Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

A novel multiplex PCR assay for the detection of 18 bacterial, yeast and viral causes of meningitis  (#322)

John Melki 1 , Shoo Peng Siah 1 , Kiran Kaur 1 , Jiny Nair 1 , William Rawlinson 2 , Doug Millar 1
  1. Genetic Signatures, Sydney, NSW, Australia
  2. Director of Virology, Prince of Wales Hospital, Randwick, NSW, Australia

Objective: Meningitis is a central nervous system (CNS) infection that can have serious clinical manifestations. Viral meningitis as a result of infection with enteroviruses is very common, usually self-limiting and does not require antibiotic treatment. Bacterial meningitis can be life threatening and patients should be treated with the appropriate antibiotics as a matter of urgency. 

Aim: To produce real time PCR assays that could detect the presence of 18 bacterial, viral and yeast agents associated with meningitis in less than 2 hours. In addition, dual labelled real time probes were used to increase the multiplexing capability and reduce the number of individual amplification reactions required.

Methods: One-hundred and fifty microliters of Central Spinal Fluid (CSF) was diluted 1:1 with sample buffer and heated at 95oC for 15 minutes to allow for lysis. Sample purification can be completed using a number of platforms including the EZ1 or Qiasymphony (Qiagen), Kingfisher (ThermoFisher), Magnapure (Roche) and the Easymag (Biomerieux) automated nucleic acid extraction systems. Real time PCR analysis can be performed on the LC480 (Roche), 7500 fast (Applied Biosystems), RotorgeneQ (Qiagen) and the Smartcycler™II (Cepheid) instruments.

Results: Using purified genomic DNA and RNA samples from the target organisms of interest the sensitivity of all components of the multiplex assay was shown to be in the range of 1-5 copies of target per reaction using mock clinical samples. No cross reactivity was shown using a wide range of non-target bacteria and viruses.

Conclusion: The multiplex PCR assay was successfully able to stratify viral and bacterial causes of meningitis in less than 2 hours. Thus patients can quickly and accurately be prescribed the most appropriate antibiotic regimes thus improving patient outcomes. This has the added advantage that Enteroviral infections can be quickly detected reducing the use of unwarranted antimicrobials, which is important, given the continued rise of drug resistant bacteria.