S 17

DIAGNOSTIC TOOLS IN DETECTION OF INVASIVE FUNGAL INFECTIONS

C. Lass-Flörl
Institute for Hygiene, University of Innsbruck, Austria

National Nosocomial Infections Surveillance (NNIS) System data show that fungi account for 9% of all nosocomial infections. From 1980 through 1990, the nosocomial fungal infection rate increased from 2.0 to 3.8 infections per 1,000 patient discharges. The most common fungi reported were Candida spp. (85.6%), followed by Aspergillus spp. (1.3%). C. albicans accounted for 76% of all Candida spp. infections. Other fungal pathogens (e.g., Malassezia, Trichosporon, Fusarium, and Acremonium) represented 11% of the nosocomial fungal pathogens. Increasing secular trends in nosocomial fungal infection rates were seen for urinary tract, and bloodstream infections and pneumonia in the intensive care units (ICUs) reporting to the NNIS System from 1986 through 1996. The crude mortality from opportunistic fungal infections exceeds 50% in most studies and has been reported as high as 95% in bone marrow transplant recipients with Aspergillus spp. infection. The attributable mortality for patients with candidemia has been estimated at 38%.
These infections are difficult to diagnose because cultures are often negative, or they become positive late in the disease. If a culture is positive, accurate identification of the organism is laborious and time-consuming, relying on macroscopic and microscopic morphologic characteristics, biochemical tests, and serotyping.
Therefore, several alternative diagnostic methods have been developed. Some of these have been very successful, such as the cryptococcal and histoplasma antigen detection methods, which have become diagnostic standards due to their availability and diagnostic performance. Some, on the other hand, have been frustrating and laid to rest, such as Candida antibodies and metabolites.
More recent advances include the detection of genetic material of organisms by polymerase chain reaction (PCR) and detection of the fungal cell components such as galactomannan and beta-glucan. Fungal PCR has been a venue with intense research. Aspergillus has been by far the most explored fungus, but assays are now being developed for multiple organisms. There is now a multitude of techniques that include both quantitative and qualitative methods, real-time PCR, and a combination of PCR and enzyme-linked immunosorbent assay (ELISA). These assays can be carried out on blood and other fluids, such as bronchoalveolar lavage. Once the techniques are fully standardized, the primers decided on, and the tests readily available in clinical laboratories, this area holds promise as the standard for diagnosis of these infections.
The galactomannan assay for Aspergillus has shown repeated good performance in a variety of settings and hosts. Yet, drawbacks persist, for example, these tests often must be performed on multiple sera over time in order to achieve acceptable sensitivity, and could be false positive in case of food uptake (eg tea, milk).
At present, highly specific and sensitive tests are not available for early and rapid diagnosis of candidiasis and aspergillosis, the two most common and clinically important opportunistic fungal diseases in immunocompromised hosts.

S 18

NEW TECHNIQUES IN MOLECULAR BIOLOGICAL DIAGNOSTICS

H.-J. Burkardt
Roche Diagnostics, Rotkreuz, Schweiz

Since the commercial introduction of PCR in diagnostics in 1992, a very rapid evolution of this technology happened. The original PCR recipes mostly consisted of an effective, but cumbersome sample preparation (e. g. with DNA extraction by phenol/chloroform technique), an amplification in a thermal cycler and detection of amplification products by gel electrophoresis. In instances of diagnoses with important consequences (like in the case of an HIV infection), confirmation had to be done by southern blotting or similar methods. To make PCR a routine method in daily laboratory life, the procedures had to be simplified and preferably to be automated. The steps undertaken in the evolution in those directions were in chronological order:
(1) replacement of gel electrophoresis by hybridisation methods in a micro titer plate format,
(2) combined automation of amplification and detection,
(3) automation of sample preparation,
(4) replacement of heterogeneous PCR test formats by homogeneous ones ("real time PCR") and
(5) for multi-parameter testing combination of PCR with micro-array techniques ("DNA chips").
This presentation will deal with and go into detail of automated sample preparation (example: COBAS AmpliPrep), real time PCR (example: COBAS TaqMan) and Chip technology (example: Affymetrix chips) and report the current status and what can be expected in the near future.