Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis.
J Microbiol Methods
; 106: 8-15, 2014 Nov.
Article
in En
| MEDLINE
| ID: mdl-25102109
ABSTRACT
The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Bacteria
/
Blood
/
Sepsis
/
Molecular Diagnostic Techniques
/
Multiplex Polymerase Chain Reaction
/
Real-Time Polymerase Chain Reaction
Type of study:
Diagnostic_studies
/
Evaluation_studies
/
Prognostic_studies
/
Risk_factors_studies
Limits:
Humans
/
Newborn
Language:
En
Journal:
J Microbiol Methods
Year:
2014
Document type:
Article