Gliotoxin and bis(methylthio)gliotoxin are not reliable as biomarkers of invasive aspergillosis.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) remains a life-threatening opportunistic infection, but can be difficult to diagnose. New biomarkers are therefore needed. Gliotoxin (GT), a secondary metabolite of Aspergillus fumigatus, and bis(methylthio)gliotoxin (bmGT), a degradation product of GT, have been proposed as potential biomarkers. However, these findings have yet to be confirmed. OBJECTIVES: To identify the diagnostic potential of GT and bmGT in serum and bronchoalveolar lavage fluid (BALf) in haematology patients compared to galactomannan (GM). MATERIALS AND METHODS: We prospectively collected culture supernatant, serum and BALf from patients with culture-positive IPA and measured GT and bmGT concentrations using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Galactomannan was detected using a commercially available enzyme immunoassay. RESULTS: We included 18 patients with proven (n = 6) and probable (n = 12) IPA, all with positive cultures for Aspergillus fumigatus. BmGT was only detected in serum from one patient (5.6%), whereas GM was positive (optical density ≥ 0.5) in 11/18 patients (61.1%, P = 0.002). We could not find GT in any serum sample. In BALf, bmGT was detected in 8/18 patients (44.4%) and GT in 9/18 patients (50%), compared to GM (optical density ≥ 1.0) in all patients (100%). CONCLUSIONS: Gliotoxin and bis(methylthio)gliotoxin had a very poor performance for diagnosing IPA. As other biomarkers are more sensitive and easier to detect, we would not recommend serum or BALf GT/bmGT to be used in the diagnosis of IPA.

Development of a Fluorescently Labeled Aptamer Structure-Switching Assay for Sensitive and Rapid Detection of Gliotoxin.

Gliotoxin, one of the most toxic metabolites produced during the growth of Aspergillus fumigatus, can cause direct damage to the immune system and results in infection and spread of Aspergillus, or even leads to invasive aspergillosis. Accurate, rapid, and sensitive detection of the disease-specific marker gliotoxin, particularly in serum, urine, or other body fluids, is therefore an important approach to achieving early and rapid diagnosis of Invasive Aspergillus Fumigatus Infection (IAFI). In this study, aptamers that specifically bind to gliotoxin were successfully obtained using immobilization-free GO-SELEX technology. Furthermore, the performance of the aptamer, including binding affinity, targeting specificity, and structural stability, was further improved by optimizing through truncation and mutation. Finally, the optimized aptamer APT8T1M was used to develop a novel fluorescently labeled aptamer structure-switching assay (FLASSA) for the detection of gliotoxin. The method exhibited a good linear range from 0.1 nM to 100 nM of gliotoxin, with a lower detection limit of 0.05 nM. Moreover, FLASSA was applied to the detection of gliotoxin in spiked serum and urine samples. A good mean recovery of 98.76-110.85% and a low coefficient of variation (5.45-14.59%) were obtained, indicating a high degree of selectivity for gliotoxin, good reproducibility, and stability. These results show that the developed FLASSA has significant potential and offers an alternative to the traditional analytical methods for the rapid, sensitive, and efficient detection of gliotoxin, thus, providing an effective tool for the early and rapid diagnosis of IAFI.

Prospective evaluation of a combination of fungal biomarkers for the diagnosis of invasive fungal disease in high-risk haematology patients.

We prospectively evaluated a combination of fungal biomarkers in adult haematology patients with focus on their clinical utility at different time points during the course of infection. In total, 135 patients were monitored once to twice weekly for serum (1-3)-ß-d-glucan (BG), galactomannan (GM), bis-methyl-gliotoxin and urinary d-arabinitol/l-arabinitol ratio. In all, 13 cases with proven or probable invasive fungal disease (IFD) were identified. The sensitivity of BG and GM at the time of diagnosis (TOD) was low, but within 2 weeks from the TOD the sensitivity of BG was 92%. BG >800 pg/mL was highly specific for IFD. At a pre-test probability of 12%, both BG and GM had negative predictive values (NPV) >0.9 but low positive predictive values (PPV). In a subgroup analysis of patients with clinically suspected IFD (pre-test probability of 35%), the NPV was lower, but the PPV for BG was 0.86 at cut-off 160 pg/mL. Among IFD patients, 91% had patterns of consecutively positive and increasing BG levels. Bis-methyl-gliotoxin was undetectable in 15 patients with proven, probable and possible IA. To conclude, BG was the superior fungal marker for IFD diagnosis. Quantification above the limit of detection and graphical evaluation of the pattern of dynamics are warranted in the interpretation of BG results.

Disseminated aspergillosis in an immunocompetent patient with detectable bis(methylthio)gliotoxin and negative galactomannan.

BACKGROUND: Disseminated invasive aspergillosis is an exceptional finding in immunocompetent hosts. As in immunocompromised patients, it has high mortality rates. Early diagnostic methods are required in order to properly manage the patient. Bis(methylthio)gliotoxin (bmGT) is a novel biomarker, useful in onco-hematological patients. CASE REPORT: A 70-year-old male, with non-insulin dependent type II diabetes mellitus and a past surgery history of aortic valve replacement with coronary by-pass five years ago, was seen in the emergency department with blurred vision. Three days later, endogen endophthalmitis was diagnosed in the ophthalmology clinic. During admission for the vitrectomy, he suffered an ischemia of the right lower limb. A thoracic computed tomography revealed a mycotic aneurysm of the ascending thoracic aorta and parietal thrombus. The ascending aorta was replaced and abundant brittle material of infectious appearance, found between the aortic valve graft and the aneurysm, was removed. Aspergillus fumigatus sensu stricto grew in both vitreous and aorta cultures. BmGT was detected in two serum samples obtained prior to intravenous antifungal treatment, which was then reduced after voriconazole treatment was started. CONCLUSIONS: Disseminated invasive aspergillosis is a severe disease regardless of the immune status of the patient. This case report suggests that bmGT could be a suitable early diagnostic biomarker, not only in neutropenic patients, but also in immunocompetent hosts.

Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis.

Early and accurate diagnosis of invasive aspergillosis (IA) is one of the most critical steps needed to efficiently treat the infection and reduce the high mortality rates that can occur. We have previously found that the Aspergillus spp. secondary metabolite, bis(methylthio)gliotoxin (bmGT), can be detected in the serum from patients with possible/probable IA. Thus, it could be used as a diagnosis marker of the infection. However, there is no data available concerning the sensitivity, specificity and performance of bmGT to detect the infection. Here, we have performed a prospective study comparing bmGT detection with galactomannan (GM), the most frequently used and adopted approach for IA diagnosis, in 357 sera from 90 episodes of patients at risk of IA. Our results, involving 79 patients that finally met inclusion criteria, suggest that bmGT presents higher sensitivity and positive predictive value (PPV) than GM and similar specificity and negative predictive value (NPV). Importantly, the combination of GM and bmGT increased the PPV (100 %) and NPV (97.5 %) of the individual biomarkers, demonstrating its potential utility in empirical antifungal treatment guidance and withdrawal. These results indicate that bmGT could be a good biomarker candidate for IA diagnosis and, in combination with GM, could result in highly specific diagnosis of IA and management of patients at risk of infection.

Development and validation of an HPLC-MS/MS method for the early diagnosis of aspergillosis.

Invasive aspergillosis is an opportunistic infection that is mainly caused by Aspergillus fumigatus, which is known to produce several secondary metabolites, including gliotoxin, the most abundant metabolite produced during hyphal growth. The diagnosis of invasive aspergillosis is often made late in the infection because of the lack of reliable and feasible diagnostic techniques; therefore, early detection is critical to begin treatment and avoid more serious complications. The present work reports the development and validation of an HPLC-MS/MS method for the detection of gliotoxin in the serum of patients with suspected aspergillosis. Chromatographic separation was achieved using an XBridge C18 column (150 × 2.1 mm id; 5 mm particle size) maintained at 25 °C with the corresponding guard column (XBridge C18, 10 × 2.1 mm id, 5 mm particle size). The mobile phase was composed of a gradient of water and acetonitrile/water (95:5 v/v), both containing 1 mM ammonium formate with a flow rate of 0.45 mL min(-1). Data from the validation studies demonstrate that this new method is highly sensitive, selective, linear, precise, accurate and free from matrix interference. The developed method was successfully applied to samples from patients suspected of having aspergillosis. Therefore, the developed method has considerable potential as a diagnostic technique for aspergillosis.

Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis.

The virulence factor gliotoxin (GT) and its inactive derivative, bis(methylthio)gliotoxin (bmGT), are produced by pathogens of the genus Aspergillus. Here we report the detection of GT and bmGT in serum of humans at risk of invasive aspergillosis (IA) as well as in cultures of fungal isolates derived from patients with proven infection with A. fumigatus. Although both compounds are readily recoverable from spiked human serum or plasma, only bmGT is retained in whole blood, indicating that bmGT may be the better marker for in vivo detection. Accordingly, bmGT was found more frequently than GT in samples from patients at risk of IA and incultures of clinical isolates of A. fumigatus. In some cases, bmGT was detected before mycologic evidence ofinfection was gained. Importantly, neither GT nor bmGT was found in serum from healthy donors or from neutropenic patients without any sign of infection. Thus, bmGT presence might provide a more reliable indicator of A. fumigatus infections than GT. Due to its simplicity and sensitivity, a diagnostic technology based on this test could be easily adopted in clinical laboratories to help in the diagnosis of this often fatal fungal infection.

Development of an HPTLC-based diagnostic method for invasive aspergillosis.

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.

Mechanism of intestinal-derived fungal sepsis by gliotoxin, a fungal metabolite.

BACKGROUND/PURPOSE: Gut barrier dysfunction resulting from fungal overgrowth may be caused by the interaction of gliotoxin (GT), a fungal metabolite, with enterocytes. The goal of this study was to determine the mechanisms by which gliotoxin (GT), a fungal metabolite, causes enterocyte apoptosis. METHODS: The authors measured enterocyte apoptosis, caspase-3 activity, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage in GT-exposed IEC-6 cells, a rat intestinal cell line. RESULTS: GT induced apoptosis in IEC-6 cells. The pan-caspase inhibitor ZVAD suppressed this GT-mediated apoptosis. GT induced a 15-fold increase in caspase-3 activity over media control. The authors detected PARP cleavage by after GT exposure. DTT pretreatment decreased apoptosis compared with GT alone. CONCLUSIONS: This study supports the concept that fungal overgrowth may lead to gut barrier dysfunction by the local release of gliotoxin and the induction enterocyte apoptosis.

Rat CNS cell culture. Enhancement of neuronal survival and delay of glial proliferation by serum from patients with multiple sclerosis. A morphological study.

The addition of serum from multiple sclerosis (MS) patients to the culture medium of dissociated cells from cerebral hemispheres of rat embryos caused a delay in glial proliferation and an enhancement of neuronal survival. Sera from normal individuals and patients with other neurological diseases failed to show this effect. These morphological observations are interpreted as the outcome of inhibition of in vitro gliogenesis.