We here report on the development of a novel multiplex PCR with product detection in a Luminex 100 suspension array system. The assay covers the nine most important bacterial and viral pathogens found in Danish meningitis patients. The microorganisms include Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Streptococcus agalactiae, herpes simplex virus types 1 and 2, and varicella-zoster virus. The study was based on 1,187 samples, of which 55 were found to be positive by PCR. The assay was found to have an excellent sensitivity and an excellent specificity compared to the results of a "gold standard," defined by routine laboratory tests, for the two most important pathogens, S. pneumoniae (95 and 99.1%, respectively) and N. meningitidis (100 and 99.7%, respectively). The method provides a valuable supplement to the traditional microscopy and culture of cerebrospinal fluid (CSF) samples in a routine diagnostic setting, and results can be available within 1 workday. The method is suitable for use for the initial screening and identification of nine important microorganisms in CSF samples from patients with suspected meningitis. Compared to microscopy and culture of CSF, this rapid and sensitive method will support physicians with the selection of the appropriate antimicrobial agents and the initiation of timely treatment in the absence of live microorganisms in the CSF.Bacterial meningitis is an infectious disease with a high rate of mortality and many cases of severe sequelae (4,5,23). Since the early administration of antibiotics correlates with reduced rates of morbidity and mortality, it is of crucial importance to initiate relevant treatment as soon as possible (10,11,13,19,22). The administration of antibiotics to patients with suspected meningitis before hospital admission and the collection of a cerebrospinal fluid (CSF) sample have become common practice (19). This practice may correspondingly impair microbiological diagnosis by culture of the bacteria responsible for the infection. The focus in recent years has therefore been on the development of alternative methods that can be used to obtain an etiological diagnosis, e.g., PCR. The use of broadrange 16S rRNA gene PCR with subsequent sequencing has been reported (1,20,24). This approach can be difficult to handle in a routine laboratory due to the presence of interfering bacterial or viral DNA, which may easily be added during sample handling, or, importantly, due to the presence of traces of bacterial DNA in the enzymes and mixtures used for PCR. The method also lacks speed because of the requisite sequencing step. Alternatively, species-specific real-time PCR (3, 21) has the advantage that rapid and high throughput is possible, but due to the limited number of detection channels of most real-time instruments, only a very limited number of different targets can be analyzed at a time.We chose to develop a system that uses a species-specific multiplex PCR focused on the bacterial and vira...