Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus  Algae and fungi have coexisted and coevolved for at least 400 million years, as deduced from fossil records showing macroalgae parasitized by fungi , as wellMicroorganisms are often subject to rapidly changing biotic and abiotic conditions in their natural habitats. To cope with these environmental changes, they often produce natural products that are low molecular mass metabolites , 8. TheyStreptomyces rapamycinicus and its closest relative S. iranensis  the presence of A. nidulans increases the growth rate and overall biomass of C. reinhardtii. We show that the algal–fungal association itself is a defense mechanism against toxins.Co-existance of filamentous fungi, streptomycetes and other producers of natural products often triggers the production of these effector chemicals . For insranensis –18. Sincnhardtii  and lichnhardtii , 12, 14,e system . We founMicrobial strains and plasmids used in this study are listed in Supplementary Table S. iranensis the method described by Netzker et al. [For deleting genes of r et al.  was esseS. iranensis, 50 mL of TSBY [8 spores and the bacteria were incubated for 4 days at 28 °C at 180 rpm. The entire culture was centrifuged to separate the biomass from the culture supernatant. After lyophilisation of the biomass, the content of the pellet was extracted twice with 50 mL methanol at 60 °C. Both extraction solutions were combined and evaporated to dryness under reduced pressure and dissolved in 2 mL 75% (v/v) aqueous methanol (MeOH(aq)). The extract was subsequently loaded onto an N-vinylpyrrolidone-divinylbenzene copolymer resin SPE column , washed with 50% (v/v) MeOH(aq) and eluted with 70% (v/v) MeOH(aq) to obtain purified azalomycin F complex. The eluent was reduced to dryness, dissolved in pure MeOH, and 5 μL were loaded onto an ultrahigh-performance liquid chromatography-mass spectrometry for analysis. The chromatographic system consisted of an UltiMate 3000 binary rapid-separation liquid chromatograph with photodiode array detector . The MS was performed on an LTQ XL linear ion trap mass spectrometer  equipped with an electrospray ion source. The sample was analyzed on a 150 mm by 4.6 mm Accucore reversed-phase (RP)-MS column with a particle size of 2.6 μm  at a flow rate of 1 mL/min with the following gradient: 0.1% (v/v) HCOOH-MeCN/0.1% (v/v) HCOOH–H2O 0/100, increased to 80/20 in 15 min and then to 100/0 in the following 20 min, held at 100/0 for 2 min, and reversed to 0/100 in 2 min, with detection range at 190–400 nm. For the azalomycin F-deficient mutants, cultivation, extraction, and analysis of azalomycin F are described in the Supplementary Methods Section To obtain samples of the azalomycin F complex from  of TSBY  were inoS. iranensis monoculture. For co-cultivation, 1.5 mL 1% (w/v) TAP agar was spread onto one half of the ITO glass slide and allowed to solidify. The other half was covered with TAP agar, containing submerged C. reinhardtii. For this purpose, a C. reinhardtii culture was centrifuged at 3500 × g for 5 min and the supernatant was discarded. The pellet was resuspended in liquid TAP agar with a temperature of about 35 °C to yield an OD750 of 2. This suspension was then added to the other side of the slide and allowed to solidify. Finally, 15 µL S. iranensis mycelium, prewashed in PBS, was set between both agar sides. The slides were incubated at 26 °C and a light intensity of 30 µE m−2 s−1 in petri dishes for 5 days and subsequently dried in a hybridization oven at 37 °C for 48 h. For MALDI-IMS analysis, see Supplementary Methods MALDI-IMS was carried out on indium tin oxide (ITO) covered glass slides . Slides were sterilized over a Bunsen burner and 3 mL of 1% (w/v) TAP  agar werC. reinhardtii cells that migrated into glass capillaries loaded with a potential attractant [®, Drummond Scientific, Broomall, PA, USA) were loaded with TAP supplemented with 10 g/L glucose, 0.5 g/L MgCl2∙7H2O, 1 mL/L trace elements, 10 mL/L 0.5 M arginine, 3 mL/L 3 mM FeSO4 and 3 mL/L 0.1% (w/v) p-aminobenzoic acid (PABA) as control. Equally supplemented supernatant of an A. nidulans culture grown in TAP medium served as experimental sample and as a negative control PBS was used. The capillaries were placed upright into a 2 mL reaction tube. For each sample, three technical replicates were tested. 100 µL of a C. reinhardtii suspension with an OD750 of 4 were placed at the bottom of each 2 mL reaction tube. When attracted, the C. reinhardtii cells had to swim against gravity. Incubation was carried out at 26 °C in the dark for 5 h. After incubation, the capillaries were cleaned with a paper towel to remove attached cells, and the content was transferred to 27 µL of PBS + 4% (v/v) paraformaldehyde. C. reinhardtii cells which had migrated into the capillaries were counted using a Neubauer counting chamber.Chemotaxis was measured by counting tractant , 24. ThrC. reinhardtii suspension and to estimate the viability of the cells 24 h after treatment with azalomycin F. Each well was inoculated with 100 µL TAP supplemented with glucose, MgSO4, PABA, FeSO4, trace elements, and arginine  on a Tecan Infinite M200 pro microplate reader . To allow C. reinhardtii to migrate into the mycelium, the plate was incubated for 5 h at 26 °C, 120 rpm and 30 µE m−2 s−1. Then, 200 ng azalomycin F were added and the fluorescence was measured again to determine the values for the start of the treatment. Incubation was continued for 24 h before measuring fluorescence using an excitation wavelength of 480 nm and measuring emission at 684 nm.The assay was performed in order to assess the chlorophyll content within a 4, PABA, FeSO4, trace elements, and arginine . After 24 h, 12.5 mL of 20% (w/v) glucose, 0.5 g/L MgSO4∙7H2O, 750 µL of 0.1% (w/v) PABA, 750 µL of 10 mM FeSO4, and 2.5 mL of 0.5 M L-arginine were added to each half of the co-cultivation chamber. A. nidulans dry mass was determined by filtering the mycelium after the experiment and drying it for 7 days at 60 °C.Co-cultivation chambers were assembled as previously reported by Paul et al. . The culsn-glycero-3-phosphoglycerol (DPPG), 16:0-i15:0 phosphatidylcholine (PC), d18:1/18:0 C18 glucosyl(β)ceramide (GC), and extract of polar lipids of Saccharomyces cerevisiae (YE) were purchased from Avanti Polar Lipids . DPPG, CL, and GC were dissolved in 95:5 methanol:water to obtain a 0.5 mg/mL stock solution. PC was dissolved in 95:5 methanol:water to obtain a 4 mg/mL stock solution. YE was purchased as a 25 mg/mL solution in chloroform. For application of the lipids to C. reinhardtii, the solvent was evaporated under a gentle stream of nitrogen. The lipids were dissolved in a culture of C. reinhardtii with an OD750 of 2. The final concentrations of the lipids were chosen similar to Xu et al. [®  at 30 °C and rapid shaking for 30 min. Afterwards, the cultures were incubated in 24 well plates at 26 °C, 120 rpm and 30 µE m−2 s−1. Autofluorescence was measured after 24 h of incubation as described for the chlorophyll quantification assay.Phosphatidylinositol sodium salt from soybean was purchased from Sigma-Aldrich . Cardiolipin (CL), 1,2-dipalmitoyl-u et al. . To dissA. nidulans and the green alga C. reinhardtii quickly assemble in co-culture to form an algal–fungal association [C. reinhardtii and S. iranensis to identify possible effects of the bacterium on the alga. When co-cultured with S. iranensis under light, C. reinhardtii lost its green color in liquid medium  and cc4385 (psbD deletion)  all significantly lowered the detrimental effect of azalomycin F on C. reinhardtii. Since 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and the sphingolipid d18:1/18:0 C18 glucosyl(β)ceramide (GC) did not affect the activity of azalomycin F  to C. reinhardtii treated with azalomycin F restored the autofluorescence to the level of untreated controls. Thus, binding of azalomycin F to membrane polar lipids is required for its antibacterial activity [Based on these findings, we hypothesized that a polar lipid extract of ner Fig. . Additioactivity , 36 as wactivity  and algiC. reinhardtii from azalomycin F. Figure A. nidulans and C. reinhardtii. To address the question whether this assembly is actively or passively established, a chemotaxis assay was performed. Figure P ≤ 0.01) more C. reinhardtii cells swam into a glass capillary loaded with supernatant of an A. nidulans culture than into a capillary filled with fresh medium  and higher overall cell density compared with the monoculture (Fig. A. nidulans produced a lower biomass in co-culture, compared with the monoculture at the end of the experiment (Fig. A. nidulans in co-culture with C. reinhardtii. Therefore, we measured the expression of polar lipid biosynthesis genes of A. nidulans in presence of C. reinhardtii by qRT-PCR. As shown in Supplementary Fig. C. reinhardtii excluding both possibilities. Taken together, our findings suggest a chemotactically driven attraction of the alga to the fungus, which ultimately provides benefit to the alga by protecting against toxins.In order to measure any effects on algal or fungal growth, co-cultivation chambers  were appure Fig. . In content Fig. . In the Although algal–fungal co-cultures have been successfully used for biotechnological processes , as wellS. iranensis kills the green alga C. reinhardtii when both organisms were grown in co-culture. The algicidal compound was identified as the polyketide azalomycin F that also has antifungal and antibacterial activity against Gram positives [We have demonstrated that ositives , 31. TheClostridium puniceum uses the polyphenolic metabolite clostrubin to be able to grow in an oxygen-containing environment and to defend its habitat against competitors [S. rapamycinicus induced the production of the novel compound fumigermin in A. fumigatus. The compound inhibits the germination of the inducing S. rapamycinicus [For only a few natural products their ecological meaning has been elucidated. For example, terpenoids serve various functions ranging from chemical communication to defense against predators in plants . The anapetitors . Bacteripetitors , to immopetitors . A recenycinicus . Here, wPhaeocystis globosa [C. reinhardtii were effectively killed by azalomycin F. In addition, the significance of this observation for an ecological setting is unclear, as C. reinhardtii will almost always be illuminated to some extent in its natural habitat. It should be noted, however, that C. reinhardtii has at least 18 photoreceptors that absorb light in the UV as well as blue and red visible spectrum [Streptomycetes are known to synthesize algicidal compounds such as NIG355, a nigericin derivative that inhibits the growth of a dinoflagellate  and the  globosa . The obsspectrum , and thespectrum , 34. AltS. iranensis in monoculture but the majority of the compound is associated with the bacterial cells and not in the supernatant. Only co-cultivation of S. iranensis with C. reinhardtii triggered the release of azalomycin F, which, in turn, led to the killing of the alga. It is yet unclear whether azalomycin F resides inside the cell and is exported, or, whether it is attached to the bacterial membrane and simply detached from it upon a certain signal.Azalomycin F is produced by C. reinhardtii is killed in conditions that prevent cellular contact.Typically, biosynthesis of an antibiotic is directly coupled to its export to avoid self-intoxication. For example, the exporters of the gyrase inhibitors simocyclinone  or of acC. reinhardtii is protected against bacterial azalomycin F by taking shelter in the mycelium of A. nidulans. Furthermore, we provide evidence that lipid binding is a means of neutralizing azalomycin F