The diagnosis of zygomycosis is usually made histologically, and the demonstration of fungal elements from cytologic preparations is complicated by the difficulty of extracting fungal elements from invaded tissues.11 The poor sensitivity of sputum culture (<25%) makes diagnosis of pulmonary zygomycosis challenging.³⁸ The yield of BAL is not higher,^38,67 but direct microscopy of BAL together with transbronchial biopsy may increase the yield.⁴³ A positive finding from BAL from a neutropaenic or immunocompromised host would be highly suggestive of infection, and should be treated as such.⁴³ Even though, on microscopy, Mucorales have been classically described as having broad (10 to 50?m), ribbon-like aseptate hyphae with right-angle branching, the hyphae are actually pauciseptate, and the angle of hyphal branching can vary from 45° to 90°, reinforcing the importance of obtaining material for culture.68 About 80% of disseminated infections with S. prolificans are associated with positive blood cultures, but this proportion is much lower with S. apiospermum infections.⁴⁵

All tissues from patients with suspected infection should be stained with fungal stains in parallel with regular stains. The practice of assessing H&E stains of tissues before deciding whether to use specialised stains for fungi frequently introduces fatal delays for patients. Reporting of specimens containing any fungal elements should always include the presence and absence of yeast forms, hyphae and whether or not they are septate, if it is possible to tell, and whether there is any melanin present.⁵⁷ However, while the demonstration of Aspergillus in tissue is the reference standard for diagnosis of invasive aspergillosis, a definitive diagnosis is possible only after identification of the fungus cultured from that tissue.⁶⁹ So, part of the biopsed tissue or other surgical specimen should be sent to the microbiology laboratory (not in formalin), in addition to the pathology laboratory. Immunohistological staining using polyclonal fluorescent antibody reagents can distinguish Aspergillus spp. from Fusarium spp.⁷⁰ In situ hybridisation may also help to distinguish Fusarium spp. from Aspergillus and Pseudoallescheria in tissue sections.⁷¹

New Diagnostic Tools

Galactomannan

Galactomannan is a cell wall polysaccharide released by Aspergillus spp. during fungal growth in tissue.^69,72,73 A commercially available sandwich ELISA (Platelia Aspergillus, BioRad) detects galactomannan by use of a rat monoclonal antibody. The test has been validated for serum specimens only and has a detection limit of ~1ng/mL, which is 10 to 15 times lower than the limit of the latex agglutination test used previously (Pastorex Aspergillus, Biorad).^73–76 Circulating galactomannan may be detected at a median of five to eight days before clinical manifestation of aspergillosis.^72,77–80 The concentration of circulating galactomannan corresponds with the fungal tissue burden^81,82 and may therefore be used to monitor the response to treatment.^77,78
Studies evaluating the role of galactomannan assay in the diagnosis of invasive aspergillosis have largely been conducted with leukaemia patients or haematopoietic stem-cell transplantation (HSCT) recipients.^{77,78,82–89} After initial clinical studies suggested a high sensitivity and specificity,^{73,76,78,79,90,91} further studies revealed high rates of false-positive results among paediatric patients and neonates.^81,92,93 In one study, rates of false-positive results as high as 83% were observed ⁹³ that may be related to cross-reactivity with Bifidobacterium bifidum, found in large inocula in the guts of breast- and formula-fed infants.⁹⁴ The presence of a damaged gut endothelium may increase the absorption of dietary galactomannan.⁹⁵ Specificity was also lower in adult allo-HSCT recipients than in adult auto-HSCT recipients or non-transplant patients.⁹² The rate of false-positive results is high in the first 30 days following bone marrow transplantation and 10 days after starting cytotoxic chemotherapy.^96,97 The use of galactomannan as a surveillance tool in transplant recipients has been associated with a positive predictive value of only 10%.⁹² The results also suggest that routine ELISA tests are not useful in patients with febrile neutropaenia with no clinical or radiological signs suggestive of a pulmonary infection.92 Otherwise, all high-risk patients with a respiratory tract infection or suspected extrapulmonary aspergillosis should be repeatedly tested with galactomannan ELISA, as the predictive positive value of the assay was highest in these groups.⁹²

Cross-reactivity of Platelia Aspergillus galactomannan with Penicillium spp. has been noted96 but is deemed to be of little clinical relevance since Penicillium spp. are rarely pathogens in humans. In addition, drugs of fungal origin, such as antibiotics, may be associated with a falsepositive test, including ampicillin-sulbactam, piperacillin-tazobactam, and amoxicillin-clavulanic acid.^98–102 The timing of collection of the sample may influence the test results, with reactivity being less likely in samples collected at trough levels or prior to the administration of the dose.103 ELISA cross-reactivity has been observed with other fungi, including Paecilomyces variotii, and Alternaria spp.⁹² False-positive reactions have also been seen in bacteraemic patients,⁹⁶ and in those with autoreactive antibodies.^64,88

In a study of 3,924 serum samples in cancer patients, the overall sensitivity of the ELISA galactomannan test was only 29.4% (64.5% when only patients with proven invasive aspergillosis were analysed).⁹² Low sensitivity for the ELISA assay has also been reported in allo-HSCT recipients (60%), lung transplant recipients (30%),⁸⁹ liver transplant recipients (56%),and in patients with various other conditions including non-malignant diseases (52%).^83,93 While a positive test in a lung transplant recipient with a clinical illness compatible with invasive aspergillosis may be considered highly suggestive of this infection, a negative test does not rule out aspergillosis.⁶⁴ The sensitivity of the test has been typically lower in non-neutropaenic patients (15% to 30%) and may be related to lower circulating galactomannan levels.⁶⁴ The use of both prophylactic and empiric antifungals may also lower antigen levels by decreasing the fungal load.64