In Vitro Release by Aspergillus fumigatus of Galactofuranose Antigens, 1,3-β- d -Glucan, and DNA, Surrogate Markers Used for Diagnosis of Invasive Aspergillosis

Abstract

Aspergillus markers are becoming increasingly important for the early diagnosis of invasive aspergillosis. The kinetics of release of these surrogate markers, however, is largely unknown. We investigated the release of ␤-(1-5)-galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-␤-D-glucan (BG) (Fungitell), and DNA (PCR) in an in vitro model of Aspergillus fumigatus. The results showed that release is correlated to the growth phase of the fungus, which depends on available nutrients. Whereas galf antigens and BG are released during logarithmic growth, DNA is released only after mycelium breakdown. During early logarithmic growth, galf antigens seem to be released somewhat earlier than BG. Furthermore, galf antigen concentrations of more than 120,000 times the serum cutoff value (0.5 ng/ml) can be measured, while BG concentrations reach a value only 978 times the serum cutoff value (60 pg/ml). During lytical growth, release of galf antigens further increased to a maximum level, which depended on pH. After that, the concentration of galf antigens stayed high (pH 7.4) or decreased to zero within 4 days (pH 5.0). In contrast to galf antigens, BG concentration decreased after 1 day of growth. The decrease of galf components seems to be due to the enzyme ␤-galactofuranosidase, which is able to destroy galf epitopes and whose activity fluctuates in the culture filtrates in parallel with galf antigen concentration. Fungal DNA seems to be released only due to autolysis caused by nutrient limitation. In conclusion, several factors clearly influence the release of surrogate markers in vitro. These same factors might also play a role at the infection site of Aspergillus disease in humans.Invasive aspergillosis (IA) has become a leading cause of death among immunocompromised hosts, including transplant patients, those treated for hematological malignancy, and those treated with high-dose corticosteroids (41, 49). In addition, IA is increasingly observed in the nonneutropenic phase after hematopoietic stem cell transplantation and in nonclassic settings, such as intensive care units with critically ill patients (13, 16). The high mortality is due partly to the difficulty in establishing a diagnosis at an early stage of infection, since presenting symptoms are nonspecific and sensitivity of cultures is low. Techniques to improve timely diagnosis have focused on the detection of circulating surrogate markers released by the fungus (43,45,50). With the development of nonculture-based methods, such as PCR and antigen detection, circulating markers can be detected at an early stage of infection in patients with invasive disease (12, 27, 28).The commercially available sandwich enzyme-linked immunosorbent assay (ELISA) (Platelia Aspergillus [PA]; Bio-Rad, Marnes-la-Coquette, France) is based on the rat immunoglobulin M monoclonal antibody EB-A2, which binds the ␤-(1-5)-galactofuranosyl (galf) side chains of the Aspergillus galactomannan (GM) molecule (24,50,51). In addition to GM, fungal glycoproteins also react with the EB-...

Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species

“…By degrading GM epitopes, the enzyme may interfere with the immunoassay by blocking the antigen-antibody complex formation. The demonstration that A. fumigatus produces 2-20% of the β-D-galactofuranosidase produced by A. niger 6 suggests that A. fumigatus could react more avidly in the GM EIA test than A. niger, as found in the current study. The influence of the β-D-galactofuranosidase on GM levels is not completely clear, since similar enzyme activity has been observed for patients with IA caused by A. fumigatus in spite of serum GM levels 5 .…”

PCR in Diagnosis of Invasive Aspergillosis: a Meta-Analysis of Diagnostic Performance

“…circulate in the blood. Of note, many studies suggest that most of the circulating Aspergillus DNA is present not in the form of conidia but rather as free DNA (67,68). This provides an explanation for the fact that blood cultures have such a low sensitivity for aspergillosis (69), as well as our finding that whole-blood testing is not superior to serum testing.…”

Evaluation of Real-Time PCR, Galactomannan Enzyme-Linked Immunosorbent Assay (ELISA), and a Novel Lateral-Flow Device for Diagnosis of Invasive Aspergillosis

“…A previous study has shown that GM is released during active growth of Aspergillus fumigatus, whereas DNA is released through hyphal damage autolysis or host defenses and will not be released concomitantly with GM (19). The extracellular glycoprotein targeted by the LFD is also released during active hyphal growth, and greater concordance with GM positivity could be predicted (11).…”