algicidal activity [A. nidulans and C. reinhardtii [We observed that activity , 36. We activity –55, is anhardtii , 57, is nhardtii . We thusnhardtii , 60. ThuS. macrospora could not protect C. reinhardtii from azalomycin F despite attracting the alga. This coincides with susceptibility of S. macrospora against azalomycin F that is not observed for S. cerevisiae and A. nidulans. It is tempting to speculate that only fungi resistant against azalomycin F have the capability to protect C. reinhardtii. The reason for the susceptibility of S. macrospora against azalomycin F awaits further clarification.It is interesting to note that Morchella crassipes fosters growth of associated Pseudomonas putida bacteria that in turn were also used as a carbon source by the fungus [A. nidulans enhanced the growth of C. reinhardtii.Fungal hyphae can provide various benefits to partner organisms, as exemplified by the symbiosis of fungi and terrestrial plants in the Mycorrhiza. Here, the fungus gains fixed carbon products from the phytobiont, in exchange provides nitrogen, phosphate, and increased water supply to the land plant . The fune fungus . Anothere fungus . SimilarAspergillus, once was lichen forming but the recent fungi have lost this ability during the course of evolution [C. reinhardtii readily migrated into A. nidulans and S. macrospora hyphal shells in liquid and formed macroscopic structures, in which the algal cells gathered around fungal hyphae. Likewise, it was reported that auxotrophic mutants of A. nidulans unable to assimilate nitrite formed an obligate mutualism with C. reinhardtii. The algal partner was able to provide nitrogen to the fungus [A. nidulans and C. reinhardtii may well have retained the capabilities of their lichen-forming ancestors to form a kind of algal–fungal mutualism. For C. reinhardtii, this is not surprising because species of the genus Chlamydomonas have been previously identified as mycetobiont algae in lichen-like symbiotic associations of wood-decaying fungi [Lutzoni et al. proposed that the common ancestor of the modern Eurotiales, including the genus volution . Howevere fungus . A. nidung fungi .Aspergillus species likewise might have already possessed the genes promoting symbiosis. Indeed, it has been suggested that the fungal spread on land and the radiation of terrestrial plants are linked to their preexisting ability to establish symbioses [A. nidulans and C. reinhardtii may thus have retained some genetic traits allowing for algal–fungal mutualism that provides a selective advantage over noncooperators, although both microorganisms no longer contribute to the formation of stable lichens and live solitarily.With reference to the co-evolution of fungi, algae, and terrestrial plants, Delaux et al.  postulatymbioses . A. niduC. reinhardtii cells located within the A. nidulans mycelium exhibited increased survival rates when treated with azalomycin F compared with C. reinhardtii that was prevented from entering the fungal mycelium. Presently, abiotic factors, such as water availability and radiation, are primarily considered as triggers for tight interactions [ractions , 70. It ractions , 13, 14 ractions  were sucSupplemental Information


Treatment of patients with solid tumors and KRAS mutations remains disappointing. One option is the combined inhibition of pathways involved in RAF‐MEK‐ERK and PI3K‐AKT‐mTOR.Patients with relapsed solid tumors were treated with escalating doses of everolimus (E) 2.5‐10.0 mg/d in a 14‐day run‐in phase followed by combination therapy with sorafenib (S) 800 mg/d from day 15. KRAS mutational status was assessed retrospectively in the escalation phase. Extension phase included KRAS‐mutated non–small‐cell lung cancer (NSCLC) only. Pharmacokinetic analyses were accompanied by pharmacodynamics assessment of E by FDG‐PET. Efficacy was assessed by CT scans every 6 weeks of combination.Of 31 evaluable patients, 15 had KRAS mutation, 4 patients were negative for KRAS mutation, and the KRAS status remained unknown in 12 patients. Dose‐limiting toxicity (DLT) was not reached. The maximum tolerated dose (MTD) was defined as 7.5 mg/d E + 800 mg/d S due to toxicities at previous dose level (10 mg/d E + 800 mg/d S) including leucopenia/thrombopenia III° and pneumonia III° occurring after the DLT interval. The metabolic response rate in FDG‐PET was 17% on day 5 and 20% on day 14. No patient reached partial response in CT scan. Median progression free survival (PFS) and overall survival (OS) were 3.25 and 5.85 months, respectively.Treatment of patients with relapsed solid tumors with 7.5 mg/d E and 800 mg/d S is safe and feasible. Early metabolic response in FDG‐PET was not confirmed in CT scan several weeks later. The combination of S and E is obviously not sufficient to induce durable responses in patients with KRAS‐mutant solid tumors. The study investigated the combination of sorafenib and everolimus in patients with solid tumors  and KRAS mutated NSCLC (expansion part). Although the trial showed manageable safety profile of sorafenib and everolimus, it failed in providing long terms responses for patients with solid tumors and KRAS mutated NSCLC. The novelty of the study was the early pharmacodynamics assessment using FDG‐PET implemented in a phase‐I. KRAS mutations occur in several types of solid tumors, most frequently in lung adenocarcinoma (17%), colorectal cancer (19%), and pancreatic cancer (60%).Alternative treatment with immune checkpoints inhibitors provided promising response rates and prolonged PFS and OS in NSCLC as monotherapy in patients with high PD‐L1 expression and in combination with chemotherapy independent of PD‐L1 status.Combined inhibition of the RAS‐RAF‐MEK‐ERK‐ and the PI3K‐AKT‐mTOR pathways might be a conceivable strategy to inhibit KRAS downstream signaling more effectively. Sorafenib is a multikinase inhibitor targeting the extracellular receptors VEGFR/PDGFR/c‐kit (upstream of KRAS) and the intracellular RAF kinase.Everolimus is an immunosuppressive drug exerting also an antiproliferative activity by inhibition of mTOR.In NSCLC, it has been shown that molecular imaging with FDG‐ und FLT‐PET can help to detect responses to targeted therapy early and simultaneously enable to identify patients with treatment failure.Here we present results of a phase I study in patients with solid tumors treated with everolimus and sorafenib. In the dose escalation part, tumor samples were retrospectively analyzed for KRAS mutation. In the expansion phase, only patients with KRAS mutated NSCLC were enrolled. FDG‐PET was used as a pharmacodynamic tool during the run‐in phase with everolimus. As this was a phase I study, the number of patients remained limited.1.1Adults with solid tumors were enrolled in the study after failure of standard treatment. In the escalation part, patients without any predefined genetic alteration and for escalation part, patients with NSCLC and KRAS mutations were recruited.Further key inclusion criteria included measurable disease according to RECIST1.1, performance status ECOG 0‐2, adequate blood count, and normal renal and hepatic functions. Key exclusion criteria comprised any concomitant uncontrolled condition. Patients with brain metastases were excluded, if they required permanent treatment.In this monocentric study, patients were treated at University Hospital Cologne, Germany.All patients signed written informed consent before enrolment. The trial was designed and conducted according to the principles of GCP/ICH, the study protocol and the informed consent form were approved by the Ethics Committee at the University Cologne (EudraCT‐number: 2008‐005440‐16).1.2No central screening was required for KRAS mutation. KRAS mutation was assessed retrospectively in the dose escalation and prospectively in the dose expansion part. The molecular diagnostics was done either by local pathology or on the central diagnostic platform of the Network Genomic Medicine (NGM) in Cologne. In NGM, KRAS (exons 2 and 3) were analyzed using high resolution melting and positive samples were confirmed by dideoxy (“Sanger”) sequencing as reported previously.1.3This phase I study was designed as a monocentric, open‐label one arm trial with the primary objective of determining a safe and tolerable dose for the combination treatment with E and S in patients with advanced solid malignancies  and NSCLC patients harboring KRAS mutation (expansion part). Secondary objectives included safety of E and S and their combination, pharmacokinetics (PK), pharmacodynamics (PD) using FDG‐PET, and objective response.1.4The maximum tolerated dose (MTD) of E was determined in a dose‐finding phase by sequential dose escalation on levels 2.5, 5, 7.5, and 10 mg given orally once daily following a 3 + 3 design with predefined criteria for dose‐limiting toxicities (DLT).1.5In order to assess the predictive value of FDG‐PET, PET scans were performed at baseline and on days 5 and 14 after start of treatment and evaluated according to the PERCIST criteria.1.6Wherever clinically feasible, repeated blood samples for PK analyses were scheduled during a 24 hours period on days 5 (E), 14 (E), and 29 (E + S) of therapy. The PK profile was completed by additional samples at regular study visits. Concentrations of both substances were quantified by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). The lower limits of quantification were 1.052 ng/mL blood (E) and 10.0 ng/mL plasma (S), respectively. Mean precision and absolute accuracy were better than 15% for all analytes. Population pharmacokinetic models were developed for E and S using NONMEM 7.4  and related software1.7For safety and tolerability analyses, all patients who received any study medication were included (safety set). For efficacy analyses, only patients compliant with the protocol were included (per protocol set). The definition of per protocol set is identical with definition of intention to treat (ITT) set in terms of data analysis.Maximum tolerated dose was estimated as the highest dose level where ≤ 1 of 6 patients experienced toxicity in terms of DLT. The safety of everolimus and sorafenib was documented using CTCAE version 4.0. Response rates were measured in accordance with RECIST 1.1 criteria. Correlation between KRAS mutation status and response was provided descriptively. Secondary endpoints PFS and OS were analyzed descriptively using the Kaplan‐Meier method. The predictive value of PET response from baseline to days 5 and 14 with respect to PFS and OS was assessed using Cox regression. In an exploratory analysis, the relationships of median individual everolimus and sorafenib clearance and identified covariates for pharmacokinetic parameters were evaluated with respect to PFS and OS using likelihood‐ratio tests. Correlation between kinetic rate constants for FDG transport (K1) and FDG metabolism (Ki) from dynamic FDG PET with SUVs from static FDG‐PET were reported descriptively.22.1Thirty‐seven patients with advanced solid malignancies gave informed consent and were screened for eligibility from October 2009 to December 2013. Six of these patients were screening failures. Thirty‐one patients were enrolled and evaluable in terms of the primary objective , 16 of them suffering from NSCLC (51.6%), and 15 (48.4%) from other malignancies. The mean age of all enrolled patients was 58.5 (41‐77) years with a balanced distribution of male  to female patients . Most patients with NSCLC were enrolled in the extension phase of the trial , all of them with lung adenocarcinoma harboring mutation of KRAS as defined per study protocol. The overall percentage of detected KRAS mutations in all patients (dose finding and extension phase) was 48.4% (15 patients) while the KRAS status was unknown in 35.5% (12 patients). Four patients had wild‐type KRAS. Of 19 patients with known KRAS status, in 18 patients KRAS was tested within the NGM and in 1 patient by local pathology. Of 18 patients tested within the NGM, KRAS was assessed using Sanger sequencing in 17 patients and NGS panel in 1 patient. Most patients presented in fairly good to moderate general condition with a majority of patients showing ECOG 0   and 800 mg/day S; further 17 patients were treated within the extension phase at the determined MTD of 7.5 mg E (dose level III) and 800 mg S. Overall, 20 patients (64.5%) were treated at dose level III (Table 2.3Extensive E blood concentration profiles (up to 9 data points) scheduled for days 5, 14, and 29 of treatment (with up to 1 day deviation) were available for 4, 3, 19, and 4 patients on 2.5, 5, 7.5, and 10 mg daily doses, respectively. S plasma concentration profiles with at least four data points (median 5 data points) on day 29 were obtained for 15 patients. Altogether, 480 data points for E and 122 data points for S were available.