbouring county in Romania, Arad, using ABTS method38 in Bihor and 1.05 mM TEAC/g40 in Cluj, Arad, and Alba neighbouring zones.The DPPH assay signals similar antioxidant capacity values for 50 and 70% ethanolic solutions Fig. . The 2,8j standing for the extract, and i for the component. The antioxidant capacity, as determined by the ABTS method, was used in real units.PLS statistical modelling gave good correlations between the 21 compounds identified in the extracts and the antioxidant capacity of aqueous and ethanolic solvents, with the aid of six PLS components for water and 25% ethanol, and 7 PLS components for 50% and 70% ethanol . Flavonoids with complex structures, such as chrysin, pinocembrin, and quercetin, credited with high antioxidant capacity2, are present in very small concentrations in water, but in 25% ethanol they have significant contributions in the differentiation of extract composition and antioxidant capacity. In more concentrated ethanolic solutions, the levels of chrysin, quercetin, pinocembrin or CAPE are sufficiently high, therefore their important contribution in the variation of antioxidant capacity is expected and confirmed.The compounds with the largest contribution in the correlation were selected for each specific solvent, based on their VIP score Table  online. ga function, for the minimization of objective function, Eq.  was tested first for the dependency of extracts antioxidant capacity on the total phenolic acids and flavonoids levels. The regression analysis over the experimental data was carried out in Matlab® R2015 , using genetic algorithms, implemented as The antioxidant capacity increases with the growth of total phenolics concentration, with a steep increase in the 25–50% ethanol region. This may be due to the increased contribution of flavonoids beginning with 50% ethanol. The model slope decreases drastically for 50–70% ethanol, reflecting the experimental values’ oscillations around 15,000 μg TEAC/mL. According to this model, the increase above 50% ethanol content in the extractant does not bring important changes in the antioxidant capacity. This agrees with the propolis-selling beekeepers’ practice; they use at most 40% alcoholic solutions (mainly 30%) to extract bioactive principles from propolis. Still, some intensification techniques could be beneficial, to increase the polyphenols and flavonoids content, thus, the antioxidant capacity.The antioxidant capacity of the extracts was also corelated with the concentration of some components which: (a) were present in larger amounts in the extracts, (b) proved to have important contribution in the PLS model . A dual mechanism52 explains its bactericidal activity: disruption of bacterial cell’s outer membranes and inhibition of cellular functions through binding to bacterial genomic DNA, ultimately leading to cell death. This activity is higher for G − bacteria53 (diderm bacteria with outer and inner membranes), much more susceptible to p-coumaric acid, due to their content of lipopolysaccharides, contrary to G + (monoderm bacteria), with lower liposaccharides content. The PLS analysis also identified the important contribution of p-coumaric acid to the antioxidant capacity.Table p-coumaric acid on Candida albicans is the result of its action upon the cell membrane and ergosterols synthesis54. Banskota et al27 were among the first to link the anti-fungal activity of ethanolic propolis extracts to pinocembrin, p-coumaric, and caffeic acids, and little other information on antifungal effects of water extracts is available. Even if the present aqueous extracts do not contain flavonoids, their proven anti-fungal effects are to be related to the presence of caffeic and p-coumaric acids. Data in Table The antifungal effect of B. subtilis (MIC < 805 μg/mL), along with the demonstrated significant contribution to the antioxidant capacity.Phenolics profile of 25% ethanol extracts is like the aqueous ones, except for the quercetin content. Quercetin levels could explain the increase in the antimicrobial effect upon the G + B. subtilis, in line with the reports of Mocanu et al55.The 50% ethanolic extracts contain, in addition to phenolic acids, large quantities of chrysin and galangin, flavonoids with already proven high antioxidant contribution. The high flavonoids levels seem responsible for the better reaction towards Candida spp., by repressing the biofilm formation57. The present experimental data indicates that the optimal concentration of phenolic acids and flavonoids is reached after 3 days extraction in 50% ethanol  does not bring further increase, neither in the antimicrobial, nor in the anti-fungal activity. Quercetin, chrysin, and pinocembrin, in the company of comparable levels of ferulic and The antimicrobial and anti-fungal investigation of the propolis extracts demonstrated that 70% ethanol does not bring advantages compared to the 50% ethanol extracts, a conclusion similar in terms of antioxidant capacity.Antimicrobial activity enhances the results of antioxidant capacity, proving the tight bound and effect displayed by the composition of different types of Romanian propolis extracts.The profile of phenolic acids and flavonoids extracted from propolis proved determinant for both the antioxidant and antimicrobial activity. Its characteristics are regulated by operational parameters. Contribution of phenolic acids to antioxidant capacity is present in all studied extracts. Higher ethanol concentration asymptotically favoured the extraction of flavonoids, which, in turn, increased free radicals scavenging ability and activity against the G + and yeast strains used.Bioactive principles in high concentrations are responsible for the registered antioxidant capacity in 50 and 70% ethanolic extracts and the inhibitory effect on the growth of all tested strains, highest in 50% ethanolic extracts.Statistical modelling results gave important insight into the relation between the polyphenolic profile and antioxidant capacity, in good agreement with theoretical considerations.Supplementary Information.


A flattening of the oxygen uptake–work rate relationship at severe exercise indicates the achievement of maximum oxygen uptake  Based on this finding the authors [−1 or 2.1 mL min−1 kg−1: “…there is small chance of making an error in deciding that the maximal oxygen intake had been reached”.The first quantitative approach for the diagnosis of the r et al. . They pe−1 the mean difference in work rate between adjacent 30 s sampling intervals is 15 W leading to a mean increase in −1 [−1 cut-off by Taylor et al. [The cut-offs of Taylor et al.  are stiliagnoses , 25, desing bout , 38. Insing bout  cut-offsresponse , 39–41. response  and testrotocols . For exas or not , 43–45. mL min−1 , 46. Herr et al.  may leadi et al.  and Marsommended , 38. Howi et al. . Based o2, may remain undetected , which means that several slopes were fitted into different 30 W intervals in the submaximal intensity domain to check for the rate/risk of false-positive diagnoses. 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Infinition , 51) repd BLCmax , 51, 109finition , 19, 49)finition , 110–112It is widely believed that children are less likely to demonstrate a entclass1pt{minimao adults , 113. Tho adults , 110, whr et al. . As descr et al.  is causeiagnoses , 52. Comiagnoses , 49–52 ts in age , 115. Acs in age  there wa in V̇E)  or fixed in V̇E) , 114. AsBeside of age, several studies observed the effect of height, body mass, body mass index, and sex on the probability of the e adults . All othncidence , 117. De unclear , 117, th2pl occurrence are not correlated with 2pl [2pl incidences after a blood donation compared to the pre-blood donation test. Despite some of these studies used It is partly believed that (endurance) trained athletes are more likely to show a plateau at entclass1pt{minima}V˙O2max , 118. Th}V˙O2max . Howevericipants . This stmentclasspt{minima cohorts , 43, 110a V̇O2pl , 50. Fura V̇O2pl  found a mentclasspt{minimavalidity , 45, 110Since energy demand above capacity , 112. Thcurrence , 119–122ate-test , 122. Ho}V˙O2max , 124. Co}V˙O2max , 122. Thexercise –127.Based on these findings Gordon et al.  firstly ent}V˙O2 , 129. Thachieved , 128–133However, both studies , 134 usea et al.  did not The ability to sustain high-intensity exercise for a long duration is additionally determined by kinetics , 136. \dntensity . The fasexercise . Consequexercise –143. Furkinetics , 81.2-kinetics. As schematically shown in Fig. The effect of recently . This stamp test .Fig. 6ThSince y domain , 86, thimp tests . Howeveriagnoses , 144. FuIn conclusion, evidence from a comparison of participants with and without a exercise  or dietaexercise .Another factor that has been considered as a major determinant of the ability to sustain high intensity exercise is the relation of the work rate at which lactate begins to accumulate and the work rate that elicits }V˙O2max –149. Thi}V˙O2max .