Multiplex PCR assay in spinal fluid to identify simultaneously bacterial pathogens associated to acute bacterial meningitis in Chilean children

Abstract

Acute bacterial meningitis (ABM) is a serious disease that needs rapid diagnosis for an accurate treatment. The most important etiological agents are: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type b. Overall pathogen detection rate in patients with ABM in Chile is 83%. To evaluate a Polymerase Chain Reaction (PCR) protocol for simultaneous detection of several pathogens in patients with ABM. We designed and evaluated a multiplex PCR protocol for simultaneous specific genes identifications of S pneumoniae ( inverted exclamation markytA and ply genes), N meningitidis (ctrA, crgA) and H influenzae (bexA) in cerebrospinal fluid (CSF) samples from pediatric patients with suspected diagnosis of ABM. Sensitivity, specificity and minimum detection levels of DNA were determined. Amplifications ofrDNA 16S gene was done to confirm extraction of bacterial DNA. Ninety nine CSF samples were studied, 90 from children with fever and negative CSF culture, and 9 from ABM and positive culture patients. The PCR protocol had a sensitivity of 89%, specificity of 100%, positive predictive value 100% and negative predictive value 99%. We observed a high concordance (89%) between bacteriological cultures and the PCR protocol results. This diagnostic tool could increase identification of agents in specific settings such as patients previously treated with antibiotics.