−1, central and peripheral volumes of distribution 46.6 (29.9‐100.1) L and 612 (488‐790) L, intercompartmental clearance 63 (53‐72) L/h, and initial systemic clearance 15.4 (11.7‐19.2) L/h, respectively. Shrinkage of E systemic clearance was 5%. Exposure to E increased essentially linearly with the dose. Interestingly, E concentrations decreased during the course of treatment. To account these changes, data points were sorted into three bins , and interoccasion variability was tested for PK parameters. E clearance for the second and third period increased by 23 (10‐48) % and 81 (53‐125)%, respectively, compared to the first period ) of pharmacokinetic parameters were absorption constant 0.74 0.58‐1.27) h7 h−1, ceF) and the fraction of cleared S subject to enterohepatic circulation (Fent) were estimated from S plasma concentrations of this study for all patients with at least four data points on day 29. Volumes of distribution and clearances were scaled allometrically with body weight , which allowed a reasonable fit of the model to the observed concentrations. Shrinkage of S systemic clearance was 12%. The estimated Fent was 0.58 and bioavailability varied with a coefficient of variation of 49%. Exposure in terms of AUCτ values for the dosing interval studied  based on individual empirical Bayes estimates was (geometric mean) 131 (CV 56%) μg*h/mL in subjects receiving only doses of S 400 mg BID until day 29. Three subjects received at least one dose of 200 mg just before day 29 (mean ± SD of sorafenib doses: 395 ± 32 mg), in these subjects AUCτ values were 85 μg*h/mL (CV 28%).For S, the limited number of available data points precluded the development of an independent pharmacokinetic model. To provide an estimate of exposure, structure and parameter estimates from a published model2.4On day 5, PET was performed in 30 patients. In one patient, new metabolic active lesions were detected as marker of progressive disease. Five patients reached partial metabolic response (PMR), of those four patients had KRAS mutation. Twenty‐three patients showed stable metabolic disease (SMD) and one patient had progressive metabolic disease (PMD).On day 14, 25 patients received PET, 5 of them showed PMR: 4 with KRAS mutation and 1 with unknown KRAS mutational status. One patient presented with PMD and 19 patients showed SMD Figures  and 4. NA more pronounced reduction in PET metrics from baseline to day 14 was linked to a significant decrease in hazard of progression (hazard ratio [95% CI] for a unit‐decrease in PET response of 0.82 [0.68‐0.97]) and death (0.79 [0.64‐0.97]). However, this depended on two influential patients with KRAS‐mutated NSCLC with the most distinct observed decreases in PET response . When these two patients were excluded, no significant link of PET response and OS/PFS remained.P = .16). Median individual E clearance was significantly related to PFS (0.97 [0.95‐1.00]) while no significant relationship was observed for OS (P = .85). Sorafenib clearance was neither significantly related to PFS (P = .08) nor OS (P = .66).Higher concentrations of baseline bilirubin were significantly related to a higher hazard of death (15 [1.3‐160]), but not of progression  the SD was confirmed, 5 patients showed PD Figures . No patiFive patients (16.1%) discontinued treatment without evidence of progression: three patients due to adverse events , 1 patient due to individual decision, and 1 patient due to newly diagnosed concomitant colorectal cancer (investigator decision).P = .26; Figure Thirty‐one patients were included in ITT population, 26 patients had progressive disease at time point of data cutoff. Progression‐free survival of 26 patients ranged from 32 to 497 days after start of treatment in Kaplan‐Meier analysis. Median PFS was 3.25 month     Figure .3The results of this trial describe a safe and feasible treatment schedule with 7.5 mg E and 800 mg S for patients with advanced solid malignancies.The MTD established in our trial is higher than published before: with 35 mg/week everolimus + 400 mg/day sorafenib,Furthermore, we observed slightly different toxicities than previously published. The dose limiting toxicities in the previous combinations trials were hand‐foot syndrome and asthenia.Exposure to both E and S was similar to other reports.This might also explain the observed relationship between PFS and everolimus clearance. Since an increase in everolimus clearance causes a decreased drug exposure, the observed increase in PFS seems contradictory. However, the observed increase in PFS might result from improved overall health and thus increased biotransformation, suggesting that reduced exposure is of subordinate importance. This is supported by the relationship between OS and baseline bilirubin, which suggests that improved liver function outweighs the potentially disadvantageous effects on pharmacokinetic in terms of a more rapid drug elimination. However, this evaluation was only exploratory since it was based on a limited selection of pharmacokinetic parameters. For example, time‐varying PK and the variability in volumes of distribution were not considered. The complex interactions between PK of everolimus and sorafenib and overall health require further evaluations, including a more extended PK/PD modeling.In terms of efficacy of treatment, the imaging procedures demonstrated heterogeneous findings. Although the PET response rates in terms of PMR on days 5 and 14 were 17% and 20%, respectively, no patient experienced objective partial response as defined by RECIST 1.1 in CT scan later. Furthermore, no correlation between PMR and PFS or/and OS was found in Cox regression analysis.The early PET results in our study are not consistent with data published previously, indicating a good predictive value of early PET scan for further treatment efficacy with erlotinib or everolimus in patients with NSCLC.This may underline recently published preclinical and translational data demonstrating that in many cases KRAS mutation represents a subclonal mutation occurring with several other oncogenic drivers.It should be noted that individual patients have experienced surprisingly long periods of stable disease in our trial, 1 patient with neuroendocrine carcinoma of unknown origin with the longest time on treatment of 17.1 months and fairly good quality of life. This seems to be consistent with previously published data showing the efficacy of E in treatment of patients with advanced pancreatic NEC.However, based on low number of patients in this study, the results are limited. The low number of patients is mainly caused due to the design of a phase I study. So far, only 17 patients were treated on maximum tolerated dose and only 13 patients had KRAS mutated lung cancer. This number is surely too low to answer the question if the combination of S and E is sufficient to induce responses in KRAS mutated NSCLC. However, as we saw almost no durable tumor shrinkage in these patients, we concluded that the combination is possibly not enough effective in these patients. Overall, this study demonstrates a safe and feasible schedule for the combination treatment with E and S at a higher dose (7.5mg+800mg) than previously published. In general, the treatment with 800mg S and 7.5mg E was well tolerated. The observed increase of E clearance during treatment suggests that tolerability limits may need to be reassessed according to the duration of treatment. Although everolimus treatment showed early metabolic responses, these responses could not be transferred in later CT findings. Thus, our study did not significantly improve treatment of KRAS‐mutated patients with solid tumors by using the combination of E and S. Our results also question the role of early FDG‐PET for predicting CT‐confirmed objective responses or even a longer PFS and OS, at least in this patient population. To improve treatment of patients with KRAS mutation, new drugs or drug combinations should be evaluated in clinical trials with thoroughly conducted translational analyses to understand the biological mechanism underling response and treatment failure.LN has received honoraria/travel expenses and/or had an advisory role by Pfizer, Celgene, Novartis, Bayer, Roche, Boehringer Ingelheim, Janssen, BMS, and Takeda. MS has had an advisory role and/or paid talks by Boehringer Ingelheim, Mediolanum Biosciences, Novartis, Roche, and Takeda. He has received a scientific support from AMGEN and travel support from Boehringer Ingelheim, Mediolanum Biosciences, and BMS. SMB has received grant from Novartis and personal fees from BMS, Novartis, Pfizer, Roche Pharma, and AstraZeneca. AAS is a current employee of AbbVie Deutschland GmbH, and is a holder of AbbVie stock. RNF has had an advisory role by BMS and Roche and has received Honoraria from BMS, MSD, Roche, Boehringer Ingelheim, and AstraZeneca. JW has had an advisory role, received honoraria and travel fees from Abbvie, AstraZeneca, BMS, Boehringer‐Ingelheim, Chugai, Ignyta, Lilly MSD, Novartis, Pfizer, and Roche. DSYA has had an advisory role and received lecture fees from BMS, Boehringer‐Ingelheim, MSD, Novartis, Roche, Healthcare Consulting Cologne, Abbvie. DSYA has received travel funding from AstraZeneca, BMS, Boehringer‐Ingelheim, MSD, Novartis, Roche, and Abbvie. LJ worked during the study conduction at Institute for Pharmacology at University Cologne. Currently she works at IQWiG in Germany . The institution of LN, MS, RNF, ML, SM, DSYA, and JW has received a research funding from Novartis, Pfizer, BMS, MSD, Roche, Boehringer Ingelheim, Bayer, and Janssen.Authors contributed to the study and the article as follows: Conceptualization: LN, CM, MS, MLS, JW, MG, CK, JF, HB, CS, and UF. Data curation: LN, CM, MS, MTa, TP, CK, JF, HB, FS, UF, and JW. Formal analysis: LN, CM, MTa, TP, CK, SMB, JF, HB, CS, FS, DO, UF, RB, and JW. Funding acquisition: JW and LN. Investigation: LN, CM, MS, MTa, MG, MLS, SM, RNF, DSYA, TP, CK, SMB, HB, RS, DB, BK, ME, CS, YT, LJ, AAS, SF, DR, DO, UF, RB, and JW. Methodology: LN, CM, MS, JF, JW, MLS, TP, CK, SMB, JF, HB, CS, FS, UF, and RB. Project administration: MTh, LN, CM, MS, and MG. Resources: UF, JF, MTa, TP, SMB, JF, CS, FS, JW, and RB. Software: UF, JF, MTa, TP, SMB, JF, HB, CS, FS, UF, vRB. Supervision: LN, JW, UF, and RB. Validation: LN, JW, UF, CM, MS, MTa, MG, TP, JF, HB, CS, FS, UF, and RB. Visualization: LN, MS, JF, UF, HB, CS, and FS. Writing—original draft: LN, JW, UF, MS, CM, RNF, and JF. Writing—review and editing: LN and MS.The design of the trial and partial results were presented as posters at ASCO annual meetings between 2012 and 2017.