−1 lactate threshold occurred at a significantly higher percentage of Lacour et al.  analysedt}V˙O2pl .2 deficiency or loss of homeostasis [Based on low s Noakes , 24, 33 eostasis . The exteostasis , 153.Nevertheless, Noakes , 24, 33 2-deficit from the beginning of an incremental exercise test due to a lagging behaviour of However, the assumption that the absence of a ent}V˙O2 , 132. Fuent}V˙O2 , 154–156abolites , 148. ThAs described in the introduction section one can be certain that Nevertheless, even if ertainty , 19. Furrdiology , 158. Thentclass1pt{minimaexertion , 25. Ther et al.  and Wagnr et al. , 75 and r et al. , 62, 99.excluded . Anotherulations , 99, butulations . HoweverBased on this and the previous sections we recommend the following approach: For most studies in which criteria , 19. Forulations , 99 In cad tests –97.A substantial fraction of studies used inappropriate Subsequent studies should take more attention to the use of valid


To investigate the association between brachial‐ankle pulse wave velocity (baPWV) and cardiovascular and cerebrovascular disease (CVD) in different age groups.A total of 39 417 people, receiving Kailuan physical examination, completing baPWV examination from 2010 to 2017, with no history of CVD and atrial fibrillation, were selected as the observation objects. The population was categorized into one age group per 10 years, namely the <50, 50–59, 60–69, 70–79, and ≥80‐year‐old groups, and the total population, and each group was further assigned into three classes according to the triple quartiles of baPWV. Kaplan–Meier method helped to calculate the cumulative incidence of CVD in different age groups. The effect of baPWV on CVD in different age groups was evaluated using the Cox proportional hazards regression model.p < .05) in the cumulative incidence of CVD among the whole population, <50, 50–59, and 60–69‐year‐old groups, while the cumulative incidence of end‐point events among the baPWV subgroups of 70–79 and ≥80‐year‐old groups exhibited no statistical significance (p > .05). Compared with baPWV in the Q1 group, hazard ratio value  of CVD in the Q3 group was 4.14 (95% CI: 2.98–5.75) in the total population, 2.98 (95% CI: 1.08–8.21) in <50‐year‐old population, 4.49 (95% CI: 2.89–7.00) in 50–59‐year‐old population, 2.78 (95% CI: 1.76–4.39) in 60–69‐year‐old population, 1.39 (95% CI: 0.86–2.24) in 70–79‐year‐old population, and 1.15 (95% CI: 0.55–2.41) in ≥80‐year‐old population.Kaplan–Meier survival curve indicated statistical significance (CVD risk attributed to increased arterial stiffness reduces with age. Thereafter, the second, third, fourth, fifth, and sixth health check‐ups were conducted in 2008–2009, 2010–2011, 2012–2013, 2014–2015, and 2016–2017, respectively. Some of the observation subjects underwent baPWV determination at the third, fourth, fifth, and sixth health check‐ups. This present study included those candidates who completed the concurrent health inspection and baPWV determination for analyses. Inclusion criteria: (i) those who underwent Kailuan physical examination in 2010–2011, 2012–2013, 2014–2015, and 2016–2017; (ii) those who completed baPWV examination at the same time; (iii) those who consented to participate in this study. Exclusion criteria: (i) those with severe physical disabilities and failed to undergo the examination; (ii) those who did not agree to participate in this study; (iii) those with a previous history of CVD, atrial fibrillation, and those with incomplete data of blood pressure. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Kailuan General Hospital.2.22. Smoking was defined as an average of ≥1 cigarette/day in the last year. Physical activity was defined as exercise ≥3 times per week, each lasting ≥30 min.Epidemiological investigations and biochemical and anthropological measurements were detailed in the published literature.2.3We collected baPWV values using a BP‐203 RPE III networked arterial stiffness detection device produced by Omron Health Medical Co., Ltd.2.4After the completion of baPWV determination, that is, the starting point of follow‐up, trained medical staff reviewed the inpatient diagnosis and recorded the end‐point events of the observation objects in the Affiliated Hospitals of Kailuan Group and the Designated Hospitals for Medical and Health Insurance of China every year. The end‐point events were defined as CVD during the follow‐up, including stroke and myocardial infarction . Based on the inpatient medical records, professional doctors confirmed all diagnoses. The time and event of the first event were considered as the end‐point for those with ≥2 events, and the final follow‐up date for those without events was December 31, 2019.2.5χ2 test was performed for the intergroup comparison. The incidence of CVD and stroke in the whole population and different age groups of baPWV was computed by the Kaplan–Meier method. The log‐rank test was utilized to compare the differences of CVD and stroke between the whole population and different age groups of baPWV. Meanwhile, the Cox regression multiplication model was applied to observe the interaction between age‐based groups and baPWV tertiles. Furthermore, the present study also calculated the hazard ratio (HR) and 95% confidence interval (CI) of CVD in the whole population and different age groups (with the first percentile as the control), and the HR and 95% CI of CVD in baPWV increased by 1 SD. Owing to the high risk of mortality on cardiovascular events, the death competitive risk models were explored for patients over 70 years old. For the sensitivity analysis, a Cox regression analysis of the effect of baPWV on CVD was done after excluding study subjects with an ankle‐brachial index (ABI) < 0.9 to avoid a low (ABI) that might affect baPWV values. All data were analyzed statistically with the help of the SAS 9.4 software, and p < .05 was considered a statistically significant difference (two‐sided).The measured data followed the normal distribution and were expressed as mean ± SD and a one‐way analysis of variance was adopted for the intergroup comparison. The measurement data of nonnormal distribution were represented as M, and the study groups were compared by Kruskal–Wallis rank‐sum test. Frequency and percentage were employed to express classified variables, and the 33.1p < .05; Table A total of 41 121 people who received physical examination for baPWV determination in Kailuan General Hospital from 2010 to 2017 were considered as research objects. The present study collectively excluded 523 cases with a previous history of CVD, 226 with a history of atrial fibrillation, and 955 with incomplete blood pressure data, and finally included 39 417 cases for statistical analyses. The study population encompassed 28 571 (72.48%) males and 10 846 (27.52%) females, with a mean age of 51.65 ± 13.28 years and a mean baPWV of 1602.43 ± 362.70 cm/s. The enrolled subjects were classified into three groups by trichotomies based on baPWV, and statistically significant differences were observed in age, gender, baPWV, SBP, DBP, MAP, triglyceride, high sensitivity C reactive protein, total cholesterol, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, fasting blood glucose, BMI, hypertension, smoking habit, physical exercise, diabetes, and antihypertensive medication among the three groups (3.2p < .05). Kaplan–Meier survival analysis revealed that the cumulative incidence of CVD was 0.19%, 0.56%, and 3.22% in the subgroup <50‐year‐old from Q1 to Q3 group, 1.30%, 3.80%, and 9.56% in 50–59‐year‐old group, 3.82%, 7.74%, and 11.47% in 60–69‐year‐old group, 11.93%, 10.80%, and 12.28% in 70–79‐year‐old group, and 7.58%, 10.39%, and 11.74% in the subgroup ≥80‐year‐old, respectively. Based on the log‐rank test, the cumulative incidence of end‐point events exhibited statistically significant difference among the subgroups of <50, 50–59, and 60–69‐year old groups (p < .05), while the difference was statistically insignificant in the cumulative incidence of end‐point events among the subgroups of 70–79 and ≥80‐year old groups . Compared to baPWV in the Q1 group, the HR values (95% CI) of CVD