Utility of Aspergillus Antigen Detection in Specimens Other than Serum Specimens

Department of Medical Microbiology, University Medical Center St. Radboud, Nijmegen, The Netherlands.
Clinical Infectious Diseases (Impact Factor: 8.89). 12/2004; 39(10):1467-74. DOI: 10.1086/425317
Source: PubMed


The detection of circulating galactomannan in serum is an important tool for the early diagnosis of invasive aspergillosis.
A commercial enzyme-linked immunosorbent assay (Platelia Aspergillus; BioRad) was shown to be both highly sensitive and specific for detection of galactomannan in serum samples. Despite the fact
that this assay is validated for serum samples, specimens of other body fluids are increasingly used for detection of galactomannan,
including urine, bronchoalveolar lavage fluid, and cerebrospinal fluid. Review of the literature shows that galactomannan
can be detected in each of these samples from patients with invasive aspergillosis with higher sensitivity than is the case
with culture, as well as early in the course of infection. However, the evidence thus far is based on case reports—predominantly
retrospective studies—that often include heterogeneous patient populations and limited numbers of cases of proven infection.
Clearly, well-designed prospective studies with systematic sampling and use of consensus case definitions are needed to compare
the performance of antigen detection in samples other than serum specimens with that in serum specimens.

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    • "Galactomannan is relatively specific for Aspergillus species45, and can be detected in urine, bronchoalveolar lavage fluid cerebrospinal fluid and other specimens with enzyme immunoassay. Various sensitivity rates from 30 to 100 per cent, and similarly wide-ranging specificities from 38 to 98 per cent have been reported for GM4647. Factors that limit the specificity of this test are immune reactivity with other fungi such as Penicillium spp. "
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    • "We next tested the affinity of EB-A2, a monoclonal antibody that binds Galf residues with high specificity, towards galactomannoproteins extracted from the disruptant strains (Fig. 2B). Clinical screening with EB-A2 is considered to be a highly sensitive and specific assay for early detection of invasive aspergillosis (Klont et al., 2004), and the specificity of EB-A2 for Galf antigen is well characterized (Stynen et al., 1992; Yuen et al., 2001; Leitao et al., 2003; Morelle et al., 2005). The main epitope of EB-A2 is reportedly a tetrasaccharide of β1,5-linked Galf (galactofuran side-chain) (Stynen et al., 1992); however, EB-A2 also reacts with a single terminal nonreducing Galf residue in N-glycans and terminal β1,5- linked Galf residues in O-glycans (Yuen et al., 2001; Leitao et al., 2003; Morelle et al., 2005). "
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    ABSTRACT: The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan, and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple galactofuranose monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in galactofuranose (Galf) antigen biosynthesis. Disruption of gfsA reduced binding of β-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro galactofuranose antigen synthase assay revealed that GfsA has β1,5- or β1,6- galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-D-galactofuranose as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature, and limited formation of conidia. Several gfsA orthologs were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β-galactofuranosyltransferase, which was shown to be involved in galactofuranose antigen biosynthesis of O-glycans in the Golgi.
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    • "There is evidence that PCR can detect Aspergillus DNA in patient samples in the absence of GM antigenaemia (Cuenca-Estrella et al., 2009). Early detection of Aspergillus fumigatus in patient cerebrospinal fluid (CSF) has been reported using the Platelia Aspergillus EIA kit for GM (Bio-Rad; Klont et al., 2004) and by nested PCR (Hummel et al., 2006). There is still a need for tools that enable earlier diagnosis of infection and that will allow optimal treatment with the prospect of improving the outcome of CNS aspergillosis. "
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    ABSTRACT: The central nervous system (CNS) is the most common site of dissemination during Aspergillus infection. PCR has the potential to facilitate early diagnosis of CNS aspergillosis, which could assist in reducing disease mortality. In two experiments, neutropenic CD-1 male mice were infected intracranially with 5×10⁶ conidia of Aspergillus fumigatus. At time points up to 120 h after infection, mice were euthanized and samples of blood, brain, spinal cord and cerebrospinal fluid (CSF) were taken. The brain fungal burden was determined by quantitative culture, and fungal DNA was detected by quantitative PCR. Plating for A. fumigatus from the brain confirmed that all mice had burdens of log₁₀>3 from 4 to 120 h after infection. A. fumigatus DNA was detected in blood (88 %), brain (96 %), CSF (52 %) and spinal cord (92 %) samples. The brain and spinal cord contained the highest concentrations of fungal DNA. Adapting the extraction protocol to maximize yield from small sample volumes (10 µl CSF or 200 µl blood) allowed PCR detection of A. fumigatus in infected mice, suggesting the use of CSF and blood as diagnostic clinical samples for CNS aspergillosis.
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