Angiostrongylus vasorum, Crenosoma vulpis, Capillaria aerophila and Aelurostrongylus abstrusus, are a cause of concern in the scientific and veterinary community, potentially causing significant disease in domestic animals. To investigate the potential of wild carnivores as reservoir hosts to these parasites, a total of 1041 animals from seven regions of Denmark were sampled: 476 raccoon dogs (Nyctereutes procyonoides), 367 red foxes (Vulpes vulpes), 123 American mink (Neovison vison), 31 beech martens (Martes foina), 30 Eurasian otters (Lutra lutra) and 14 polecats (Mustela putorius). Hearts and lungs were collected and examined for cardiopulmonary parasites. Capillaria aerophila was identified using morphology, whereas A. vasorum and C. vulpis were identified by a duplex real-time PCR, and A. abstrusus by conventional PCR. This is the first Danish report of A. vasorum and C. vulpis infections in raccoon dogs, mink and polecats, and of C. aerophila in raccoon dogs and beech martens. In addition, this is the first time A. vasorum and C. vulpis have been identified in wild animals from the island of Bornholm, just as it is the first report of C. vulpis in American mink, and C. vulpis and A. vasorum in polecats in Europe. The prevalence of A. vasorum appears to have increased in red foxes in Denmark compared to previous studies, while C. vulpis and C. aerophila prevalences are lower. Our data show that several wild carnivores can serve as reservoir hosts for A. vasorum, C. vulpis and C. aerophila in Denmark, and that A. vasorum appears more abundant than previously reported. It is speculated that the A. vasorum increase might relate to increased snail abundance, which may be due to a rise in mean yearly temperatures in Denmark.The cardiopulmonary nematodes   •C. vulpis in American mink.The first report of •C. vulpis and A. vasorum in polecats.The first report of •A. vasorum in raccoon dogs, mink, and polecats from Denmark.The first report of •C. vulpis in raccoon dogs, mink, and polecats from Denmark.The first report of •C. aerophila in raccoon dogs and beech martens from Denmark.The first report of  Aeluros Denmark .A. vasorum in red foxes and domestic dogs for decades  and American mink  have never been recorded in Denmark) are abundant wildlife species in Denmark and could contribute to substantial contamination with environmental stages of cardiopulmonary nematodes. Red foxes are native to Denmark and the most abundant species distributed throughout the country  with a population size of approx. 40,000–60,000 individuals (Martes foina) and polecats (Mustela putorius) are believed to be less common.Red foxes, raccoon dogs , red foxes (n = 367), mink (n = 123), beech martens (n = 31), Eurasian otters (n = 30) and polecats (n = 14) were examined for cardiopulmonary nematodes .Table 1NThe carcasses were submitted for necropsy between January 2017 and March 2018, as a part of the national wildlife surveillance program. The carnivores were hunted, found dead or euthanized for animal welfare reasons. All animals were transported in sealed plastic bags and subsequently stored at −80 °C for min. 4 days prior to necropsy to inactivate potential zoonotic parasites. The wild carnivores were divided into seven regional groups depending on their origin: North Jutland (NJ), Middle Jutland (MJ), South Jutland (SJ), Funen (FU), North Zealand including Copenhagen (NZ), South Zealand (SZ) and Bornholm (BO) . Funen, At necropsy, hearts and lungs were collected and stored at −20 °C until further examination. Occasionally, hearts or lungs were unsuitable for examination due to traumatic injury, predation or decomposition. Individual data on regional origin, and gender were recorded when possible.2.2C. aerophila, from individual animals were collected in 2 mL eppendorf tubes and stored at −20 °C. Adult worms of C. aerophila were identified morphologically based on their easily identifiable characteristics, as described by C. aerophila and other cardiopulmonary parasites were subjected to DNA extraction and molecular analysis.The heart was separated from the lungs by transecting all the arteries and veins proximal to the base of the heart. The ventricles and major blood vessels were opened using scissors and the heart was examined microscopically for adult worms using a stereomicroscope  at 80–100× magnification. The pulmonary blood vessels, trachea, and bronchial tree were dissected using scissors and inspected macroscopically for adult worms. Parallel incisions were made throughout the lung lobes approximately 1–2 cm apart. Subsequently, the heart and lungs were flushed with 500 mL tap water into a 500 mL conical glass. The fluid was left to sediment for 20 min. After which, the supernatant was removed, and the sediment transferred to microscopic slides, for examination using a light microscope  at 100–400× magnification. All worms and larvae, except adult worms of 2.3From each positive animal, up to 30 larvae or two adult worms or worm fragments were distributed into 1.5 mL eppendorf tubes, i.e. some samples were distributed into >1 tube and all tubes were analysed for each animal. From a pilot study, it was verified that >30 larvae or >2 adult worms could saturate the DNA extraction columns. Genomic DNA was extracted from all eppendorf tubes using the QIAmp DNA Mini Kit  according to the manufacturer's instructions. Extracted DNA was eluted in a final volume of 100 μL AE Buffer  and stored at −20 °C.2.42.4.1Angiostrongylus vasorum and C. vulpis were identified using a modified method by 2, 2.0 μL DNA, and dH2O. PCR reactions were performed in a Rotor-Gene Q rt-PCR cycler  using the following conditions: 94 °C for 2 min, followed by 45 cycles of 94 °C for 15 s, 55 °C for 60 s, 1 min with fluorescence detection in the green (FAM) and yellow (HEX) channel. DNA from 15 A. vasorum first stage larvae (L1) obtained from a German domestic dog, and DNA from 15 C. vulpis L1 obtained from a Swedish domestic dog were used as positive controls.2.4.2A. abstrusus was performed by conventional PCR according to Taq ReadyMix , 5.0 μl template DNA, and dH2O. Amplifications were performed using a Biometra T3 Thermocycler using the following conditions: 94 °C for 7 min followed by 40 cycles of 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 45 s, and a final extension for 10 min at 72 °C. DNA from 15 A. abstrusus L1 obtained from a Danish domestic cat was used as a positive control.Examination for Amplicons were electrophoresed in a 2% agarose E-Gel™  and lengths were sized by comparison with a 100-bp DNA Ladder .2.4.3A. vasorum and C. vulpis DNA in samples containing ≥2 and ≥5 L1, respectively. The conventional PCR consistently detected A. abstrusus in samples containing ≥1 L1.For each parasite species, the detection limits were established using a duplex dilution series of MQ water spiked with 1, 2, 5, 10, 15, and 30 L1 isolated from the same material that was used for positive controls , of which 19 were co-infected with other cardiopulmonary parasites. These were subjected to DNA extraction together with other parasites from a further 119 carnivores (n = 138). In total, DNA was successfully extracted from 119/138 samples and determined to belong to A. vasorum and C. vulpis. The remaining 19 samples have been designated as positive for unidentified cardiopulmonary parasites. The prevalences and 95% CI of A. vasorum, C. vulpis, C. aerophila and unidentified nematodes by carnivore species and region of origin are listed in Angiostrongylus vasorum and C. vulpis were identified from red foxes, raccoon dogs, mink and polecats, while C. aerophila was identified from red foxes, raccoon dogs and beech martens. Otters were negative for all parasite species. None of the carnivores were positive for A. abstrusus.A. vasorum and C. vulpis were observed in 11 carnivores (six raccoon dogs and five red foxes), four were co-infected with A. vasorum and C. aerophila (one raccoon dog and three red foxes) and three were co-infected with C. vulpis and C. aerophila (three red foxes).Co-infections with 4A. vasorum, C. vulpis and C. aerophila in red foxes, but also provided novel evidence of these parasites from other wildlife species, confirming their potential as reservoir host for domestic animals in Denmark. Thus, we report, for the first time, infection with A. vasorum and C. vulpis in raccoon dogs, mink and polecats, and C. aerophila infection in raccoon dogs and beech martens from Denmark. Additionally, this is the first report of A. vasorum and C. vulpis in wild carnivores from the island of Bornholm. Lastly, this is, to our knowledge, the first report of C. vulpis infection in mink, and C. vulpis and A. vasorum infections in polecats worldwide. Aelurostrongylus abstrusus was absent from all examined animals.This study confirmed the occurrence of Angiostrongylus vasorum and C. vulpis prevalences in raccoon dogs from Denmark  were similar to those observed for red foxes from the same regions . Similarity in the regional prevalence of A. vasorum and C. vulpis was observed for both red foxes and raccoon dogs. Both parasites have previously been identified in raccoon dogs in other European countries  compared to beech martens (6.7%) and raccoon dogs (1.9%) suggesting that red foxes are the most important reservoir host. In contrast to A. vasorum and C. vulpis, C. aerophila prevalences in red foxes were similar (10.8%–13.8%) in four out of five regions. These findings concur with previous reported prevalences for red foxes in South Jutland and North Zealand  in North Zealand compared to red foxes from Jutland (1.7–2.3%), which is in line with previous reports in this area (35.9–80.0%)  . North Z decades . Originan France . Thereafse areas . Our res 5 years , in Hung 5 years , and 2.6 5 years . This initanicus , a commo vasorum . Moreove vasorum . In Denm vasorum . Moreoveinfected .A. vasorum, C. vulpis was more prevalent in red foxes from North Zealand (18.8%) compared to those from Jutland (1.7–7.6%). This contrasts findings from a previous Danish study where C. vulpis prevalence in red foxes from South Jutland and Copenhagen metropolitan area  was equal (22.9%)  (C. vulpis and C. aerophila compared to previous studies.As for  (22.9%) . Thus, C%–53.8%) . Besides%–53.8%) . The met%–53.8%) . All of Aelurostrongylus abstrusus was absent from our study sample population, suggesting that wild carnivores can be considered unimportant as reservoir hosts for this parasite. This confirms that the parasite is rather species-specific (Aelurostrongylus spp. in mink (specific , even th in mink .C. vulpis and 1.2% with A. vasorum. Interestingly, mink from other regions were not infected with these parasites. No mink were infected with C. aerophila. As Bornholm is an island, parasites were likely introduced through importation of infected animals, notably domestic dogs. As prevalences were low in mink, their role as reservoir hosts can be considered negligible.Mink is the only carnivore present on the island of Bornholm. In total, 8.2% of these was infected with 5A. vasorum and C. vulpis, and that raccoon dogs and beech martens were infected with C. aerophila. Our findings confirm that other wild carnivores in addition to red foxes can act as reservoirs for cardiopulmonary nematodes in domestic animals. Furthermore, prevalences of A. vasorum have increased in red foxes in most areas compared to previous studies, while quantitatively and epidemiologically, the role of these reservoir hosts is unknown.Our findings showed for the first time that raccoon dogs, mink, and polecats from Denmark can be infected with None.