in the Q3 group were 4.14 (95% CI: 2.98–5.75), 2.98 (95% CI: 1.08–8.21), 4.49 (95% CI: 2.89–7.00), 2.78 (95% CI: 1.76–4.39), 1.39 (95% CI: 0.86–2.24), and 1.15 (95% CI: 0.55–2.41) in the total population, <50‐year‐old population, 50–59‐year‐old population, 60–69‐year‐old population, 70–79‐year‐old population, and ≥80‐year‐old population, respectively. Relative to the Q1 group, the HR values (95% CI) of stroke in the Q3 group were 4.22 (95% CI: 3.04–5.86) in the total population, 3.80 (95% CI: 3.04–5.86) in <50‐year‐old group, 4.54 (95% CI: 2.91–7.06) in the 50–59‐year‐old group, 3.13 (95% CI: 2.11–4.66) in the 60–69‐year‐old group, 1.61 (95% CI: 0.96–2.69) in the 70–79‐year‐old group, and 0.86 (95% CI: 0.38–1.92) in ≥80‐year‐old group. By every one increase of SD of baPWV, the HR values (95% CI) of CVD were 1.30 (95% CI: 1.25–1.36), 1.44 (95% CI: 1.22–1.70), 1.21 (95% CI: 1.08–1.36), 1.32 (95% CI: 1.14–1.53), 1.08 (0.97–1.20), and 1.13 (95% CI: 0.77–1.66) in the total population, <50, 50–59, 60–69, 70–79, and ≥80‐year‐old groups, respectively, whereas the HR values (95% CI) of stroke events were 1.28 (95% CI:1.23–1.34), 1.40 (95% CI: 1.21–1.68), 1.26 (95% CI: 1.21–1.31), 1.24 (95% CI: 1.10–1.41), 1.27 (95% CI: 1.08–1.47), and 0.92 (95% CI: 0.65–1.32), respectively  in the 70–79 and ≥80‐year‐old groups  or baPWV.The presence of lower limb arterial occlusive lesions contributed to the inaccuracy in the measured baPWV values.The present study concluded that increased arterial stiffness‐induced risk of CVD and stroke decreased or even disappeared with age, but the mechanism perplexed us. Younger individuals exhibit fewer risk factors for CVD compared to older individuals, and the increased arterial stiffness, as a single risk factor, contributes more to CVD and stroke than older individuals, whereas older individuals have more cardiovascular risk factors and the synergistic effect of multiple risk factors may diminish the risk effect of single arterial stiffness. Another possible mechanism is the so‐called “ceiling” effect.Our findings emphasized higher risk due to arterial stiffness in younger individuals, which is independent of traditional risk factors. These results significantly claimed the potential of baPWV determination as a screening method for younger individuals at risk. The geriatric population should also undergo routine baPWV testing. Prolonged exposure to multiple cardiovascular risk factors results in increased arterial stiffness, justifying the necessity for the regulation of multiple risk factors in older individuals with increased arterial stiffness. Moreover, in the absence of effective drugs to improve arterial stiffness, it is thus imperative to maintain a healthy lifestyle and control risk factors, including hypertension, at the individual or group level to reduce or delay the progression of arterial stiffness.5Despite the large sample size observed in this study and the adjustment for various confounding factors to ensure the reliability of the results, there are several limitations to our study. First, a larger proportion of males observed in this study thus indicated the presence of selection bias. Second, arterial stiffness was measured using baPWV rather than the gold standard cfPWV.6The arterial stiffness is an independent risk factor for compound cardiovascular events, however, the risk of CVD due to increased arterial stiffness tended to decrease with increasing age.The authors declare that there are no conflict of interests.Supplementary information.Click here for additional data file.


Consumption of maize posed adverse health effects in all age categories of the locations studied since the calculated MOE values were less than 10,000.Mycotoxin contamination of foodstuffs is a serious food safety concern globally as the prolonged ingestion of these toxins has the tendency to worsen the risk of hepatocellular carcinoma. This study aimed at estimating ochratoxin A (OTA) and aflatoxin (AF) levels above international  and local  standards as well as the health risks associated with the consumption of maize (n = 180) sampled from six (6) regions representing the agro-ecological zones of Ghana. OTA and AF were measured with High-Performance Liquid Chromatography with a Fluorescence detector. Out of the 180 samples analyzed for total aflatoxins , 131/180 tested positive and 127 (70.50%) exceeded the limits of EFSA and ranged 4.27–441.02 µg/kg. While for GSA, 116 (64.44%) of samples exceeded this limit and ranged between 10.18 and 441.02 µg/kg. For OTA, 103/180 tested positive and 94 (52.22%) of samples between the range 4.00–97.51 µg/kg exceeded the tolerable limit of EFSA, whereas 89 (49.44%) and were in the range of 3.30–97.51 µg/kg exceeded the limits of GSA. Risk assessment values for total aflatoxins  ranged between 50 and 1150 ng/kg bw/day, 0.4–6.67, 0–0.0323 aflatoxins ng/kg bw/day and 1.62–37.15 cases/100,000 person/year for Estimated Daily Intake (EDI), Margin of Exposure (MOE), Average Potency, and Cancer Risks respectively. Likewise, ochratoxin (OTA) values were in the ranges of 8.6 × 10 Zea mays L), a principal cereal extensively consumed1 around the globe is exceedingly prone to fungal infection by many toxigenic fungal species. This subsequently leads to mycotoxins  production3 due to its ideal nutrient composition4. This situation is particularly distressing since maize (Zea mays) accounts for 40% of the cereal production in Sub-Saharan Africa (SSA), where more than 80% is used as food5. The crop provides at least 30% of the total calorie intake of people in Sub-Saharan Africa. Maize is consumed as a staple in the African region where intake ranges from 52 to 450 g/person/day6 and so is an easy channel for mycotoxin contamination. Mycotoxins, which are natural toxins from fungi, contaminate maize grains and render them potentially dangerous. Mycotoxins represent one of the main global foodborne risks for human health, and are considered an important issue in the situation of food safety, due to their acute and chronic toxic effects on animals and humans8. Noteworthy, mycotoxins of maize include aflatoxins (AFs), ochratoxin A (OTA), and Fumonisins (FM)11. Attendant contamination could either be single or occur in multiple combinations with other mycotoxins. Ochratoxins are fungal metabolites produced from the growth of some notable species including Aspergillus ochraceus, A. carbonarius, A. niger, and Penicillium verrucosum. Ochratoxins occur in A, B, C, and D types, of which the most common and toxic type is Ochratoxin A (OTA). OTA is assumed to be one of the five most important mycotoxins in agriculture12. Although not well known in Africa and the world at large compared to aflatoxins, ochratoxins are described as one of the most commonly occurring, mycotoxins in the world due to their presence in a wide variety of foodstuffs such as potatoes, pulses, nuts, spices coffee, cacao, beer, and wine10. Nonetheless, notable among the main foodstuffs are cereals and cereal products, which constitute 60% of its total exposure to man and animals according to13 (JECFA).Maize  is aptly classified as a possible human carcinogen (2B agent) according to the International Agency for Research on Cancer (IARC)20. Aflatoxins are also fungal metabolites produced by strains of Aspergillus flavus and A. parasiticus. They are by and large the most well-known mycotoxins owing to their persistence in the environment and the ubiquitous nature of their contaminants. Aflatoxins occur in five different types; aflatoxins B1, B2, G1, G2, and M1 produced primarily in cow milk by cow-eating contaminated silage. Prolonged ingestion of aflatoxins has been reported to cause impaired immune function, hepatotoxicity, neurotoxicity, malnutrition, and stunted growth in children, teratogenic, mutagenic disabilities, and eventual death22. The International Agency for Research on Cancer (IARC) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) named it as a class 1 carcinogen due to its high potency24.The toxicological profile of OTA has been investigated and reviewed expansively in numerous studies25. These permissible levels are almost excruciating especially in developing countries in Sub-Saharan Africa. Bankole and Adebanjo26 noted when the worldwide directive of mycotoxins in food is under consideration, the impact seems to be greater on low to medium-income countries. It was projected that enforcement of strict regulations regarding aflatoxin contamination by the European Union would result in the rejection of 64% of imports of