Design and validation of a low density array (Nosochip) for the detection and identification of the main pathogenic bacteria and fungi responsible for nosocomial pneumonia.
The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.
Burteau
S
S
URBC, FUNDP, 61 Rue de Bruxelles, 5000, Namur, Belgium.
Bogaerts
P
P
de Mendonça
R
R
Irenge
L
L
Berhin
C
C
Hiffe
J
J
de San
N
N
Beyne
P
P
Hamels
S
S
Glupczynski
Y
Y
Struelens
M
M
Gala
J-L
JL
Remacle
J
J
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003428:Cross Infection / Q000382:microbiology*; D017931:DNA Primers; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015345:Oligonucleotide Probes; D011014:Pneumonia / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S