The range from 0.5 to 500 µg/L is observed for the determination of MDMA with the MDL down to 0.25 µg/L. The practical applicability of the method is performed with the recovery ranging from 85.3% to 94% for MA and from 86.9% to 95.5% for MDMA. At the different concentrations of drugs, the relative standard deviations (RSD) for both MA and MDMA are lower than 5.7%. The method was applied to analyse 1995 blood samples that had been collected from the Forensic Medicine Centre of Ho Chi Minh City. The results showed 1.75% positive with MA and 0.25% positive with MDMA. These two drugs take 10% of the total drugs positive samples. By using deuterium-labelled methamphetamine-d5 and 3,4-methylenedioxy-N-methamphetamine-d5 as the internal standards in the determination and the use of MS/MS in multiple reaction monitoring mode signal readout, the method exhibits robustness specificity and can be applied in simultaneous determination of MA and MDMA in blood with high selectivity and sensitivity.A liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been validated for the simultaneous determination of methamphetamine (MA) and 3,4-methylenedioxy-N-methamphetamine (MDMA) in the blood sample. Under the optimal experimental conditions, the concentration of MA can be determined in the range from 1  In recent decades, the illicit use of methamphetamine, 3,4-methylenedioxy-N-methamphetamine, and other psychostimulant drugs has dramatically increased in many countries . MethampµL), this method still suffers from some drawbacks such as multistep in sample preparation and the narrow range in quantitation (0.003–0.050 mg/L). With the toxic blood concentrations of MA and MDMA ranging up to 40 mg/L for MA and 0.5 mg/L for MDMA, this method cannot be applied to evaluate and classify the effect level of these drugs in the blood sample  [For diagnosis and therapy to the person who is drug-addicted, assessment MA and MDMA level in blood is an indispensable step. Gas chromatography-tandem mass spectrometry (GC/MS) had been used for the analysis of these drugs for many years , 13, butl level) –19. Focu4OH 25%, KH2PO4, Na2HPO4, and LiChrolut® RP-18 (40–63 µm) 500 mg 3 ml standard PP-tubes were purchased from Merck . All solutions were prepared and diluted using ultrapure water (with an electric resistivity >18.3 MΩ cm) produced by a Millipore Milli Q system . Standard and internal standard working solutions (100 mg/L) were prepared from the stock standard solution (1000 mg/L), and these solutions were stored at 4°C for use in 3 months.All reagents used in this work were of analytical grade. Methamphetamine 1 mg/mL in methanol, 3,4-methylenedioxy-N-methamphetamine 1 mg/mL in methanol, methamphetamine-d5 1 mg/mL in methanol, and 3,4-methylenedioxy-N-methamphetamine-d5 1 mg/mL in methanol were purchased from Sigma-Aldrich (Singapore). Acetonitrile (ACN), methanol, acetone, dichloromethane, 2-propanol, NHµL MA-d5 2 mg/L and 250 µL MDMA-d5 2 mg/L into a 15 mL centrifuge tube, and this mixture was vortexed for 15 minutes and then left for 10 minutes. After centrifuging the mixture at 8000 rpm for 5 minutes, the analytes in the supernatant were cleaned by C18 extraction column in four steps: (1) the column is activated by 1 mL methanol, 1 mL distilled water, and 1 mL phosphate buffer solution (pH = 6); (2) 1 mL of the prepared sample is loaded within 1 minute; (3) the column is washed with 5 ml distilled water/acetone , waiting for rinse solution completely running out of the column in 5 min; and (4) the analytes are eluted by 1 mL methanol. The obtained solution was evaporated and rediluted in 500 μL MeOH and then filtered through a 0.45 μm filter paper. 5 µL final solution was analysed by HPLC-MS/MS under operating conditions shown in Blood samples were prepared according to the previous reports , 21, briIn order to confirm the accuracy in the determination of MA and MDMA in blood, the specificity, linearity, method detection limit (MDL), accuracy, and precision were evaluated according to the guidance in ASB Standard 036–2017 and ANSI/ASB Standard 072–2019 , 23.The characterization of MA and MDMA was verified by analysis of the standards. The chromatography spectrum was observed in multiple reaction monitoring modes (MRM) with mass fragments shown in Figures µg/L MA to blank sample, the single peak appeared at ∼0.441 min . In contrast, when spiking 0.2 .441 min . A simil.441 min . This coA sample . All theµL MA-d5 2 mg/L and 250 µL MDMA-d5 2 mg/L before extracted and analysed according to µg/L to 5000 µg/L with the coefficient correlation (R2) of 0.998. The concentration that can be analysed for MDMA is in a range from 0.5 to 500 µg/L with R2 ～0.999. This is an extensive range, and a low concentration of MA and MDMA in the blood can be determined. The stability of the linearity is also observed in three different days. The coefficient correlation for both MA and MDMA is obtained stable from 0.998 to 0.999. These results demonstrate that both calibration curves are available for the determination of MA and MDMA in the blood sample.The matrix-matched calibration was used in order to minimize the matrix effect to the analytical signal. The blank matrix was simultaneously spiked with proper amounts of MA and MDMA, 250 µg/L and was lower than 0.25 µg/L for MDMA , and the MQL was as ten times of SD. MDL for MA determination was lower than of 0.31 for MDMA . At the µg/L MA and MDMA on three different days by two analysts. The recovery of MA and MDMA was from 85.3 to 94% for MA and 86.9 to 95.5% for MDMA . The recovery assay results of samples spiked at three levels: 10, 100, and 300 for MDMA . The recµg/L, respectively. RSD at the MDMA concentration of 17, 137.3, and 314.6 µg/L were 5.7%, 4.3%, and 4.8%, respectively. According to the ABS standard 036–2017, this was much lower than the maximum RSD values acceptable (<20%). Therefore, it can be stated that the method exhibited highly accurate and precision for MA and MDMA determination in blood.The repeatability of the method was also evaluated in six repetitive analyses of three blood samples. The relative standard deviations were 5.2%, 4.5%, and 4.3% at the MA level of 24.8, 102.6, and 281.9 The method was applied for analysing 1995 blood samples. These samples were collected from the Forensic Medicine Centre of Ho Chi Minh City and were analysed for MA, MDMA, and other drugs such as morphine, codeine, acetaminophen, and ketamine. The percentage of total drugs positive sample was 10.02%, in which MA positive sample was 1.75%, and MDMA was 0.25%. Calculating within each kind of drug, the positive sample from the female was 25.7%, 40%, and 5.6% for MA, MDMA, and other drugs, respectively. The related abundance of MA, MDMA positive blood sample, was also compared within the total drug positive sample. The percentage of MA, MDMA, and other drugs was 17.5%, 2.5%, and 80%, respectively .The results were also classified in the range of drug concentration that differs impacts on human health. For MA, the range of concentration in therapeutic, toxic, or comatose-fatal was 0.01–0.2 mg/L, 0.2–10 mg/L, and >10 mg/L. The range for MDMA was 0.1–0.35 mg/L, 0.35–0.5 mg/L, and >0.4 mg/L –19. Compµg/L). The desirable precision and accuracy were also obtained with relative standard deviations lower than 5.7%. And the recovery ranges from 85.3 to 95.5%. The practical usefulness of this work was demonstrated by evaluating MA and MDMA in real samples. In 1995 human blood samples, the MA and MDMA positive case was 1.75% and 0.25%, respectively. In comparison with other drugs positive in the blood sample, MA and MDMA occupied 10%; this showed that these drugs were used popularly. Most MA and MDMA positive samples were in the level of therapeutic or toxic, and there was not positive sample in the level of comatose-fatal. Taken together, this work supplies a supersensitive and accurate method for the determination of MA and MDMA in the blood sample.In summary, a sensitive method has been validated for the simultaneous determination of MA and MDMA in blood samples based on deuterium-labelled internal standards and liquid chromatography-tandem mass spectrometry. The method exhibits supersensitivity and selective in the determination of MA and MDMA in the blood sample. Based on this method, the calibration curves were obtained in an extensive range and very high correlation coefficient (>0.998) with an extremely low method detection limit (～0.3