cereals, dried fruits, and nuts from African countries with an estimated trade loss per year of approximately US $670 million26. In addition to setting regulatory limits for mycotoxins, it is also imperative to conduct health risk assessments in the population due to dietary exposure. A low-dose extrapolation approach introduced by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1997 and the margin of exposure (MOE) method proposed at the 64th JECFA meeting in 200527 were both recommended and have been widely used worldwide29 to assess the risk of dietary exposure to mycotoxins.The evidence that mycotoxins can have adverse health effects on humans and animals has led many countries to set up standards for maximum levels of total aflatoxins and ochratoxins in food products intended for both human and animal consumptionThere is a paucity of data on the combined occurrence of ochratoxin A and aflatoxin contamination in foodstuffs in Ghana. Previous works by national institutions such as the Food and Drugs Authority (FDA), Ghana, Ghana Standards Authority (GSA), and Council for Scientific and Industrial Research (CSIR), Ghana have conducted surveillance programmes to monitor contamination by only aflatoxins in foodstuffs. To the best of our knowledge, not a single work is published on the exposure assessment of the co-occurrence of OTA and AFs as well as their risk characterization. This study is the only study in Ghana that assessed the levels, exposure and risk characterization of ochratoxin A and aflatoxins through the consumption of maize in different age group samples from six (6) different regions representative of the different agro-ecological zones of Ghana by using MOE and quantitative liver cancer risk approaches.30.To collect a representative data set, we first obtained the list of villages in each district from the Regional Directorate of the Ministry of Agriculture. From each district, an average of 5 villages Table  was then33).The analytical standard of OTA was supplied by Sigma-Aldrich . All solvents used for the preparation of the mobile phase were HPLC grade and obtained from Merck . Methanol and hexane used for extraction were of analytical grade supplied by Sigma-Aldrich. All homogenized mixtures and eluates were filtered through Whatman no. 4 and 0.45 mm membrane filters respectively . De-ionized water was obtained with a Millipore Elix Essential purification system . OCHRA PREP immunoaffinity columns were supplied by R-Biopharm, Rhone limited, and used for SPE and cleanup. These columns have a concentration capacity of 100 ng/mL with at least 90% recovery. Phosphate-buffered saline (PBS) was prepared by dissolving PBS tablets (Sigma-Aldrich) in distilled water. Sodium chloride (≥ 99.0%) was sourced from Sigma-Aldrich. Six-point calibration was made using the pure Ochratoxin A standard at concentrations of 5 µg/kg, 10 µg/kg, 15 µg/kg, 20 µg/kg, 25 µg/kg and 30 µg/kg. Linearity was accepted at 0.99 or 99% for the regression curve  was added to the groundnut mixture and the samples homogenized for 3 min . The groundnut mixture generated two organic layers (the hexane upper layer and methanol lower layer). The lower methanol layer of the groundnut mixture and the maize mixture was filtered through Whatman number 4 filter paper. Ten milliliters (10 mL) of filtrate were used for Ochratoxin A solid-phase extraction and cleanup. Exactly 150 mL of phosphate buffer saline (PBS) was added to 10 mL of filtrate and the mixture stirred. Immuno-affinity columns specific for Ochratoxin A were pre-conditioned and antibodies in the column were activated by eluting 10 mL of phosphate buffer saline through columns at a flow speed of 3 mL/min. Exactly 50 mL of the 160 mL filtrate-PBS mixture was loaded onto pre-conditioned immune-affinity columns specific for Ochratoxin A and allowed to drain by gravity. The columns were washed three times with 5 mL PBS and allowed to elute at a flow rate of 5 mL/min. Using a vacuum pump, the air was blown through the columns to get rid of all wash solvent molecules. Ochratoxin A was eluted in two steps into a 5 mL volumetric flask by first eluting with 1 mL of methanol (highest grade) followed by another 1 mL of methanol after one minute. Air was blown through the column to collect all eluates. Aqueous acetic acid (1%) was used to make up the volume of eluate to 4 mL and eluate vortexed, after which 2 mL was pipetted into HPLC vials for quantification.Ochratoxin A was determined based on CEN official method EN14123 (2007)33).Agilent high-performance liquid chromatography system (HPLC 1260 infinity series) with a quaternary pump and fluorescence detection was used for OTA quantification. Data acquisition and quantification were done using Chem station (Open Lab edition). The Agilent HPLC equipped with a fluorescence detector was set at an excitation wavelength of 333 nm and an emission wavelength of 467 nm and the column compartment temperature regulated at 30 °C. The mobile phase was a mixture of 5 mM sodium acetate with acetic acid (pH 2.4): methanol: acetonitrile at ratios of 40:30:30, respectively, and an isocratic delivery mode employed at a flow rate of 1 mL/min with an injection volume of 10 µl. The run time was set at 10 min  official method EN1412333 for aflatoxin extraction. Methanol in water (200 mL) (8 + 2) and 5 g NaCl were used to extract 25 g of sample. Hexane (100 mL) was added to samples containing more than 50% fat. The mixture was homogenized for 3 min at 3000 rpm (2 min) and 3500 rpm (1 min). The extracts were filtered and 10 mL of the filtrate added to 60 mL of phosphate buffer saline (PBS) for solid-phase extraction using a preconditioned immune-affinity column specific for, AFB1, AFB2, AFG1, and AFG2. The 70 mL filtrate-PBS mixture was loaded onto the preconditioned column and allowed to elute by gravity at a flow rate of 1 mL/min. This was followed by a cleanup with 15 mL distilled water at a flow rate of 5 mL/min. Aflatoxins were eluted in two steps into a 5 mL volumetric flask with 0.5 mL followed by 0.75 mL of methanol (HPLC grade) and allowed to elute by gravity. Deionized water was used to make up the volume of eluate to 5 mL and eluate vortexed and 2 mL pipetted into HPLC vials for quantification.AFB(4.6 mm × 25 cm), 5 μm particle size, 250 A pore sizeLOD = Limit of detectionLOQ = Limit of quantificationACN = AcetonitrileMeOH = MethanolLOD calculation = 3 * standard deviation/slopeLOQ calculation = 3 × LODSupplier of Column R- Biopharm, Block 10 campus, West ScotlandScience Park, Acre Road, Glasgow, Scotland G20 OXAInjection volume: 10 μl flow rate: 1 mL/min, column temperature: 35 ℃, excitation wavelength: 360 nm, emission wavelength: 440 nm, mobile phase composition: water/acetonitrile/MeOH (65:15:20 v/v/v), post-column derivatization: Kobra cells. HPLC Column Specification Spherisorb ODS1- ExcelAspergillus flavus and A. parasiticus) levels in the samples were determined according to the CEN official method EN1412333 by High-Performance Liquid Chromatography HPLC  , USA with fluorescence detector and post-column derivatization using Kobra cells to generate bromine electrochemically at the CSIR- Food Research Institute, Ghana.The aflatoxins  of the HPLC were estimated by making a calibration curve around the standard used for spiking, 5 µ/kg . Blank did not produce any signal, so the LOD and LOQ were calculated as;Spiking of pure aflatoxin standard solution was done to ensure the measurement accuracy of the analysis. Three levels of spiking were done at the lower, mid, and upper concentration range of the calibration curve concentrations . Spike volumes of pure standards were calculated as;Spike volumes were distributed evenly on aflatoxin free sample (blank) and the spiked sample analyzed for percentage recovery which was calculated as;33).Repeatability and intermediate precision analyses of an internal reference material (IRM) were used to ensure the measurement precision of the method. For repeatability analysis, 10 parallel extractions of the IRM were done by the same analyst at the same time using the same HPLC and the relative standard deviation between the results was calculated. For intermediate precision, 10 extractions of the IRM were done on different days by different analysts, and the relative standard deviation between the results was calculated. The relative standard deviations were calculated as; [Standard deviation/mean] * 100  and 0.98 (G2).LinearityIntermediate Precision: Relative standard deviation among results obtained under intermediate precision conditions