Diagnosis of zygomycosis remains difficult. There are no specific clinical or radiological signs for zygomycosis and the relative low frequency of the disease explain why the diagnosis is not always suspected. A definite diagnosis could be done by histopathology or direct examination of infected tissues, by demonstration of hyphae characteristic of a zygomycete (Figure 7.2). In tissue sections, hyphae are broad, ribbon-like with irregular diameter, non- or rarely septated, and branched at right angles (Ribes et al., 2000). These morphological characteristics are different from those of hyphomycetes (e.g. Aspergillus spp.). Demonstration of fungal elements could be done after routine histopathological staining but are better visualized with fungal-specific staining such as Gomori methenamine-silver (GMS) or periodic acid-Schiff. Direct examination of tissue or other samples such as

Figure 7.2 Typical morphology of zygomycetes in tissues (Gomori methenamine-silver stain). Characteristics of hyphae are identical for all species. In tissue sections, hyphae are broad, ribbonlike with irregular diameter, non- or rarely septated, and branched at right angles

bronchoalveolar lavage, preferably after specific fungal stain (e.g. GMS, Calcofluor white), should also be performed in the microbiology laboratory before culture. Demonstration of typical hyphae is of prime importance for at least two reasons. First, because mucorales are common laboratory contaminants and the signification of a positive culture from a non-sterile site is difficult to interpret when histology or direct examination is negative or has not been performed. Second, culture of infected samples is often negative. Probably due to their coenocytic nature, the hyphae seem to lose viability very easily. In this respect, it is not recommended to grind tissues before culture (Dromer & McGinnis, 2002), but alternatively to place small pieces of intact tissues directly on the culture medium. As zygomycetes are sensitive to cycloheximide, the culture medium should not contain this antimicrobial agent. Although, zygomycetes have the capability to invade vessels and to disseminate, blood cultures are usually negative, even in case of disseminated disease. There are no serological methods or antigen detection tests currently available. PCR and other molecular-based diagnostic methods have been recently evaluated but are not yet standardized and are not commercially available.

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