Multiplex-PCR for diagnosis of bacterial meningitis

Abstract

Considering the great lethality and sequels caused by meningitis, rapid diagnosis and prompt treatment initiation have a great impact on patient outcome. Here, we developed a multiplex-PCR for simultaneous detection of the four most prevalent bacterial pathogens directly in CSF samples. The multiplex-PCR was designed to detect the following genes: fbsA (Streptococcus agalactiae), lytA (Streptococcus pneumoniae), crtA (Neisseria meningitidis), p6 (Haemophilus influenzae), and 16S rRNA (any bacterial agent). The multiplex-PCR showed a DNA detection limit of 1 pg/μL. Among 447 CSF samples tested, 40 were multiplex-PCR positive, in which 27 and 13 had positive and negative bacterial culture, respectively. Our multiplex-PCR is fast, reliable, and easily implementable into a laboratory routine for bacterial meningitis confirmation, especially for patients who previously started antimicrobial therapy. Our molecular approach can substantially improve clinical diagnosis and epidemiological measures of meningitis disease burden.

Keywords: Bacterial pathogens; Cerebrospinal fluid samples; Meningitis; Multiplex-PCR.

Fig. 1

Multiplex-PCR to determine bacterial pathogens related to meningitis. Electrophoretic separation of amplicons from multiplex-PCR developed for the detection of four bacterial species: Streptococcus agalactiae, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. 1—molecular weight marker of 100 bp, 2—Streptococcus agalactiae (fbsA gene, 529 bp and 16S rRNA gene, 283 bp), 3—Streptococcus pneumoniae (lytA gene, 395 bp and 16S rRNA gene, 283 bp), 4—Neisseria meningitidis (crtA gene, 110 bp and 16S rRNA gene, 283 bp), 5—Haemophilus influenzae (p6 gene, 177 bp and 16S rRNA gene, 283 bp), 6—Escherichia coli (16S rRNA gene, 283 bp), 7—Listeria monocytogenes (16S rRNA gene, 283 bp). bp, base pairs

Fig. 2

Multiplex-PCR performed with different microorganisms. Lysates of different bacterial species and CSF samples positive for Candida albicans and Cryptococcus neoformans were tested by the multiplex-PCR. The electrophoretic separation was performed in agarose gel. 1—molecular weight marker of 100 bp, 2—Enterococcus faecalis, 3—Streptococcus pyogenes, 4—Cryptococcus neoformans, 5—Candida albicans, 6—Staphylococcus aureus, 7—Escherichia coli, 8—Salmonella typhi, 9—Pseudomonas aeruginosa, 10—Shigella flexneri, 11—Listeria monocytogenes, 12—Streptococcus agalactiae, 13—Klebsiella pneumoniae, 14—Citrobacter diversus, 15—Neisseria gonorrhoeae, 16—Neisseria meningitidis, 17—Streptococcus agalactiae, 18—Haemophilus influenzae, 19—Streptococcus pneumoniae, 20—molecular weight marker of 100 bp. Amplicons size correspond to Streptococcus agalactiae—fbsA gene (529 bp), Streptococcus pneumoniae—lytA gene (395 bp), 16S rRNA gene (283 bp), Haemophilus influenzae—p6 gene (177 bp), Neisseria meningitidis—crtA gene (110 bp). bp, base pairs

Fig. 3

Sensitivity test of the multiplex-PCR performed with serial dilutions of DNA extracted by Kit. Different dilutions of bacterial DNA were tested in the reaction of multiplex-PCR to evaluate the sensitivity of the method. The electrophoretic separation was performed in agarose gel. The figure illustrates the testing of (A) Neisseria meningitidis (crtA gene, 110 bp and 16S rRNA gene, 283 bp), (B) Haemophilus influenzae (p6 gene, 177 bp and 16S rRNA gene, 283 bp), (C) Streptococcus pneumoniae (lytA gene, 395 bp and 16S rRNA gene, 283 bp), and (D) Streptococcus agalactiae (fbsA gene, 529 bp and 16S rRNA gene, 283 bp). The gel columns represent different DNA concentrations, as specified in the figure, with the following concentrations from the left to right: 1000 pg/μL, 500 pg/μL, 100 pg/μL, 10 pg/μL, 1 pg/μL, 0.1 pg/μL. bp, base pairs