Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition.
BACKGROUND
Pathogen identification in clinical routine is based on the cultivation of microbes with subsequent morphological and physiological characterisation lasting at least 24 hours. However, early and accurate identification is a crucial requisite for fast and optimally targeted antimicrobial treatment. Molecular biology based techniques allow fast identification, however discrimination of very closely related species remains still difficult.


RESULTS
A molecular approach is presented for the rapid identification of pathogens combining PCR amplification with microarray detection. The DNA chip comprises oligonucleotide capture probes for 25 different pathogens including Gram positive cocci, the most frequently encountered genera of Enterobacteriaceae, non-fermenter and clinical relevant Candida species. The observed detection limits varied from 10 cells (e.g. E. coli) to 10(5) cells (S. aureus) per mL artificially spiked blood. Thus the current low sensitivity for some species still represents a barrier for clinical application. Successful discrimination of closely related species was achieved by a signal pattern recognition approach based on the k-nearest-neighbour method. A prototype software providing this statistical evaluation was developed, allowing correct identification in 100 % of the cases at the genus and in 96.7 % at the species level (n = 241).


CONCLUSIONS
The newly developed molecular assay can be carried out within 6 hours in a research laboratory from pathogen isolation to species identification. From our results we conclude that DNA microarrays can be a useful tool for rapid identification of closely related pathogens particularly when the protocols are adapted to the special clinical scenarios.
Wiesinger-Mayr
Herbert
H
Molecular Diagnostics, Austrian Research Centers GmbH - ARC, Mendelstrasse 1, A-2444 Seibersdorf, Austria. herbert.wiesinger@arcs.ac.at
Vierlinger
Klemens
K
Pichler
Rudolf
R
Kriegner
Albert
A
Hirschl
Alexander M
AM
Presterl
Elisabeth
E
Bodrossy
Levente
L
Noehammer
Christa
C
D016470:Bacteremia / Q000382:microbiology; D001419:Bacteria / Q000145:classification* / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D001769:Blood / Q000382:microbiology*; D002175:Candida / Q000145:classification* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D016469:Fungemia / Q000382:microbiology; D006801:Humans; D025202:Molecular Diagnostic Techniques; D016533:Mycological Typing Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes
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