should be less than 20%Repeatability: Relative standard deviation;B1 = 5.5%; B2 = 6.7%; G1 = 7.4%; G2 = 12.1% and Total aflatoxins = 5.2%.Intermediate Precision (Reproducibility): Relative standard deviation;B1 = 13.2%; B2 = 13.4%; G1 = 13.7%; G2 = 12.2% and Total aflatoxins = 11.9%.: Percent recovery of measurement procedure;RecoveryLow concentration: B1 = 107%; B2 = 87.2%; G1 = 113.4%; G2 = 112.8% and Total aflatoxins = 108.2%.High concentration: B1 = 102.6%; B2 = 101.6%; G1 = 104.2%; G2 = 104.4% and Total aflatoxins = 103.3%.Linearity: Linearity of the regression curve;33).B1 = 0.991; B2 = 0.997, G1 = 0.994; G2 = 0.995  was considered by using the mean quantities of mycotoxins (ochratoxins or aflatoxins) derived from the cereal samples, the number of samples consumed daily, and the average body weight. The EDI for mean aflatoxin was premeditated according to the following formula  and expr2 is approximately 0.107 kg/day (39.3 kg/year).The daily intake of maize in Ghana according to MOFA-IFPRI (2020) in Kortei et al.36 and their corresponding estimated average weights in Ghana used in this study were done as follows; Infants- 2.9 (2.5–3.2) kg38, Toddler- 9.8 (7–12.6) kg40 , Children- 26 (24–28) kg42, Adolescents- 46.25 (38.5–54) kg43 , Adults- 60.7 kg44.The different age categories according to EFSA45.Genotoxic compounds such as aflatoxins and ochratoxins have their risk assessments fittingly computed based on the Margin of Exposure (MOEs) approach, which was estimated by dividing the Benchmark dose lower limit (BMDL) for aflatoxins is 400 ng/kg bw/day by toxin exposure46 and 120 ng/kg bw/week47 resulting in an average of 125 ng/kg bw/week (17.86 ng/kg bw/day) by toxin exposure as expressed in Eq.  for ochratoxins—130 ng/kg bw/weekA public health alarm is raised in instances where MOEs are less than 10,000 for both ochratoxin A and aflatoxins.49. Therefore, liver cancer risk estimation for Ghanaian adult consumers was calculated for aflatoxins45. This involved estimating the population cancer risk per 100,000, which is a product of the EDI value and the average hepatocellular carcinoma (HCC) potency figure from individual potencies of Hepatitis B surface antigen (HBsAg) (HBsAg-positive and HBsAg-negative groups).The ingestion of aflatoxins, likewise ochratoxins, can be linked to the onset of liver cancer50 estimated potency values for AF/OTA which corresponded to 0.3 cancers/year/ 100,000 population/ng/kg bw/day (uncertainty range: 0.05–0.5) in HBsAg-positive individuals and 0.01 cancers/year/100,000 population/ng/kg bw/day (uncertainty range: 0.002–0.03) in HBsAg-negative individuals49 were adopted for this calculation. Moreover, the average HBsAg + prevalence rate of 7.74%  for Ghana52 was adopted and 92.26% (100–7.74%) was extrapolated for HBsAg-negative groups. Hence, the average potency for cancer in Ghana was estimated as follows according to Eq.  was estimated using the following formula in Eq. 2,45:8\doThe use of plants in the present study complies with international, national, and/or institutional guidelines.1, and Ochratoxins) were determined using Kendall’s tau_b correlation test.The ochratoxins and aflatoxin concentrations were calculated using regression analysis from the curves generated from the standards of ochratoxins/aflatoxins with Excel for Microsoft Windows (version 10). SPSS 22  was used in the analysis of data. Descriptive analysis was performed to describe the concentration of ochratoxins/aflatoxins in maize samples by using the mean ± standard deviation. Deterministic risk assessment model calculations for ochratoxins and aflatoxins dietary exposure (Estimated Dietary Intake), MOE values, Average potency, and cancer risk were calculated. The results are summarized as standard error, median, standard deviation, variance, skewness, standard error of skewness, kurtosis, and standard error of kurtosis and mean values (range from the 25th percentile to the 75th percentile). Associations of mycotoxins  within the tested range. For the recovery analysis, samples previously tested to guarantee the nonappearance of the studied mycotoxins were used in the validation procedure. For ochratoxins, the limit of detection was 0.83 µg/kg while the Limits of Detection for AFB1 and AFB2, likewise AFG1 and AFG2, ranged between 0.13 and 0.15 µg/kg. The limit of Quantification for ochratoxins was 2.49 µg/kg. Aflatoxins ranged between 0.39 and 0.45 µg/kg, respectively, for both  were in the range of 0–441.02 µg/kg. While for ochratoxins (OTA), 103 samples tested positive. OTA levels were also observed to be lesser than AFB1, AFB2 but more than AFG1 and AFG2 and ranged between 0 and 97.51 µg/kg. There were significant (p < 0.05) differences observed in all categories of the tested samples. For the Upper East region representing the Sudan Savanna zone  correlation between AFB1 and AFtotal in this data set (Table p < 0.01) correlations established between AFB1 and AFtotal as well as AFtotal and OTA (Table p < 0.01) correlations were established between AFB1 and AFtotal, and likewise AFtotal and OTA (Table p < 0.01) correlations were recorded between AFB1 and AFtotal, and likewise AFtotal and OTA (Table p < 0.01) correlated with AFB1 and OTA (Table 1 and OTA were found to be significant (p < 0.01)  Table .Figure 153 and Ghana Standards Authority (GSA)54 regulatory limits for total aflatoxins  used were 4 and 10 µg/kg respectively. While ochratoxins (OTA) limits used were 4 and 5 µg/kg, respectively, for the two institutions55  within the range of 4.88–62.50 µg/kg and 17 (56.67%) of the range 11.86–62.50 µg/kg tested positive for EFSA and GSA respectively. For Northern, 22/30 were positive with total aflatoxin values of 21 (70%) in the range 9.73–285.31 µg/kg. For OTA, 23/30 were positive with values of 18 (60%) which ranged between 4.70 and 46.77 µg/kg, and 17 (56.67%) of 3.30–46.77 µg/kg were recorded as positive for EFSA and GSA respectively. In the Ashanti Region, recorded 23/30 were positive with total aflatoxin values of 21 (70%) within the range of 7.98–230.00 µg/kg and 18 (60%) within the range of 19.40–230.00 µg/kg were recorded. OTA showed 10/30 positive, while 10 (33.33%) within the range of 5.80–76.05 µg/kg and 10 (33.33%) in range 5.80–76.05 µg/kg for EFSA and GSA. In the Eastern Region, 17/30 were positive and total aflatoxin values of 16 (53.3%) within a range of 4.72–441.02 µg/kg and 12 (40%) within a range of 10.70–441.02 µg/kg were recorded. OTA levels of 16/30 were positive and 15 (50%) with a range of 5.50–97.51 µg/kg were recorded for EFSA while 16 (53.33%) within a range of 5.50–97.51 µg/kg was recorded for GSA. The Central Region recorded 20/30 as positive, while total aflatoxin values of 20 (66.7%) within the range of 8.86–139.71 µg/kg and 19 (63.33%) within the range of 15.84–139.71 µg/kg were recorded. OTA showed 12/30 as positive while 11 (36.67%) within the range of 4.00–38.34 µg/kg and 8 (26.67%) in range 5.98–85.90 µg/kg for EFSA and GSA. Lastly, the Western Region recorded 24/30 positive samples and total aflatoxin values of 24 (80%) within the range of 4.27–217.41 µg/kg and 23 (76.67%) within the range of 10.18–217.41 µg/kg were recorded. OTA showed 22/30 positive and 22 (73.33%) within the range of 5.0–85.90 µg/kg and 21 (70.0%) in the range 5.0–85.90 µg/kg for EFSA and GSA respectively (Table The European Food Safety Authority (EFSA)55 Table  in this −1 bwday−1 and produced cancer risks of 19.70, 5.81, 4.52, 2.58 and 1.94 cases/100,000 person/year respectively (Table The Estimated Daily Intakes (EDI) of total aflatoxins in the maize samples from the Upper East Region were 610, 180, 140, 80, and 60 ng/kg bw/day for infants, toddlers, children, adolescents, and adults respectively. The Margin of Exposure (MOE) values recorded were 0.66, 2.22, 2.86, 5.00, and 6.67, respectively. The average potency of the aflatoxins was 0.0323 aflatoxins ngkgly Table . The Norly Table . For thely Table . In the ly Table . Lastly,ly Table .Table 16–3. Likewise, MOE values recorded were 0.20, 0.60, 0.89, 1.79, and 2059.97. The average potency was the same while Cancer Risks were 2.91, 0.97, 0.65, 0.32, and 2.78 × 10–4 cases/100,000 person/year (Table Ochratoxins A (OTA) in the maize samples obtained from various regions produced varied results. In the Upper East Region, the Estimated Daily Intake (EDI) values recorded were 340, 100, 70, 40, and 30 ng/kg bw/day for infants, toddlers, children, adolescents, and adults, respectively. The Margin of Exposure (MOE) values recorded were 0.05, 0.18, 0.26, 0.45, and 0.60, respectively, for