Aeromonas hydrophila, an inhabitant in the aquatic ecosystem is considered as an important foodborne bacterial zoonotic pathogen in aquaculture. The present study aimed to identify virulent A. hydrophila from naturally infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan. Rimler Shotts (RS) and blood agar medium identified the presence of pathogenic Aeromonas sp. from the infected C. striata. A. hydrophila Ah17 was identified using 16S rRNA nucleotide sequence. Extracellular enzymes such as amylase, lipase, and protease were screened in A. hydrophila Ah17. Antibiotic susceptibility tests showed A. hydrophila Ah17 was highly resistant against β‐lactam, glycopeptide, macrolides, phosphonic, fucidin, and oxazolidinone classes of antibiotics. Virulent genes such as hemolysin (aer and hly), heat‐labile enterotoxin (act), cytotonic heat‐stable enterotoxin (ast), elastase (ahyB), and lipase (lip) were identified. Growth and the viable cell population of virulent A. hydrophila Ah17 were significantly reduced in a dose‐dependent manner against shrimp chitosan (CHS) from Penaeus indicus (P. indicus). Thus, the present study isolated virulent A. hydrophila Ah17 from the environmental source and characterized in vitro with shrimp chitosan. Virulent Aeromonas hydrophila was isolated from the river stream and characterized by chitosan, an in vitro approach. The suspension was serial diluted and plated on RS agar medium (supplemented with novobiocin (5mg/L)) and incubated at 37ºC. After overnight incubation, isolates were patched on tryptic soy agar (TSA) medium for further analysis.Ulcerated regions (skin‐lesions/muscle) of infected 2.3β‐hemolysis activity, isolates were plated on blood agar medium containing 5% (v/v) defibrinated sheep blood and the activity was observed after 24 hr at 37ºC . Quality and quantity of genomic DNA were measured using Nanodrop™ (Thermo Fisher Scientific) and resolved using 0.7% agarose gel electrophoresis. Details of the primers and their product size are provided in Table Escherichia coli DH5‐α. Transformants were selected on Luria Bertani (LB) agar ampicillin (50 µg/ml) plate by Blue‐white selection method and confirmed by colony PCR. All the clones were sequenced in automated DNA sequencer .16S rRNA region was amplified for all the  Goodman , and A. 2.5://blast.ncbi.nlm.nih.gov/). The molecular phylogenetic tree was constructed using the 16S rRNA sequence of A. hydrophila Ah17 (GenBank Accession no: KY646209), by Neighbor‐Joining method  from NCBI database; KC150866, AM992197, KC793904, JN559379, JN561162, KX980436, KX980452, KC800792, X60404, DQ095200, DQ095201, AY538658, LC200778, KC812104, KC812105, KC812106, KF146349, KF146350, EU913851, EU913854, EU913855, EU913856, HM991866, AB473043, KU570318, GU204971, KU605548, KU605549, KU605564, KU605566, KU605575, KU605578, KU605581, FN997627, KM396315. Horizontal branch lengths were drawn to scale the bar indicating 10 nucleotide replacements per site.The following 16S rRNA nucleotide sequences of 2.6A. hydrophila Ah17. Briefly, for amylase activity, the isolate was patched on starch agar medium (HiMedia) and incubated at 37ºC. After incubation, the surface of the culture was flooded with Gram's iodine (HiMedia), and appearance of the zone of clearance around the colonies was indicated as amylase‐positive isolate  containing 10 ml of tributyrin and incubated at 37°C. The appearance of the zone of clearance around the colonies was indicated as lipase‐positive isolate  and incubated at 37ºC. The appearance of the zone of clearance around the colonies was indicated as protease‐positive isolate  medium (HiMedia) and kept for drying. Antibiotic discs were placed on the MHA medium and incubated at 37°C for 24–48 hr. The diameter of the zone of inhibition was measured, and susceptibility was categorized according to the manufacturer's protocol.Antibiotic susceptibility profile for 2.9A. hydrophila Ah17 were identified using PCR‐based approach. Briefly, virulent genes such as cytolytic heat‐labile enterotoxin (act), cytotonic heat‐stable enterotoxin (ast), aerolysin (aer), hemolysin (hly), elastase (ahyB) and lipase (lip) were amplified by the respective gene‐specific primers. A. hydrophila ATCC 7966 was used as the positive control for this study. Sequences of primers and the respective product sizes were provided in Table Virulent factors present in 2.102.10.1Penaeus indicus and reported in our earlier study , and finally, the bacterial cell suspension was adjusted to 1 O.D (measured at 600 nm). 1 ml of A. hydrophila Ah17 cell suspension was suspended in 9 ml of CHS  and incubated at 37°C.Antibacterial activity of CHS against A. hydrophila Ah17 against CHS. Briefly, bacterial culture was taken at different time intervals , and serial dilution was performed for all the concentrations of CHS used. Finally, 10 μl of the suspension was plated on TSA medium and incubated at 37°C. Colonies were counted at different time intervals and expressed as relative viability of A. hydrophila Ah17 against CHS. 1 ml of bacterial cell suspension and 9 ml of PBS were used as the control. All the experiments were performed in triplicates, and A. hydrophila ATCC 7966 was used as the control strain for the study.Antibacterial activity of shrimp chitosan was calculated as colony‐forming unit (CFU/ml) and expressed as relative viability of 2.10.3A. hydrophila Ah17 against CHS was estimated by MTT ‐2,5‐diphenyltetrazolium bromide) assay in 96‐well microtiter plate. Briefly, MTT  stock solution of 5 mg/ml  was prepared and filtered through 0.22‐μm syringe filter. 1 O.D culture of A. hydrophila Ah17 was serially diluted in the ratio of 1:103 in tryptic soy broth (TSB). 200 μl of CHS was added to the bacterial cell suspension with the final concentration of 0.05%, 0.1%, 0.15%, and 0.2% (w/v) in 96‐well microtiter plate and incubated at 37°C for 48hr. After incubation, MTT was added (0.5 mg/ml) on each well and incubated for 4.5 hr. Finally, the formazan product was solubilized by adding 100 μL of dimethyl sulphoxide  and incubated for 30 min. After incubation, formazan product was quantified at 590 nm using microplate absorbance reader . The quantified dissolved formazan crystals are directly proportional to the number of relative cell viability of A. hydrophila Ah17 against shrimp chitosan. All the experiments were performed in triplicates, and A. hydrophila ATCC 7966 was used as the control strain for the study.The relative cell viability of 2.11p < .05 was considered as statistically significant. All the statistical analysis was performed using the GraphPad Prism 7.0 software.One‐way analysis of variance (ANOVA) and Dunnett's multiple comparison test were performed to scrutinize the data of bacterial cell viability assay. For all comparison, 33.1C. striata from the river Cauvery are represented in , five isolates were amplified  using Aeromonas sp.‐specific 16S rRNA primers , and the rest of the isolates were identified as other Aeromonas sp. such as A. veronii and A. veronii biovar sobria.Based on nucleotide sequencing and BLAST analysis, one isolate was identified as A. hydrophila strain Ah17 was aligned on the separate branch with KC150866 and AM992197 sequence id whereas, both KC150866 and AM992197 were found in the same clade of the Indian origin A. hydrophila strains. Overall, molecular evolutionary relationship of A. hydrophila Ah17 showed that the strain Ah17 aligned with Indian origin A. hydrophila strains except for JN561162 strain , lysine decarboxylase, and arginine dihydrolase were positive for A. hydrophila Ah17 . Further, A. hydrophila Ah17 conferred resistance against various classes of antibiotics such as macrolides (clarithromycin), phosphonic (fosfomycin), fucidin (fusidic acid), oxazolidinone (linezolid). Aminoglycoside , cephalosporins (cefoperazone and cefpodoxime), streptogramins (pristinamycin), tetracycline (minocycline), and nitrofurans (nitrofurantoin) showed an intermediate response against A. hydrophila Ah17.Antimicrobial susceptibility profile of β‐lactam third‐generation antibiotic cephalosporins (cefixime), aminoglycoside (streptomycin and tobramycin), tetracycline (tetracycline and doxycycline), dihydrofolate reductase (DHFR) inhibitors (trimethoprim), quinolones , rifampicin, chloramphenicol, co‐trimoxazole, and azithromycin were sensitive to A. hydrophila Ah17.Antibiotics such as fluoroquinolones (ciprofloxacin and norfloxacin), carbapenems (Imipenem), 3.4A. hydrophila Ah17  and heat‐stable cytotonic enterotoxin (ast) in A. hydrophila Ah17 were confirmed with the isolate harbours both act (232 bp) and ast (331 bp) genes  and lipase (lip) genes was confirmed in A. hydrophila Ah17 with a product size of 513 bp and 382 bp, respectively , 24 hr (0.2% and 0.15%) and 12h (0.2%) , 12 hr (0.2% and 0.15%), 24 hr , and 48 hr  in a dose‐dependent manner  and 1% (p < .01%) group when compared to the control group. No significant difference was observed in 0.05% CHS‐treated A. hydrophila Ah17 group , 0.1% (p < .01), and 0.05% (p < .5) group when compared to the control group  formed a separate branch. Overall, all the A. hydrophila strains from Indian origin except JN561162 are grouped together. Thus, using 16S rRNA nucleotide sequences (based on N‐J method), evolutionary relatedness was drawn for A. hydrophila Ah17 strain.With the help of molecular markers, identification of specific microbial taxa and their phylogeny was explored over several decades  present in almost all members of A. hydrophila and it secretes extracellular enzymes such as amylase, protease etc., which contributes pathogenicity to the eukaryotic system. Earlier studies proved that highly virulent strains of A. hydrophila such as NJ‐35, J‐1, ML09‐119, AL09‐71 produce amylase and protease which contributes pathogenicity to the host organism  and hemolysin, nonpore‐forming toxins)  and hemolysin (hly) genes and the presence of these genes evidently supports the pathogenic nature of A. hydrophila Ah17.Virulent act), and heat‐stable cytotonic enterotoxin (ast) play a major role in diarrhoeal disease . In vitro characterization demonstrated that shrimp chitosan can able to control the growth of virulent A. hydrophila Ah17 in a dose‐dependent manner. In future, it is necessary to recognize and monitor the potential reservoirs of pathogenic bacteria and ensure their control measurements in an eco‐friendly manner, which are essentially important in epidemiological and environmental studies to prevent possible health risks.In conclusion, We declare that we have no conflict of interest.This study does not involve any human or animal testing.


