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Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed., (© 2012 Blackwell Verlag GmbH.)

In the course of invasive aspergillosis, Aspergillus fumigatus is capable of penetrating any tissue of the host. Secretory proteinases of the fungus might facilitate the hyphae to grow through fibrillar proteins like elastin and collagen. However, using systemic infection models, no significantly reduced virulence could be shown with fungal mutants deficient for all known secretory proteinases. Thus, secretory proteinases might be of minor relevance for the pathogenesis of invasive aspergillosis. In addition, microscopic examination of aspergilli penetrating vessel walls did not reveal obvious lysis of wall proteins, thus emphasizing a mechanical disruption of fibrillar proteins by the growing hyphae. However, a strictly localized proteolysis at the tips of growing hyphae caused by wall associated proteinases might be involved. Candidates for such a mechanism are the activities of aspartic and serine proteinases which we have discovered in the cell wall fraction of A. fumigatus.

A method for the detection of Candida albicans from up to 15 ml of blood by polymerase chain reaction (PCR), based on the differential resistance of mammalian and fungal cells towards detergent was developed. The procedure essentially involved removal of the blood cells by sodium dodecyl sulfate (SDS) induced lysis, followed by DNA extraction after degradation of fungal cell walls by a recombinant beta-1,3-glucanase. The genes of two different aspartic proteinases from C. albicans, SAP1 and SAP2, with an overall homology of 77% in their nucleotide sequences, were chosen as targets for PCR. The oligonucleotide primers used were directed to strictly conserved regions similar in both genes. As the number of base pairs between the primers are different in the two genes, amplification products of 220 bp and 238 bp in length were obtained. This led to a characteristic double band in subsequent agarose gel electrophoresis. The detection limit for a nested PCR was less than 10 C. albicans cells ml-1 of seeded blood. The detection limit of conventional PCR from a blood volume in the 10 ml range was less than 100 yeasts ml-1. Preliminary trials with clinical blood specimens suggested, that conventional PCR from large blood samples, being less laborious and prone to contamination than nested PCR, could be suited for the detection of deepseated C. albicans mycosis.

Sporulating colonies of Aspergillus fumigatus, A. flavus, and A. niger were subjected to immunofluorescence using specific polyclonal antibodies against the aspergillopepsin PEP (EC 3.4.23.18), a secretory aspartic proteinase produced by A. fumigatus. The proteinase antigen was found mainly in developing conidiophores of aspergilli, in submerged mycelia and on the tips of growing aerial mycelia. Mature aerial hyphae and spores showed no immunofluorescence at all. Sporulating conidiophores revealed only weak activity in A. fumigatus and A. flavus. The distinct pattern of expression of the aspartic proteinase antigens suggests a role for such enzymes in the growth of hyphae and the development of conidiophores and thus for the sporulation process in aspergilli.