Value of Candida serum markers in patients with invasive candidiasis after myeloablative chemotherapy.

Invasive Candida infections are associated with a significant morbidity and mortality. Detection of circulating biomarkers has been shown to precede conventional diagnostic methods, which is important in improving outcome. We investigated the performance of multiple biomarkers using Candida antigen and anti-Candida antibody detection systems of Platelia and Serion and beta-d-glucan detection in serial serum samples from patients, treated for leukemia, with invasive candidiasis. The performance of single assays and combined detection appeared different for patients with 1 or more episodes of neutropenia and is therefore related to the phase of therapy for the underlying leukemia of the patient. These new insights may help to optimize the diagnostic strategies for the diagnosis of invasive candidiasis.

Improved detection of circulating Aspergillus antigen by use of a modified pretreatment procedure.

Detection of circulating galactofuranose (galf) antigens, including galactomannan (GM), by the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) is an important tool in the early diagnosis of invasive aspergillosis (IA). We used a modified pretreatment technique (MT) on consecutive negative PA ELISA plasma samples from IA patients in order to improve the detection of the fungal components present. Plasma samples (52) were collected from healthy donors, and 174 plasma samples with a galactomannan index (GMI) below 0.5 were collected from 25 unclassifiable and 23 IA patients. The PA ELISA reactivity of pretreated samples was determined before (conventional technique [CT]) and after (MT) filtration using a Microcon filter with a 50-kDa cutoff (Millipore). For the MT, the sensitivity of the PA ELISA increased from 42.9% (CT) to 78.6% (MT) using a cutoff for the GMI of 1.5 in the probable and proven group, whereas specificity slightly decreased from 98.7% to 96.1% in the control group. The 10-fold concentration step increased the GMI as high as 121-fold. The MT resulted not only in positive reactivity in samples that tested negative with the CT but also in the earlier detection of antigen by 2 to 17 days.

Paradoxical increase in circulating Aspergillus antigen during treatment with caspofungin in a patient with pulmonary aspergillosis.

A paradoxical increase in circulating Aspergillus antigen was observed during treatment with caspofungin in a patient with proven invasive aspergillosis. With the exception of treatment with the echinocandin, no other factors were found that might explain this clinical observation, which was supported by experiments done in vitro.

In vitro release by Aspergillus fumigatus of galactofuranose antigens, 1,3-beta-D-glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis.

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 beta-(1-5)-galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-beta-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 beta-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.

Bifidobacterial lipoglycan as a new cause for false-positive platelia Aspergillus enzyme-linked immunosorbent assay reactivity.

We previously hypothesized that a lipoglycan of Bifidobacterium bifidum subsp. pennsylvanicum cross-reacts with the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) based on the presence of galactofuranosyl epitopes in the cell wall (M. A. S. H. Mennink-Kersten, R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E. Verweij, Lancet 363:325-327, 2004). We tested this hypothesis by testing bacterial suspensions of different bifidobacterial species and other gram-positive and -negative bacteria with the PA ELISA, which is used to detect circulating galactomannan for the serodiagnosis of invasive aspergillosis. Furthermore, neonatal fecal samples were enumerated for bifidobacteria by fluorescence in situ hybridization (FISH) and tested for PA ELISA reactivity. All bifidobacteria, except B. infantis and B. adolescentis, showed reactivity 6- to 600-fold higher compared to the controls (i.e., Micrococcus luteus and Propionibacterium freudenreichii, which contain a cell wall lipomannan). Eggerthella lenta showed a 25-fold-higher reactivity. ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a beta-1,5-galactofuranosyl chain. All neonatal feces showed PA ELISA reactivity and associated numbers of bifidobacteria. Since high concentrations of bifidobacteria are present in the human gut, these bacteria or excreted lipoglycan may cause false serum PA ELISA reactivity in selected patient groups, especially neonates.