the same age categories. The average potency of the ochratoxins was 17.86 ochratoxins ng/kg bw/day which produced cancer risks of 10.98, 3.23, 2.26, 1.29, and 0.97 cases/100,000 person/year respectively  of ochratoxins was obtained and were comparable to other previous studies globally. Nalle et al.57 reported a mean value of 20.385 μg/kg in corn grains and corn products from different sources in West Timor, Indonesia. Kamala et al.58 reported a range of 16–73 μg/kg OTA concentration in maize samples from Tanzania. Values of the ranges 0.12–0.59 μg/kg and 1.70–19.5 μg/kg have been reported by Kara et al.59 in cereal flour samples from Turkey and by De Giromalo et al.60 for cereals from Italy respectively, Again, Temba et al.61 also reported levels of 19.5 μg/kg. Veldman et al.62 demonstrated a total of 16.7% of maize samples originating from Holland contaminated by OTA with an average level of 73 μg/kg. From Brazil, Sekiyama et al.63 recorded levels between the range of 0–64 μg/kg. Greater quantities of ochratoxins have been reported in maize from other studies worldwide. Recently, ul Hassan et al. 64 reported ochratoxins (OTA) levels that ranged from 2.14 to 214 μg/kg which was detected in 71% of commercially grown maize samples in Pakistan. Makun et al.65 reported that samples of maize showed the highest levels of OTA were in the range of ND to 139.2 μg/kg in Nigeria. Conversely, Iram et al.66 did not detect ochratoxins in maize samples obtained from Punjab, Pakistan. Adebajo et al.67 also detected OTA in corn-based snacks in Nigeria only at toxicologically significant levels. The co-occurrence of different mycotoxins in one commodity created by fungal genera is common68. The permissible limit of OTA recognized by FAO/WHO Joint Committee Experts on Food Additives is 100 ng/kg/week and 14 ng/kg/day, whereas European Food Safety reestablished 120 ng/kg of OTA, which is nearly 17.1 ng/kg47. Ochratoxins have been implicated in a variety of adverse health effects both in humans and in animals suggested to be reached from renal, neuro-, immuno-, and embryo toxicity to muta- or teratogenicity69. OTA was proven as a renal carcinogen in rodents70, nevertheless its transferability to humans is still not clear71. Its carcinogenicity has been adopted to be related with genetic changes leading to a new assessment so that OTA can also be considered as genotoxic/mutagenic72. The high presence of OTA might be attributed to other fungal species that have not yet been explored or due to other pieces.The discrete incidence of ochratoxins and aflatoxins in foodstuffs is quite common in cereals and is a worldwide problem during pre-and post-harvest stages67 for values between the ranges 25–770 ppb, 15–1070 ppb, and 10–160 ppb for corn, corn cake, and corn roll snack respectively in Nigeria. Recently, Kortei et al.2 also reported aflatoxins levels of 0–445.01 μg/kg from maize samples obtained from different agro-ecological zones across Ghana. Aflatoxin quantities of 692 and 945 ng/g from maize samples obtained from Fumesua and Ejura in Ghana respectively by73. From Eastern and Central Kenya, Lewis et al.74 reported greater quantities of aflatoxin > 1000 ppb in maize samples from contamination due to aflatoxin of commercial maize products during an outbreak of acute aflatoxicosis. Dadzie et al.73 recorded Total aflatoxins levels in the samples per community were in the range of limit of detection (LOD) with 692 ng/g, 23 ng/g, 945 ng/g, and 112 ng/g for Fumesua, Wenchi, Ejura, and Akomadan, respectively. Lower aflatoxin levels have been reported in other studies. de Souza et al.75 reported quantities as low as 0–16 in Brazilian maize and maize-based foods. Total aflatoxin values of 50.234, 70.102, and 30.943 ng/g were, respectively, obtained from three composite samples taken from the Ejura market was reported by76. Danso et al.77 reported aflatoxin levels of 2.9–3.4 ppb in all markets in the minor season maize samples, but levels ranging from 38.2 to 64.0 ppb were found in the major season samples. Total aflatoxin levels of 82.9 ppb, 48.9 ppb, and 48.9 ppb were recorded for maize samples stored in polypropylene sack, hermetic bags, and local crib respectively by78.Aflatoxin level recorded in this study were in the range of 0–441.02 μg/kg, which is within the range of the values reported by Adebajo et al.Aspergillus (esp. flavus), owing to their ubiquitous nature, infect maize grains in the field even before harvest80. Ghana is reliant on rain-fed agriculture which is coupled with high temperatures and unavailability of regular rains, the crop is left under stress which predisposes the crop to fungal invasion81 during the growth cycle. This may explain the comparatively greater quantities of aflatoxins on maize obtained from the Easter Region since the region is endowed with sufficient rainfall and relatively high temperatures which induce aflatoxin production. Additionally, the different water activity levels of the grains due to the drying process used before and during storage could account for the different mycotoxin levels.The variation in contamination levels as seen in the different agro-ecological zones could be attributed to speckled infection levels of the toxigenic fungus genus, 1 and AFtotal in this study. These observations corroborate the findings of some previous researchers82. The co-existence of two or more fungi and their subsequent mycotoxin production in an environment suggests a possible non-antagonistic metabolite interaction, Moreover, the probable effect of combined exposure to aflatoxins with other mycotoxins in foodstuffs could be additive or antagonistic. At variance with our observation, Imperato et al.82 reported that among food products analyzed in Italy, dried vine fruits were mainly contaminated with OTA and less with AFs. Discovered from the pertinent literature, there is an inverse correlation between AFB1 and OTA. To buttress this claim83 observed, no AFB1 was found in dried vine fruits, while OTA was detected at high levels. In addition, Dimitrokallis et al.84 reported that OTA inhibits AFB1 production by Aspergillus species in a related study.There were significant correlations between AFtotal and OTA. Likewise, AFB85 as well Garcia et al.86 emphasized mycotoxin production depends on species or/and strain, which is affected by the growth substrate and environmental conditions. de Souza et al.75 and Kortei et al.88 also explained co-existence of fungal strains on a substrate can affect both the level of mycotoxin production and the toxicity of the contaminated grains resulting in additive and synergistic effects when tested Nonetheless, data on multiple mixtures is very rare87. In this study the relatively high aflatoxin (AF) and ochratoxin (OTA) concentrations in maize grains obtained in the Eastern Region is expected as it geographically falls in a semi-deciduous zone with mean rainfall and temperatures of 1400–1900 mm and 25.9 °C respectively suggesting favorable environmental conditions for the growth of these toxigenic fungi89 implicated. Pitt and Hocking90 noted that Penicillium spp. and Aspergillus spp. grow well between 0 and 30 °C and have been found to produce greater quantities of ochratoxin A and aflatoxins respectively, this may explain the high incidence of this mycotoxin. Furthermore, Magan et al.89 also emphasized fungal contamination in cereals is influenced by two main factors. Firstly, by initial high moisture content in crops or late harvesting of crops in rural areas, and secondly, the lack of poor storage facilities characterized by poor ventilation, high temperatures, and humidity.The different categorizations of the biosphere  presage different growth conditions for the proliferation of fungi. Brzonkalik et al.91. Other options are biological control by using non-toxigenic strains to competitively displace toxigenic fungi92.Regular monitoring and pre-and post-harvest control measures can be used to control mycotoxins by enhancing the resistance of the crop to incursive fungi through plant breeding or genetic engineering, which are laborious and time-consuming. Effective, sustainable, and universally applicable preharvest intervention strategies are needed. Proper field management practices such as the use of resistant varieties, timely planting, fertilizer application, weed control, insect control, and avoiding drought and nutritional stress could reduce mycotoxins93 highlighted that risk estimations are computed to envisage the adverse health implications of mycotoxin exposure and guide food