Rapid detection of the aspergillosis biomarker triacetylfusarinine C using interference-enhanced Raman spectroscopy.

Triacetylfusarinine C (TAFC) is a siderophore produced by certain fungal species and might serve as a highly useful biomarker for the fast diagnosis of invasive aspergillosis. Due to its renal elimination, the biomarker is found in urine samples of patients suffering from Aspergillus infections. Accordingly, non-invasive diagnosis from this easily obtainable body fluid is possible. Within our contribution, we demonstrate how Raman microspectroscopy enables a sensitive and specific detection of TAFC. We characterized the TAFC iron complex and its iron-free form using conventional and interference-enhanced Raman spectroscopy (IERS) and compared the spectra with the related compound ferrioxamine B, which is produced by bacterial species. Even though IERS only offers a moderate enhancement of the Raman signal, the employment of respective substrates allowed lowering the detection limit to reach the clinically relevant range. The achieved limit of detection using IERS was 0.5 ng of TAFC, which is already well within the clinically relevant range. By using an extraction protocol, we were able to detect 1.4 µg/mL TAFC via IERS from urine within less than 3 h including sample preparation and data analysis. We could further show that TAFC and ferrioxamine B can be clearly distinguished by means of their Raman spectra even in very low concentrations.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Pharmacokinetics, Safety, and Tolerability of Cefiderocol, a Novel Siderophore Cephalosporin for Gram-Negative Bacteria, in Healthy Subjects.

Cefiderocol is a novel parenteral siderophore cephalosporin that shows potent efficacy against various Gram-negative bacteria, including carbapenem-resistant strains, in vitro and in preclinical models of infection. The aim of the present study was to evaluate the pharmacokinetics (PK), safety, and tolerability of cefiderocol after both single and multiple dosing by intravenous infusion over 60 min in healthy adult subjects. A single-ascending-dose study at doses of 100, 250, 500, 1,000, and 2,000 mg was conducted in 40 healthy Japanese males and females (6 individuals receiving the active drug and 2 individuals receiving a placebo per cohort). A multiple-ascending-dose study at doses of 1,000 (two groups) and 2,000 mg every 8 h (q8h) was conducted in 30 healthy Japanese and Caucasian males (8 individuals receiving the active drug and 2 individuals receiving a placebo per cohort). There were no serious or clinically significant adverse events (AEs) observed in either study. A single subject receiving 1,000 mg cefiderocol q8h was withdrawn due to AEs. Dose-proportional increases in the maximum plasma concentration (Cmax), the area under the concentration-time curve (AUC) from time zero to the time of the last quantifiable concentration after dosing, and the area under the concentration-time curve extrapolated from time zero to infinity were observed across the dose range of 100 to 2,000 mg. The mean plasma half-life of cefiderocol was 1.98 to 2.74 h. Cefiderocol was primarily excreted unchanged in the urine (61.5% to 68.4% of the dose). There was little accumulation of Cmax and AUC by dosing q8h, and the PK of cefiderocol did not change with multiple dosing. This study indicates that single and multiple intravenous doses of cefiderocol at up to 2,000 mg are well tolerated in healthy subjects and exhibit linear PK at doses up to 2,000 mg.

Modulation of urinary siderophores by the diet, gut microbiota and inflammation in mice.

Mammalian siderophores are believed to play a critical role in maintaining iron homeostasis. However, the properties and functions of mammalian siderophores have not been fully clarified. In this study, we have employed Chrome Azurol S (CAS) assay which is a well-established method for bacterial siderophores study, to detect and quantify mammalian siderophores in urine samples. Our study demonstrates that siderophores in urine can be altered by diet, gut microbiota and inflammation. C57BL/6 mice, fed on plant-based chow diets which contain numerous phytochemicals, have more siderophores in the urine compared to those fed on purified diets. Urinary siderophores were up-regulated in iron overload conditions, but not altered by other tested nutrients status. Further, germ-free mice displayed 50% reduced urinary siderophores, in comparison to conventional mice, indicating microbiota biotransformation is critical in generating or stimulating host metabolism to create more siderophores. Altered urinary siderophores levels during inflammation suggest that host health conditions influence systemic siderophores level. This is the first report to measure urinary siderophores as a whole, describing how siderophores levels are modulated under different physiological conditions. We believe that our study opens up a new field in mammalian siderophores research and the technique we used in a novel manner has the potential to be applied to clinical purpose.

An expedient reverse-phase high-performance chromatography (RP-HPLC) based method for high-throughput analysis of deferoxamine and ferrioxamine in urine.

The present study was planned to optimize and validate an expedient reverse-phase high chromatography (RP-HPLC) based protocol for the analysis of deferoxamine (DFO) and ferrioxamine (FO) in urinary execration of patients suffering ß-thalassemia major. The optimized RP-HPLC method was found to be linear over the wide range of DFO and FO concentration (1-90 µg/mL) with appreciable recovery rates (79.64-97.30%) of quality controls at improved detection and quantitation limits and acceptable inter and intraday variability. Real-time analysis of DFO and FO in the urine of thalassemic patients (male and female) at different intervals of Desferal®(Novartis Pharmaceuticals Corporation) injection revealed DFO and FO excretion at significantly (p < 0) different rates. The maximum concentrations of DFO (76.7 ± 3.06 µg/mL) and FO (74.2 ± 3.25 µg/mL) were found in urine samples, collected after 6 h of drug infusion while the minimum levels of DFO (1.10 ± 0.12 µg/mL) and FO (2.97 ± 0.13 µg/mL) were excreted by patients after 24 h. The present paper offers balanced conditions for an expedient, reliable and quick determination of DFO and FO in urine samples.

[Production of pili, hemolysin and siderophores in the urinary isolates of Escherichia coli].

INTRODUCTION: Escherichia coli (E. coli) are the most frequent cause of the urinary tract infections. Uropathogenic E. coli (UPEC) produce virulence factors which enable them to survive in the urinary tract and cause an infection. OBJECTIVE: The objective of this study was to determine phenotype characterization of E. coli isolated from outpatients' urine in the region of Banja Luka over three-year period. In line with the objective, the following research tasks were set up: determining the production of type 1 fimbriae, P-pili, alpha-hemolysin and siderophores. METHODS: A total of 417 urinary isolates and 100 control intestinal isolates were screened for virulence factors. Production of adhesions was confirmed by haemagglutination test. Plate haemolysis test was done for the detection of alpha-hemolysin, and siderophores production assay was carried out by using the method named chrome azurol sulfonate agar diffusion assay. RESULTS: In the group of urinary isolates, almost 60% of isolates produced two or three virulence factors; only 3.8% produced none of the virulence factors. In the group of intestinal isolates, even 43% of isolates produced none of the virulence factors while 48% of isolates produced a single virulence factor and 9% produced two virulence factors. CONCLUSION: Urinary isolates E. coli express significantly more P-pili, alpha-hemolysin and siderophore than intestinal isolates (p < 0.001).There was no significant difference in production of type 1 fimbriae among the urinary and intestinal isolates.