Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Enterobacteriaceae</it>, non-fermenter and clinical relevant <it>Candida </it>species. The observed detection limits varied from 10 cells (e.g. <it>E. coli</it>) to 10⁵cells (<it>S. aureus</it>) 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).</p> <p>Conclusion</p> <p>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.</p

Gene Expression Profiling in AGS Cells Stimulated with Helicobacter pylori Isogenic Strains (cagA Positive or cagA Negative)

To study host response to CagA, human gastric cancer cell line AGS was infected with a Helicobacter pylori type I wild-type or isogenic cagA-negative mutant. Differentially expressed genes were identified using cDNA array technology. By Northern blotting, downregulation of focal adhesion kinase and upregulation of LIM kinase mRNA in the presence of CagA were clearly verified. Furthermore, upregulation of LIM kinase, macrophage inflammatory protein-2, c-myc, and bone morphogenetic protein-1 and downregulation of transcription factor Y-box binding protein-1 and focal adhesion kinase mRNA in response to H. pylori type I infection compared to the uninfected control could be shown by Northern blotting. Hence, these findings identified new targets for further functional studies on H. pylori-associated pathogenesis

Comparison of a New Commercial Test, GLABRATA RTT, with a Dipstick Test for Rapid Identification of Candida glabrata

This study compares the performance of a 3-h dipstick trehalose test with GLABRATA RTT, a new commercially available 20-min test for the rapid identification of Candida glabrata. With the exception of blood agar, GLABRATA RTT gave reliable results with all media tested and was always superior to the dipstick test

Development of Novel Real-Time PCR Assays for Detection and Differentiation of Eleven Medically Important Aspergillus and Candida Species in Clinical Specimens

In the present study, novel real-time PCR assays targeting the fungal ITS2 region were developed for the detection and differentiation of medically important Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) and Candida species (Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) using a LightCycler instrument. The combination of a group-specific and a universal primer with five Aspergillus or six Candida species-specific biprobes in one reaction mixture facilitated rapid screening and species differentiation by the characteristic peak melting temperatures of the biprobes. Both assays can be performed either as single assays or simultaneously in the same LightCycler run. The analytical sensitivity using pure cultures and EDTA-anticoagulated blood, cerebrospinal fluid (CSF), and tissue samples spiked with A. fumigatus and C. albicans cell suspensions was shown to be at least 1 CFU per PCR, corresponding to 5 to 10 CFU/ml blood and 10 CFU/200 μl CSF or 0.02 g tissue. To assess the clinical applicability, 26 respiratory samples, 4 tissue samples from the maxillary sinus, and 1 blood sample were retrospectively tested and real-time PCR results were compared with results from culture, histology, or a galactomannan enzyme-linked immunosorbent assay (ELISA). Twenty samples (64.5%) were both culture positive and positive by real-time PCR. Six samples (19.4%) showed no growth of fungi but were positive by real-time PCR. However, all of the tissue samples were positive by both PCR and histology. The blood sample showed no growth of Aspergillus, but aspergillosis was confirmed by positive galactomannan ELISA, histology, and PCR results. The remaining samples (16.1%) were culture and PCR negative; also, no other signs indicating fungal infection were observed. Our data suggest that the Aspergillus and Candida assays may be appropriate for use in clinical laboratories as simple and rapid screening tests for the most frequently encountered Aspergillus and Candida species and might become an important tool in the early diagnosis of fungal infections in the future