regulators to set limits for these toxins in foodstuffs. Risk assessment results obtained in this study were comparable to the published findings of Blanckson and Mills-Robertson94 as they reported Total aflatoxins EDI values of 0.005–1.054 μg/kg bw/day and 0.004–0.838 μg/kg bw/day for infants and young children respectively in infant cereal-based formula and were risky for children to consume in Ghana. The Estimated Daily Intakes (EDI) of total aflatoxins in the maize samples recorded by Kortei et al.2 were 109.7, 58.8, 33.08, and 25.2 μg/kg bw/day for infants, children, adolescents, and adults with Margin of Exposure (MOE) values of 4.73, 8.79, 15.63 and 20.51 respectively. Exposure assessment carried out by Omari and Anyebuno95 showed that the minimum and maximum daily AFS exposures were 0.044 and 2.805 µg/kg bw/day, respectively for weanimix (infant complementary food prepared from maize) for rural households; these rates for complementary food purchased from urban shops were 0.014 and 0.55 µg/kg bw/day, respectively. The chances of liver cancer development would increase to 0.6 per year if infants were fed with complementary food prepared in rural households with a minimum AF level of 7.9 µg/kg.Kuiper-Goodman96 reported EDI values of range 0.02–0.04 respectively, for the age ranges of 3–6, 7–17, 18–59, and above 60 years for maize and products. All their computed MOE values were below the safe threshold of 10,000 and so the risk analysis results showed that most of the lower bound MOE values ranged from 10 to 100, indicating a concern for risk management. Age-group analysis suggested close attention were paid to the 3 ~ 6 years of age group, whose MOE value was the lowest. Their results reflected that preschool children might have the highest risk of being exposed to AF. Their results agreed with our findings. The MOE values (995–860 at mean and 336 at 95th percentile exposure) and cancer potency estimates, based on the current exposure levels indicated a potential health concern for Turkish adults was reported by97. Li et al.98 pointed from a Chinese survey data, that the average daily intake of AFB1 from maize in the high-risk area was 184.1 µg, and the probable daily intake is estimated to be 3.68 µg/kg bw/day. Chun et al.99 estimated excess cancer risk values for liver cancer incidence by ingestion of these foods for AFB1 were calculated to be 5.78 × 10–6 for individuals negative for hepatitis B and 1.48 × 10–4 mg/kg bw/day for individuals positive for hepatitis B in Korea.In Guangzhou China, Zhang et al.54 observed that aflatoxin exposures were to some extent linked to nutrient deficiency in humans following a suggestion that aflatoxin exposure expedites intestinal damage resulting in a decline in nutrient absorption.In this study, EDI were slightly greater compared with other EDI values reported globally. This implies a significant impact of aflatoxins on the nutritional status of humans and animals. Omari et al.100. Although there is no consensus on the specific dose of aflatoxins that triggers acute toxicity in humans, it is well established that such a dose is highly variable depending on many factors, including the age, gender, health and nutritional status, presence or absence of underlying factors ; and it is lowest in youngsters, as substantiated by the highest death rates of this age-group in aflatoxicosis outbreaks. A scoping review by Wu100 have shown the presence of these aflatoxins appeared in greater proportion in kwashiorkor of children and in different organs  and biological samples . This observation is particularly worrying since the Eastern Region of Ghana, is a major crop producing zone where most of the foodstuffs  used as ingredients for the production of local weaning formulas for humans and then animal feeds for poultry and livestock are produced due to favorable climatic conditions.A strong association between anaemia and aflatoxin has been reported in Ghana showing that aflatoxin exposure may contribute partly to the high iron deficiency prevalent in children in developing countries including Ghana. The consumption of aflatoxins at high levels in a single dose or repeatedly for a brief period induces acute intoxication, henceforward labeled aflatoxicosis, in humans and animals with typical symptoms, including jaundice, lethargy, nausea, edema, hemorrhagic necrosis of liver tissues, bile duct hyperplasia, and eventually death (10–60%) subsequent to severe liver damage100. Advocacy on strict compliance with good agricultural practice (GAP), good manufacturing practice (GMP), as well as good hygiene practice (GHP), which are critical ingredients to alleviate the formation of aflatoxins in the field as well as during storage of foodstuffs, must be strengthened. By impeding aflatoxins formation in foods, there is the protection of both public health and the prevention of economic losses. Monitoring foods prone to fungal infection and the presence of mycotoxins regularly is cautious to assess the public level of awareness.Various interventions have been established to combat aflatoxin biosynthesis and accumulation, ranging from preharvest to dietary interventions. Simply avoiding or reducing the consumption of foods that are frequently contaminated with aflatoxin has shown effectiveness in reducing liver cancer mortality in one population1, 71% of the samples exceeded EFSA limits while 64% exceeded GSA limits. For OTA, 94 (52.22%) of samples exceeded the tolerable limit of EFSA, while 89 (49.44%) exceeded the limits of GSA.Based on the results obtained in this study, per the permissible limits set by the European Food Safety Authority (EFSA) (2 and 4 µg/kg) and the Ghana Standards Authority (GSA) (5 and 10–15 µg/kg), it can be construed that out of the 180 samples analyzed for total aflatoxins , 68% exceeded the limits of EFSA whereas 58% exceeded GSA limits. In the case of AFBHealth risk assessment for ochratoxin A as well as aflatoxin exposure via maize consumed in different regions of Ghana by infants, children, and adolescents, and adults showed a significant adverse health risk in all age categories of humans since all calculated values for MOE were less than 10,000 for both aflatoxins and ochratoxins. This study scratches the surface of a dire situation that calls for attention by all stakeholders involved in mitigating the harmful effects associated with these hazardous mycotoxin exposures to humans.A. flavus under extreme heat and dry condition is an expected and emerging dilemma mainly in many parts of the world  where there were very low reports of mycotoxin contamination. However, surges in maize contamination were observed after prolonged hot and dry weather. Due to this, the world’s largest agricultural food exporters such as Brazil, Argentina and some parts of Asia to include China and India have been identified as hot spots for impacts of climate change. From a food security viewpoint, a more precise forecast of impacts of climate change on mycotoxins need to be addressed to prevent conceded food sustainability which possibly results in negative social consequences.Presently, the prime challenge of mycotoxins proliferation in our foods is the link with climate change. There is now a widespread consensus that the world is warming at an unparalleled rate and this is expected to seriously affect our crop production as well as the phyllosphere microflora of these crops. The irrepressible growth of Notwithstanding, some possible routes to improve the situation is the utilization of enzymatic biotransformation (purified enzymes) to degrade mycotoxins with much better precision in feeds/foods. Again the inclusion of binding agents or enterosorbents in the diet of humans or animals has been given considerable attention as a strategy to reduce foodborne exposures to mycotoxins by decreasing the mycotoxin availability thereby reducing its absorption. Recently, materials such as mycosorb (a new product which acts by binding pathogens and mycotoxins without affecting gut bacteria) and aluminosilicates have been reported to be adequately efficient in this regard. Furthermore, the use of biofuels and fermentation by-products such as distillers dried grains (DDGs) are some promising ways to drastically reduce the impacts of these mycotoxins on the gut and health of livestock and poultry. Lastly, a more practical means of curbing mycotoxicity is the diversification of our diets which include the non-adherence to a few food classes  but an expanded one to include different classes is a more promising approach.


