Detection of Neisseria meningitidis and unknown Gammaproteobacteria in cerebrospinal fluid using the two-step universal method
Barghouthi, Sameer A.
Al Zughayyar, Dima K.
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Bacterial meningitis is insufficiently diagnosed based on microscopic, cultural, and multiplex-polymerase chain reaction (M-PCR). The use of already established universal method (UM) offers the ultimate solution to the detection and potential identification of bacteria in cerebrospinal fluid (CSF) samples. We have applied the UM together with a newly established Anchored Multiplex PCR (AMD4; Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Listeria monocytogenes) to screen 130 CSF samples obtained from suspected meningitis cases for any bacterium. This two-stage UM approach was able to show that three of the samples contained bacterial DNA, only one of the three samples (K1) was shown to contain N. meningitidis whereas the other two samples (A35 and H1) were negative with AMD4. Nucleotide sequencing and BLAST analyses of 16S amplicons obtained by the UM from samples A35 and H1 showed no significant homology (<90%) to any available 16S sequence, yet indicated both bacteria (A35 and H1) to share 94.2% similarity. Both bacteria belonged to Gammaproteobacteria. The bacterium from sample K1 was isolated by culture and identified as N. meningitidis. The other two samples were negative by culture according to the clinical laboratories at both hospitals; A35 was from a patient who had received empirical antimicrobial therapy prior to sample collection. The remaining 127 samples were shown by the UM to be negative in accordance with clinical and laboratory findings. The UM can contribute significantly to the identification of bacterial meningitis cases to initiate empirical antimicrobial therapy within 3 h of sample collection. Simultaneously, bacterial meningitis can be ruled out from samples producing negative UM results. AMD4 application will detect and identify the major pathogens of bacterial meningitis whereas the UM will detect any bacterium, UM can potentially identify any bacterium as long as it is represented in the nucleotide databases; if not represented, it is labeled as unknown. We recommend the utilization of the UM in clinical testing; we also recommend culturing, characterization and identification of these unknown bacterial agents of meningitis as well as others.