Development of an HPTLC‐based diagnostic method for invasive aspergillosis

Abstract

A rapid, sensitive and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single-step extraction with chloroform. The method uses TLC aluminum plates pre-coated with silica gel 60F-254 as a stationary phase and toluene-isoamyl alcohol-methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R(f) = 0.51). The calibration curve was linear (r(2) > or = 0.994) between peak area and concentration in the tested range of 100-1000 ng spot(-1) with minimum detectable range 0.025 ng mu(-1) of serum sample. The mean +/- SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 +/- 1.555635 and 915.8 +/- 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost-effective diagnosis of IA.

RsmA Regulates Aspergillus fumigatus Gliotoxin Cluster Metabolites Including Cyclo(L-Phe-L-Ser), a Potential New Diagnostic Marker for Invasive Aspergillosis

“…Additionally, these mutants show reduced virulence in non-neutropenic murine models but not neutropenic models [6], [18]–[20]. This toxin and/or its derivatives are also considered for use for diagnosis of IA [21], [22]. Aside from gliZ, no other transcription factors have been described to regulate the gli cluster.…”

Determining Eight Biogenic Amines in Surface Water Using High-Performance Liquid Chromatography–Tandem Mass Spectrometry

“…The sample pretreatment for LC-MS is quick and easy. Other methods for determining BAs were excluded because of false positives [22]. The standard addition recovery test showed that HPLC-MS/ MS has higher sensitivity and a detection limit down to lower level compared with the current conventional liquid phase standard procedure.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…These include the surface protein Cf2, which was isolated from a human patient with proven IA and is also present in growing hypha of A. fumigatus but not in spores [107] as is Asp f6/MnSOD [108], gliotoxin, which is produced by most A. fumigatus strains and is present in plasma and serum [109] and its inactive derivative bis(methylthio)gliotoxin, which is also recoverable from blood [110], 15 immunogenic proteins of the immunosecretome [111] and secreted proteases whose activity in serum specimens might serve as a new diagnostic approach if used with reporter peptides [112].…”