Multiplex-PCR for diagnosis of bacterial meningitis
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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.
KeywordsMeningitis Multiplex-PCR Cerebrospinal fluid samples Bacterial pathogens
Conceptualization: MBM. Data curation: RCA, ACRM, SRS, and SLBR. Formal analysis: RCA, ACRM, and MBM. Funding acquisition: MBM. Investigation: RCA, ACRM, and MBM. Project administration: MBM. Resources: MBM. Supervision: MBM and ACRM. Validation: RCA and ACRM. Visualization: RCA and ACRM. Writing – original draft: RCA, ACRM, and MBM. Writing – review and editing: MBM and ACRM.
This work was funded by grants 2006/55141-4 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). A.C.R.M. was awarded scholarship grants by the Brazilian Federal Agency for Support and Evaluation of Graduate Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES).
Compliance with ethical standards
This study was approved by the Ethics Committee of the HU-USP (protocol number 45904) and the IIER (protocol number 3807).
Conflict of interest
The authors declare that they have no conflicts of interest.
- 7.Philip AGS (2009) Neonatal bacterial meningitis. In: Fetal and neonatal brain injury, 4th edn. https://doi.org/10.1017/CBO9780511581281.033
- 9.Heckenberg SGB, Brouwer MC, van de Beek D (2014) Bacterial meningitis. Handb Clin Neurol. https://doi.org/10.1016/B978-0-7020-4088-7.00093-6
- 12.Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB (2001) Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 39:1553–1558. https://doi.org/10.1128/JCM.39.4.1553-1558.2001 CrossRefGoogle Scholar
- 13.Abdeldaim GMK, Strålin K, Korsgaard J, Blomberg J, Welinder-Olsson C, Herrmann B (2010) Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis. BMC Microbiol 10:310. https://doi.org/10.1186/1471-2180-10-310 CrossRefGoogle Scholar
- 14.Sacchi CT, Fukasawa LO, Gonçalves MG, Salgado MM, Shutt KA, Carvalhanas TR, Ribeiro AF, Kemp B, Gorla MCO, Albernaz RK, Marques EGL, Cruciano A, Waldman EA, Brandileone MCC, Harrison LH, São Paulo RT-PCR Surveillance Project Team (2011) Incorporation of real-time PCR into routine public health surveillance of culture negative bacterial meningitis in São Paulo, Brazil. PLoS One 6:e20675. https://doi.org/10.1371/journal.pone.0020675 CrossRefGoogle Scholar
- 15.Chakrabarti P, Das BK, Kapil A (2009) Application of 16S rDNA based seminested PCR for diagnosis of acute bacterial meningitis. Indian J Med ResGoogle Scholar
- 18.McHugh ML (2012) Interrater reliability: the kappa statistic. Biochem Med:276–282. https://doi.org/10.11613/BM.2012.031
- 19.Wang X, Theodore MJ, Mair R, Trujillo-Lopez E, du Plessis M, Wolter N, Baughman AL, Hatcher C, Vuong J, Lott L, von Gottberg A, Sacchi C, McDonald JM, Messonnier NE, Mayer LW (2012) Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens. J Clin Microbiol 50:702–708. https://doi.org/10.1128/JCM.06087-11 CrossRefGoogle Scholar
- 21.Wagner K, Springer B, Pires VP, Keller PM (2017) Pathogen identification by multiplex LightMix real-time PCR assay in patients with meningitis and culture-negative cerebrospinal fluid specimens. J Clin Microbiol 56. https://doi.org/10.1128/JCM.01492-17
- 22.Seth R, Murthy PSR, Sistla S, Subramanian M, Tamilarasu K (2017) Rapid and accurate diagnosis of acute pyogenic meningitis due to streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitis using a multiplex PCR assay. J Clin Diagn Res. https://doi.org/10.7860/JCDR/2017/28114.10532
- 23.Khumalo J, Nicol M, Hardie D, Muloiwa R, Mteshana P, Bamford C (2017) Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting. PLoS One 12:e0173948. https://doi.org/10.1371/journal.pone.0173948 CrossRefGoogle Scholar
- 24.Billal DS, Hotomi M, Suzumoto M, Yamauchi K, Kobayashi I, Fujihara K, Yamanaka N (2007) Rapid identification of nontypeable and serotype b Haemophilus influenzae from nasopharyngeal secretions by the multiplex PCR. Int J Pediatr Otorhinolaryngol 71:269–274. https://doi.org/10.1016/j.ijporl.2006.10.009 CrossRefGoogle Scholar
- 25.Nelson MB, Munson RS, Apicella MA, Sikkema DJ, Molleston JP, Murphy TF (1991) Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms. Infect Immun 59(8):2658–2663Google Scholar
- 27.de Filippis I, de Andrade CF, Caldeira N, de Azevedo AC, de Almeida AE (2016) Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples. Braz J Infect Dis 20:335–341. https://doi.org/10.1016/j.bjid.2016.04.005 CrossRefGoogle Scholar
- 29.Whatmore AM, Dowson CG (1999) The autolysin-encoding gene (lytA) of Streptococcus pneumoniae displays restricted allelic variation despite localized recombination events with genes of pneumococcal bacteriophage encoding cell wall lytic enzymes. Infect ImmunGoogle Scholar