Examination of Aspergillosis

Laboratory
Direct examination
Clinical material, such as fluids, sputa, or tissue, is mounted in 10% KOH. Long, branching, hyaline, septate hyphae approximately 3.0 µm in diameter typify aspergillosis. The demonstration of hyphae in the clinical specimen and the repeated recovery of the same species of Aspergillus in culture is critical in supporting the diagnosis of aspergillosis. It must always be remembered that a number of other fungi can be morphologically identical to Aspergillus in tissue. On rare occasions, the hyphae of an Aspergillus sp. may have lateral conidia in tissue.

Isolation
Inoculate the clinical material onto Sabouraud glucose agar, Inhibitory Mould Agar (IMA) or other proper medium with antibiotics (gentamicin or chlorampenicol) and incubate at 30°C. The aspergilli are sensitive to cycloheximide, hence they will not grow on media containing this antimicrobial agent. Discard negative cultures after 4 weeks.
Mycology (principal fungi) : Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus group, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus group

Detection of galactomannan antigen in serum
The mortality rate of invasive aspergillosis is as high as 50-100% and definitive diagnosis by culture may take as long as 4 weeks. Thus, early diagnosis is of remarkable significance for earlier initiation of antifungal therapy and reduction of mortality rates. Detection of galactomannan antigen, an exoantigen of Aspergillus, has recently been shown to be a useful screening test for early diagnosis of invasive aspergillosis. Platelia Aspergillus EIA (Bio-Rad Laboratories) is a commercially available kit used to detect galactomannan antigen in body fluids. This method can detect as little as 1 ng/ml of galactomannan in the tested sample. Serum is the most frequently tested specimen and appears to provide highest sensitivity. Use of other samples such as bronchoalveolar lavage and cerebrospinal fluid also appears to provide promising results. Galactomannan antigen positivity is among the microbiological diagnostic criteria proposed by European Organization for Research and Treatment of Cancer (EUORTC) and Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) for diagnosis of invasive aspergillosis This tool continues to gain acceptance: the US Food & Drug Administration approved the marketing of Platelia Aspergillus EIA kit in USA on 16 May 2003. Galactomannan antigen positivity can be detected 5-8 days (average) before clinical signs develop (in 65.2% of patients), findings on chest X-ray are visible (in 71.5% of patients) and culture results become positive (in 100% of patient). The test should be used as a screening test for patients at high risk of developing invasive aspergillosis.Detection of positive results particularly in two consecutive serum samples provides strong support for the diagnosis of invasive aspergillosis. Platelia Aspergillus EIA can also be used for follow-up of clinical response to antifungal therapy. The titer of the antigen tends to decrease in case of clinical response, except for patients who are treated with an echinocandin compound.The overall sensitivity and specificity of the method were 80.7% and 89.2%, respectively, in the dataset submitted to the US FDA. False positive reactions have been observed in 1-18% of the tested samples and may be due to cross reactivity or false positive antigenemia. Cross reactivity may be due to the existence of other fungi, such as Penicillium chrysogenum, Penicillium digitatum, Rhodotorula rubra, and Paecilomyces variotii in the tested sample [2208]. The mechanism of false positive antigenemia, on the other hand, has not been fully clarified. It appears to be more frequent in children and may develop after translocation of galactomannan antigen found in various food stuff (bread, pasta, corn flakes, rice, cake, turkey, sausage, etc.) through the damaged intestinal mucosa. In addition to these, very recent data have shown that the serum samples of patients receiving piperacillin/tazobactam (Zosyn®), an injectable antibacterial combination product from Wyeth Pharmaceuticals, may also yield false positive galactomannan antigen test results. Detection of galactomannan antigen in certain batches of Zosyn® strengthened this finding. Thus, Bio-Rad, the manufacturer of the Platelia Aspergillus EIA kit, now states that positive galactomannan antigen test results in patients treated with Zosyn® should be interpreted cautiously. Since the existence of invasive aspergillosis cannot be ruled out in these patients, other methods should be used for confirmation of the diagnosis.