ABSTRACT
BACKGROUND: Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS: Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21 PM-related pathogens and could produce results within 3 h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS: The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS: Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.
Subject(s)
Bacteria/genetics , Fungi/genetics , Meningitis, Bacterial/microbiology , Meningitis, Fungal/microbiology , Oligonucleotide Array Sequence Analysis/methods , Real-Time Polymerase Chain Reaction/methods , Bacteria/classification , Bacteria/isolation & purification , Bacteria/pathogenicity , Cerebrospinal Fluid/microbiology , Child , DNA Primers/genetics , Female , Fungi/isolation & purification , Fungi/pathogenicity , Humans , Male , Meningitis, Bacterial/cerebrospinal fluid , Meningitis, Fungal/cerebrospinal fluid , Neisseria meningitidis/genetics , Neisseria meningitidis/isolation & purification , Sensitivity and SpecificityABSTRACT
Bloodstream infections (BSIs) are often life-threatening, and rapid identification is critical. Here, we developed a TaqMan array card (TAC) assay to detect pathogens in BSI specimens. The TAC included 30 primer/probe pairs targeting 27 species and 3 controls. Reverse transcription and 0.1% blue dextran 2000 increased the TAC assay efficiency. The primer/probe pairs had a limit of detection of 100-102 CFU/mL and a specificity of 100%. For whole blood specimens, the TAC assay showed a sensitivity and specificity of 79.4% and 99.69%, respectively. For blood culture samples, the TAC assay showed a sensitivity and specificity of 100% and 99.67%, respectively. The TAC assay could be a promising method for early detection of bloodstream infection.
