Conventional or molecular measurement of Aspergillus load

The quantification of organisms is a standard tool. Measurement of a hyphal organism, like Aspergillus, presents difficulties in that it is problematic to define what constitutes a cell. Growth occurs by hyphal tip extension, in which the hypha elongates and a septum is formed behind the tip to divide it in to a separate compartment. However, communication between compartments and streaming of nuclei makes defining a cell of a hyphal organism difficult and the best method for quantification of the hyphal organism remains controversial. Conventional CFU determination of fungal burden in tissue homogenates is readily done by most laboratories, but CFU recovered temporally tend to show minimal increase, and may indicate that mechanical homogenization does not cause significant fragmentation of the hyphae in the tissues. Does the lack of increase in CFU as infection worsens inadequately reflect fungal load in the tissue? Non-culture based assays including chitin determination, quantitative PCR and enzyme immunoassay (EIA) of cell wall constituents, galactomannan or -glucan overcome this difficulty in part. However, qPCR and EIA do not indicate viability, may result in false negatives. qPCR assay may represent a significant over-estimation, because it correlates with number of nuclei present; it also requires specialized equipment and reagents. Temporal studies of infection have demonstrated that qPCR and to some extent EIA reflect an increase in fungal burden not shown by CFU. Although qPCR and CFU may not correlate in those types of studies, comparative studies have shown CFU and qPCR do correlate when determining antifungal drug efficacy, where each method can clearly demonstrate differences in fungal burden; EIA methods have also been shown to reflect this difference. Overall, there remains no optimal, single method for determination of fungal load of Aspergillus and it may be that a combination of methods (e.g., CFU and qPCR) should be used.