Setting. Hebei Eye Hospital, Hebei, China.  To examine the magnitude, orientation, and age-related changes of corneal astigmatism of the eyes before cataract surgery.  A clinical-based retrospective study. p for nonlinearity <.01). With the increase of age, the astigmatism axis gradually changes from with-the-rule (WTR) to against-the-rule (ATR). Moreover, in young patients with age below 65 years, WTR astigmatism was negatively correlated with age, while ATR was positively correlated with age . However, in the old patients with age above and equal to 65 years, all types of astigmatism were positively correlated with age.  The study consisted of 5662 eyes of 5662 consecutive cataract surgery patients with a mean age of 68.26 ± 10.39 (mean ± standard deviation (SD)) years (range 40 to 97 years), and 59.86% of the patients were women. Mean corneal astigmatism was 0.98 ± 0.76 diopter (D) (range 0.00–9.61 D). Corneal astigmatism of 0.50–0.99 D was the most common range of values (30.08%), followed by 1.00–1.49 D (22.15%), ≤0.50 D (21.21%), and 1.50–1.99 D (10.28%). There was a strong U-shaped relation between corneal astigmatism and age . Cataract is the leading cause of blindness worldwide, and it is one of the most frequently performed surgeries globally. Cataract surgery has also gradually become less invasive and more precise. Its goal has changed from improving visual acuity to providing optimal vision and minimizing postoperative spectacle dependence . CornealTo date, previous studies reported the prevalence of preoperative astigmatism in different populations –16. HoweThis is a retrospective study of 5662 consecutive patients who attended the cataract surgery in the Ophthalmology Department of the Hebei Eye Hospital between January 2018 and June 2019. The study adhered to the Declaration of Helsinki and was approved by the Hebei Eye Hospital Ethical Committee. Informed consent was obtained from the subjects. Patients with a history of corneal disease  or intraocular surgery, ocular trauma, contact lens wear, irregular astigmatism, and corneal diseases that did not allow IOL Master measurement were excluded from the study.The medical and systematic histories of the patients were reviewed. The patient underwent a comprehensive eye examination, including slit lamp biomicroscopy, applanation tonometry and fundus ophthalmoscopy through dilated pupils. The data points extracted for the study included age, sex, and the magnitude and orientation of corneal astigmatism. All reported measurements of corneal astigmatism refer to values obtained from the IOL Master 500 . Experienced technicians measured keratometry data with, at least, three automatic measurements performed in each eye.Corneal astigmatism was designated as with-the-rule (WTR) when the axis of correcting minus cylinder was within 180 ± 30 degrees (the steep meridian of the cornea being within 90 ± 30 degrees in this case), against-the-rule (ATR) when the correcting minus cylinder axis was within 90 ± 30 degrees, and oblique if it was neither WTR nor ATR.χ2 test for categorical variables, respectively. The Bonferroni method was used for post hoc pairwise comparisons. Associations between corneal astigmatism and age were determined using Spearman's correlation analysis and nonlinear regression analysis. p values of <.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics Version 25 and R Version 3.6.0. Graphs were generated with GraphPad Prism 8.Data were tested for normal distribution using the Kolmogorov–Smirnov test. Descriptive statistics are the mean ± standard deviation (SD) for normally variables and median values/interquartile range for nonnormal variables. Frequency and percentages were used for categorical variables. Comparisons among the age groups were performed using nonparametric tests for continuous variables and the Z = 1.16, p=.138).A total of 5662 eyes of 5662 patients were included in the study, of which 3389 were females. The mean age of patients was 68.26 ± 10.39 years (range 40–97 years). Mean corneal astigmatism was 0.98 ± 0.76 diopter (D) (range 0–9.61 D) . The frep < .005. The astigmatism values of each age group were 0.81 (0.50–1.25) for 40–49 years of age, 0.75 (0.48–1.19) for 50–59 years of age, 0.75 (0.50–1.12) for 60–69 years of age, and 0.75 (0.50–1.25) for 70–79 years of age  s of age .p for nonlinearity <.01)  .p < 0.5) (p < 0.1)  . In younp < 0.1) .r = −0.11, p=.001; r = 0.10, p=.010, respectively). There was no association between corneal astigmatism and age in the oblique group. However, the corneal astigmatism of old patients was positively correlated with age, regardless of the type of astigmatic axis , which is comparable to 1.01 ± 0.69 by Chen et al.  and 0.98p < .01), which was consistent with conclusions of most previous articles [p < .01). In contrast, as in other published articles [p < .01). The shift might be induced by possibly changes in corneal structure [r = 0.10, p=.010; r = 0.16, p < .001, respectively). Previous studies found that ATR corneal astigmatism increased with age even after sutureless cataract surgery, and this change was similar to the aging process of the normal cornea [We also found that there was a positive correlation in the prevalence of the ATR astigmatism with age , which could benefit from toric IOL implantation. Considering the trend of magnitude, orientation, and age-related changes of corneal astigmatism, ATR corneal astigmatism should be considered more aggressively with the aim to fully correct or overcorrect in long-term astigmatism treatment. Although the majority of our study population came from patients in our hospital, these data will not only expand the database of corneal astigmatism in the Chinese population but also provide valuable and practical information to surgeons when selecting the appropriate surgical method and toric IOLs.