Giardia duodenalis is a widespread protozoan parasite affecting humans and many species of animals, including dogs and cats. Due to its zoonotic potential, it is important to know the frequency of this parasite in companion animals. The aim of this study was to determine current epidemiological status of G. duodenalis in household dogs and cats.G. duodenalis by PCR (using β-giardin gene). The animals were divided into groups depending on their age, breed and fecal consistency.In this study, 293 fecal samples from pet dogs and cats were collected from January 2017 to July 2019 and tested for G. duodenalis in 6.0% of canine and 3.9% of feline fecal samples. The highest frequency was revealed in young (under one-year old) dogs. Sequencing confirmed the presence of assemblages C and D in dogs and A and F in cats.The examination allowed for detection of G. duodenalis in dogs and cats and also revealed the occurrence of host-specific assemblages as well as zoonotic assemblage A.The study showed current frequency of  Giardia duodenalis, Cryptosporidium spp., Echinococcus spp., Dipylidium caninum or Toxocara spp.Dogs and cats are intimate companion animals of humans. According to data from 2019, there are over seven million dogs and over six million cats in Poland . DespiteGiardia duodenalis  is a unicellular protozoan parasite affecting humans and many animal species. There are two morphological stages of G. duodenalis: a trophozoite and a cyst (infective stage). The parasite can colonize the upper small intestine but it was also found in the lower small intestine, stomach, colon and biliary tract [G. duodenalis transmission occurs via fecal–oral route  [ry tract , 7. G. dviduals) . GiardiaG. duodenalis in Europe ranged from 0.8% (Switzerland) to 42% (Germany) and from 5.9% (Spain) to 20.5% (Greece) in dogs and cats, respectively [In recent years, the infection rate of ectively , 31, 42.G. duodenalis to range from 2 to 36% in dogs and from 3.2 to 15.1% in cats, depending on the examined population, geographical origin of the animal, and diagnostic methods . The assemblages A and B are further divided into sub-genotypes AI, AII, AIII, BIII and BIV. Typical genotypes in dogs are C and D, but A and B can also be found, and exceptionally even E and F ones [d F ones , 14, 20.d F ones , 21, 24.d F ones , 37.gdh), triosephosphate isomerase (tpi) or β-giardin (bg), allow for detection of Giardia DNA and also for genotyping [bg or tpi) is commonly accepted, especially where the diagnosis is extended to the sequencing of the obtained PCR products [Giardia spp. is immunofluorescence and it is considered a reference standard assay for the detection of this parasite in dogs and cats feces [Microscopic studies using fecal flotation enable detection of the cysts . Other dnotyping , 26. Altproducts , 36. Oneproducts , 23. β-gproducts . Moreovets feces , 38.G. duodenalis, it is particularly important to determine its current infection rate in domestic animals. The overall prevalence and frequency of G. duodenalis genotypes in dogs and cats can indicate the potential risk of invasion in humans. The aim of this study was to run a molecular detection of G. duodenalis in fecal samples and to assess its overall frequency broken into frequency of each genotype in household dogs and cats from Poland.Due to the zoonotic potential of A total of 293 fresh fecal samples were obtained between January 2017 and July 2019 from individual, randomly chosen household dogs (217 samples) and cats (76 samples) living in different regions of Poland. The area of Polish territory is over 312,000 square kilometers divided into 16 provinces. The examined samples came from nine provinces , which account for nearly 153,000 square kilometers (about 50% of the country area)  as per the manufacturer’s instruction. The DNA samples were stored at – 80 °C until further use.G. duodenalis in the stool samples, fragments covering β-giardin gene were amplified by nested PCR. The amplification of a 763 bp region was carried out using a forward primer G7 (5′AAGCCCGACGACCTCACCCGCAGTGC3′) and a reverse primer G759 (5′GAGGCCGCCCTGGATCTTCGAGACGAC3′). For secondary PCR, 587 bp fragment was amplified using 1 µl of the first PCR product. The secondary PCR was carried out using nested forward 511 (5′ GAACGAACGAGATCGAGGTCCG’3) and nested reverse 511 (5′ CTCGACGAGCTTCGTGTT 3′). The mixture composition and PCR conditions were described by Lalle et al. (2005) [2, 200 mM of each dNTP  10 pmol of each primer, 2.5 units of RUN DNA polymerase  and 3 µl (1 µl in secondary PCR) of purified DNA in a final volume of 25 µl. PCR was performed using a thermocycler BioRad T100™ Thermal Cycler. Primary PCR conditions were as follows: 95 °C for 5 min for 1 cycle, 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min for 40 cycles followed by 72 °C for 7 min. Secondary PCR conditions were: 96 °C for 5 min for 1 cycle, 96 °C for 45 s, 55 °C for 30 s and 72 °C for 45 s for 35 cycles followed by 72 °C for 7 min. The secondary PCR products were examined electrophoretically in 2% agarose gels and visualized after staining with Midori Green Advance DNA Stain .To identify . (2005) . PCR mixhttps://blast.ncbi.nlm.nih.gov/Blast.cgi) with sequences deposited in GenBank. Phylogenetic analysis was performed using neighbor-joining method with MEGA 4 free software. Evaluation of the reliability of the clusters was confirmed using Bootstrap values (1000 replicates). Reference sequences used in the phylogenetic tree were: AB508814.1 for assemblage A; MN270296.1, KX757753.1 for assemblage C; JN416550.1, JN416548.1, JN416559.1 and LC316659.1 for assemblage D and LC341557.1 for assemblage F.PCR products purification and sequencing was performed by Genomed (Poland) in both directions. The resulting chromatograms were visually assessed to exclude the presence of double peaks. The obtained sequences were compared by a blast search  at the level of 95% (p = 0.05), calculated according to the Wilson method. The chi-square test (χ2) with Yates correction implemented in STATISTICA ver. 12.0 software package was used to compare the differences in Giardia infection rates among the investigated groups. Differences were considered significant at p ≤ 0.05.The frequency of G. duodenalis DNA was detected in 13/217 (6.0%) canine and in 3/76 (3.9%) feline fecal samples. Higher frequency of the infection was observed in dogs under 1 year old  (p < 0.05). Among the dog breeds, Giardia infections were most frequently observed in French bulldogs , and the infection rate was significantly higher than in other breeds (p < 0.05). There were no statistically significant differences connected with age and breed in cats or fecal consistency in both species . In this study, the frequency of G. duodenalis found in fecal samples of dogs from different regions of Poland was 6.0% and this result fell within the lower limit of previous results from Poland [bg locus, a highly sensitive and widely recommended marker [Giardia noted in Germany can be connected with lower number of examined samples and this result cannot be representative for the entire country. In our study, significantly higher frequency of the parasite was noted in young dogs, under one-year old (14%). The age of the dogs seems to be an important risk factor for G. duodenalis, as previously described. A recent study conducted by Pan et al. (2018), based on amplification of bg and tpi genes, showed significantly higher detection rate of Giardia in young (16.1%) than in adult dogs (7.6%) [bg gene as a molecular marker and confirmed significantly higher prevalence in young, sheltered dogs [G. duodenalis in the dogs over one-year old, however, many studies detected the presence of this parasite also in adult dogs [G. duodenalis in symptomatic and asymptomatic dogs [G. duodenalis in household dogs with loose consistency of feces  was reported by Ulterwijk et al. (2019), but in other groups of dogs (sheltered or hunting), significant differences were not observed [The occurrence of m Poland , 34, 43.d marker , 23. Howd marker . Our stud marker , 21, 31.s (7.6%) . Shin etred dogs . We founult dogs . Among tult dogs . There atic dogs . We founobserved , 33, 41.G. duodenalis was found in 3.9% of feline fecal samples. These results were similar to those obtained in household cats from Spain (5.9%), but lower than in Germany (14%) and Greece (15.6%) [In the current study,  (15.6%) , 21, 31.In dogs, we confirmed only host-specific genotypes D (77% of positive samples) and C (23% of positive samples). This differed significantly from the results previously described for western Poland, where mostly genotype C, and just a few cases of genotypes D and B were detected . The occG. duodenalis genotypes specific for dogs or cats and also the occurrence of zoonotic genotype A in cats. The study confirmed that giardiasis in dogs is strongly connected with their age and is often diagnosed in dogs under one-year old. Moreover, G. duodenalis was found more often in French bulldogs than in the dogs of other breeds, which according to our knowledge is the first communication of such a result.In summary, fecal samples of dogs and cats from different regions of Poland were collected and PCR examination based on β-giardin locus amplification was conducted. This examination confirmed the occurrence of