Theileria equi, Babesia caballi, and a recently reported new species, T. haneyi. Infections by these apicomplexan parasites limit performance and cause economic losses for the horse industry. Equine piroplasmosis is widespread in the northern regions of Nigeria, where an increasing portion of the animal population is composed of horses. This disease has remained epidemiologically challenging, especially as the movement of horses increases across Nigeria. In this study, blood samples from 300 horses were collected in three states of northwestern Nigeria. The presence of piroplasms was screened by nested PCR targeting 18S rDNA and positive samples were analyzed using species-specific-nested PCR-targeting genes including ema1 (T. equi), rap1 , and a gene coding a protein of unknown function (T. haneyi). Species-specific-nPCR results demonstrated that the prevalence of T. equi was 13.0% (39/300), B. caballi was 3.3% (10/300) and T. haneyi was 2.7% (8/300). Mixed infections with T. equi and B. caballi was 2.7% (8/300) while T. equi, B. caballi, and T. haneyi multiple infection prevalence was 0.6% (2/300). We used 18S rDNA sequences to determine close relationships between T. equi by phylogenetic analysis and demonstrated that among 57 sequences of Theileria parasites, 28 samples belonged to clade A (49%), 13 samples were found to be clade C (22%), and 16 were clade D (28%). These results demonstrate the genetic diversity of T. equi circulating in horses from Nigeria.Equine piroplasmosis (EP) is an infectious, tick-borne disease caused by the hemoprotozoan parasites,  Theileria equi, Babesia caballi, and a recently reported new species, T. haneyi  is an infectious, tick-borne disease caused by the hemoprotozoan parasites T. haneyi, defined as a new species infective to equids that lacks ema-1, the current diagnostic test target, allows the new parasite to elude current regulatory tests . The application began at the center of the circle and moved out spirally towards the edge to result in a uniform distribution and the opportunity to isolate DNA from repeated sample punches. After applying the blood, the card was stored at room temperature for 6 h to dry. The dried card was sealed in a re-sealable bag with a pack of desiccant and stored in a cool, dry, and dark place until analysis.A sample disc approximately 4 mm in diameter was taken from each sample spot using a disposable biopsy punch with a plunger . The sample discs were placed in 0.2-mL PCR tubes. Two hundred microliters of FTA Purification Reagent  was added to each PCR tube and incubated for 5 min at room temperature. The supernatant was discarded. This procedure was repeated for a total of 3 washes with FTA Purification Reagent. The discs were suspended in 200 μl of TE buffer  and incubated for 5 min at room temperature. The same procedure was repeated for a total of 2 washes with TE buffer. The discs were then allowed to dry at room temperature for 1 h. The FTA disc was used for PCR amplification.A hemi-nested PCR (the reverse primer being used in each reaction) assay was used to amplify the hypervariable region of the 18S rRNA gene as previously described  to identify specific piroplasms present in the samples. Primer sets for T. equi (ema-1 gene), T. haneyi (gene coding protein of unknown function), and B. caballi (rap-1 gene) are shown in Table T. equi and B. caballi nPCR are as follows: the PCR external reaction was prepared with 12.5 μl 2× Platinum SuperFi PCR master mix , 1 μl of 10 μM of each primer, the FTA disc, and 9.5 μl of nuclease-free water. The PCR cycling conditions for T. equi and B. caballi were a denaturation at 98 °C for 5 min, followed by 35 cycles of 98 °C for 20 s, 59.5 °C for 20 s, and 72 °C for 20 s, with a final extension at 72 °C for 5 min. In the internal PCR reaction, 1.0 μl of each PCR product from the external PCR reaction was used as a template and added to 12.5 uL 2× Platinum Superfi master mix and 11.5 uL of water. The internal reaction consisted of denaturation at 98 °C for 5 min followed by 35 cycles of 98 °C for 5 s, 61 °C for 5 s, and 72 °C for 5 s, with a final extension at 72 °C for 5 min , 1 μl of 10 μM of each primer, the FTA disc, and 9.5 μl of nuclease-free water. The cycling condition for the external reaction of T. haneyi included an initial denaturation at 95 °C for 4 min., followed by 35 cycles of 95 °C for 20 s, 63.5 °C for 30 s, and 72 °C for 20 s and a final extension at 72 °C for 7 min  for which 18S-PCR and sequencing unexpectedly resulted in B. bovis 18S rDNA identity. The nPCR primers targeting B. bovis rap-1 gene are reported in Table Finally, a https://blast.ncbi.nlm.nih.gov/Blast.cgi) using nucleotide Blast with default settings. Reference sequences for 18S rDNA representing T. equi genotypes A–E, T. haneyi, B. caballi, and B. bovis were retrieved from GenBank for inclusion in the phylogenetic analysis , T. equi clade B (AB515310), T. equi clade C , T. equi clade D , and T. equi clade E (HM229408). Reference sequences for B. bovis (AY150059) and B. caballi (EU642512) were also included in the analysis as outgroups.The 18S rDNA nPCR products from samples that were positive for 18S nPCR but negative for a species-specific reaction were sequenced to identify other parasites infecting horses. The samples were amplified for the V4 region of the 18S rRNA gene. The PCR products were cleaned up using ExoSAP-IT reagent  before being sent for sequencing using amplifying primers . Sequencher  was used to trim raw sequences, and the resulting sequences were analyzed at the NCBI website  B. caballi, and 2.7% (8/300) T. haneyi  of horses and triple infection with T. equi, B. caballi, and T. haneyi was detected in 0.6% (2/300) horses. However, not all 18S rDNA positives could be amplified by species-specific nPCR. Interestingly, two individual horses (Thatcher198 and Castora213 RC) were 18S rDNA sequence positive for B. bovis and confirmed to be that by specific nPCR (data not shown).In the nPCR targeting the 18S rDNA gene, 33.3% (110/300) of samples were positive. To determine the specific species of organisms infecting horses, individual nPCRs and sequencing were performed using the 18S rDNA positive samples. The overall results for species-specific reactions yielded 13.0% (39/300) for T. equi was 18.7% (17/91). B. caballi and T. haneyi were not detected. In Zaria LGA (Kaduna State), 152 horses were sampled and the nPCR prevalence of T. equi was 6.6% (10/151), while B. caballi had 3.3% (5/151) and T. haneyi was 4.9% (6/151). Double infection with T. equi and B. caballi was 3.3% (5/151). In Mai’adua LGA (Katsina state), 32 horses were sampled and the nPCR prevalence for T. equi was 34.4% (11/32), B. caballi 12.5% (4/32), and T. haneyi were 6.25% (2/32). Double infection with T. equi and B. caballi were 6.25% (2/32) and triple infections with T equi, B. caballi, and T. haneyi were 6.25% (2/32). In Maigatari LGA (Jigawa state), 25 horses were sampled and the nPCR result for T. equi was 4% (1/25) and B. caballi 4% (1/25). Double infection with T. equi and B. caballi was found in 4% of the samples (1/25).In Igabi Local Government Area (LGA) (Kaduna State), 91 horses were sampled and the prevalence for Theileria that infect horses, two were B. caballi and two were B. bovis. The familiar clade members that were recognized for Theileria with the highest percent similarity to the query sequence were 28 as clade A, 13 as clade C, and 16 as clade D. All the familiar clades were found in Nigeria with exception of clades B and E. To construct the phylogenetic tree, we used 22 randomly selected Nigerian samples of which 18 were closely related to Theileria spp. that infect horses, two were B. caballi and two were B. bovis  has one of the highest numbers of polo ponies in the region, both exotic and indigenous, and annually hosts international polo tournaments, horse racing, and other horse-related activities. Zaria LGA hosts the most prestigious traditional horse procession (durbar), as well as polo and horse racing. This region has one of the largest populations of horses in Kaduna state. Hundreds of horse transits in and out of Zaria, particularly the ancient city, are coming mostly from different parts of northern Nigeria and the Niger Republic. A variety of events occur in this region including twice a year horse processions (durbar), polo events, and horse racing. These events have made Zaria an epidemiologically important niche for disease transmission, infection, and enabling possible adaptation of apicomplexans to other hosts, which could potentially result in various clinical manifestations that relate to EP that have not yet been fully recognized.Though the number of horses sampled in Zaria was higher compared to other LGAs, it was evident that the result in this area represented the genotypes and parasites found in the other areas. Katsina and Jigawa both share a substantial border with the Niger Republic; the markets were part of the network of centuries-old markets when the region played an important role as the hub of trans-Saharan trade routes. These livestock markets linked into the road networks and settlement patterns, which makes it easier to transport animals across Nigeria. The movement of horses across the northern borders of Nigeria from various neighboring countries for various purposes could have been responsible for the pattern of results seen in the study areas. Therefore, these results could be an epidemiological reflection of EP status in West African Sub-region.T. equi genotpes A C and D were identified in Kadunga and Kasinga, while genotypes A and D were only detected in Jigawa. Significantly, T. equi clades B and E were absent in all the samples analyzed. Consistent with other studies, genotype diversity of Theileria was reported in horses in South Africa, Israel, USA, and Spain (Criado-Fornelio et al. Since there is no restriction on horse importation, the current horse importation strategy in Nigeria could lead to the introduction of these clades in the future. A large number of the Argentine polo ponies were negative for EP. These negative results could be related to the routine health care management practice which involves the administration of imidocarb which could have led to the persistently low parasitemia that usually follows a fluctuating parasite-load pattern in recovered horses (Ueti et al. T. haneyi, there were questions raised on whether co-infection with T. equi and T. haneyi occurs naturally. The data obtained from this work prove that T. equi and T. haneyi can co-infect naturally and additionally with B. caballi. Experimental co-infection of T. equi and T. haneyi was reported (Sears et al. T. haneyi. It was evident that T. haneyi was not detected at the time of arrival. The failure to detect T. haneyi is no surprise; Knowles et al. (T. haneyi lacks ema-1 which allows the parasite to elude the current regulatory cELISA test which is based on EMA-1 of the immunodominant EMA superfamily and a specific monoclonal antibody (Knowles et al. T. haneyi as compared to the species-specific-nested PCR. This discrepancy is potentially associated with a reduced sensitivity to the gene coding for a protein of unknown function used to detect T. haneyi. The T. haneyi reference JQ390047 in Fig. T. haneyi reference JQ390047 has only 2 nucleotide differences with the Nigerian clade C samples yet gives the appearance of greater distinctness in the phylogram. For comparison, T. equi clade A reference JX177671 has 24 nucleotide differences compared to clade C members in the phylogram, demonstrating the distinction of a separated clade. No samples reported here were 100% identical to T. haneyi.Following the serendipitous discovery of s et al.  reportedB. caballi is not predominant in areas that are EP-prone as confirmed using molecular assays. The occurrence of B. caballi ranges from 0 to 19.3% depending on the area (Heim et al. B. caballi was reported in Mongolia (Munkhjargal et al. B. caballi detected in this study could be a result of the very low parasitemia that characterizes B. caballi infections, which rarely exceeds 1% and the removal of B. caballi over a period of time by the horse immune system (Hanafusa et al. Compared with equine theileriosis, B. bovis DNA from horses that arrived in Nigeria from Argentina. Two animals were both 18S rDNA-PCR positive with sequences closely related to B. bovis, and those samples were also B. bovis species-specific nPCR positive. Herein, we have presented that imported horses could be infected with B. bovis, where different strains could be distributed to various areas of the world, considering the human-driven global horse movement. Whether B. bovis established infection in these horses is unknown, further investigation is needed to rule out the possibility of B. bovis, and other bovine Apicomplexan parasites, to establish infections in horses. Nonetheless, if B. bovis established infection, these horses could be sources of parasite dissemination. This could necessitate testing horses frequently whenever they are moving into disease-controlled areas. This is supported by the recent identification of T. annulata, B. ovis, and B. canis species in horse blood samples in Turkey (Ozubek and Aktas T. equi and T. velifera in guard dogs kept in horse stables in Saudi Arabia (Salim et al. T. equi has been detected in clinically ill dogs in Croatia (Beck et al. An interesting finding was the detection of T. equi, T. haneyi, and B. caballi are present in Nigeria and circulating on an endemic basis among the equine species supported by the current study. The presence of infected horses may play a critical role in the spread of these pathogens via tick vectors. However, it is unknown which ticks are vector competent in Nigeria. Determination of the various competent tick vectors responsible for parasite transmission warrants additional study. Therefore, surveillance and restriction of the international movement of equids are required to prevent the introduction of infected horses into Nigeria to avoid pathogen dissemination.It is evident that