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Brazilian Journal of Microbiology

, Volume 50, Issue 2, pp 435–443 | Cite as

Multiplex-PCR for diagnosis of bacterial meningitis

  • Renata Chaves Albuquerque
  • Ana Carolina Ramos MorenoEmail author
  • Silvia Regina dos Santos
  • Selma Lopes Betta Ragazzi
  • Marina Baquerizo Martinez
Clinical Microbiology - Research Paper
  • 23 Downloads

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

Meningitis Multiplex-PCR Cerebrospinal fluid samples Bacterial pathogens 

Notes

Author contributions

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.

Funding information

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.

References

  1. 1.
    Brouwer MC, Tunkel AR, Van De Beek D (2010) Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 23:467–492.  https://doi.org/10.1128/CMR.00070-09. CrossRefGoogle Scholar
  2. 2.
    Van De Beek D, Brouwer M, Hasbun R, Koedel U, Whitney CG, Wijdicks E (2016) Community-acquired bacterial meningitis. Nat Rev Dis Primers 2:16074.  https://doi.org/10.1038/nrdp.2016.74 CrossRefGoogle Scholar
  3. 3.
    van de Beek D, Drake JM, Tunkel AR (2010) Nosocomial bacterial meningitis. N Engl J Med 362:146–154.  https://doi.org/10.1056/NEJMra0804573 CrossRefGoogle Scholar
  4. 4.
    Kim KS (2010) Acute bacterial meningitis in infants and children. Lancet Infect Dis 10:32–42.  https://doi.org/10.1016/S1473-3099(09)70306-8 CrossRefGoogle Scholar
  5. 5.
    van de Beek D, de Gans J, Tunkel AR, Wijdicks EFM (2006) Community-acquired bacterial meningitis in adults. N Engl J Med 100:37–40.  https://doi.org/10.1093/qjmed/hcl131. Google Scholar
  6. 6.
    Saez-Llorens X, McCracken GH Jr (2003) Bacterial meningitis in children. Lancet. 361:2139–2148.  https://doi.org/10.1016/S0140-6736(03)13693-8 CrossRefGoogle Scholar
  7. 7.
    Philip AGS (2009) Neonatal bacterial meningitis. In: Fetal and neonatal brain injury, 4th edn.  https://doi.org/10.1017/CBO9780511581281.033
  8. 8.
    McGill F, Heyderman RS, Panagiotou S, Tunkel AR, Solomon T (2016) Acute bacterial meningitis in adults. Lancet. 388:3036–3047.  https://doi.org/10.1016/S0140-6736(16)30654-7 CrossRefGoogle Scholar
  9. 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
  10. 10.
    Van De Beek D, Brouwer MC, Thwaites GE, Tunkel AR (2012) Advances in treatment of bacterial meningitis. Lancet. 380:1693–1702.  https://doi.org/10.1016/S0140-6736(12)61186-6 CrossRefGoogle Scholar
  11. 11.
    Mohammadi SF, Patil AB, Nadagir SD, Nandihal N, Lakshminarayana SA (2013) Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis. Ann Indian Acad Neurol 16:645–649.  https://doi.org/10.4103/0972-2327.120491 CrossRefGoogle Scholar
  12. 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. 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. 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. 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
  16. 16.
    de Zoysa A, Edwards K, Gharbia S, Underwood A, Charlett A, Efstratiou A (2012) Non-culture detection of Streptococcus agalactiae (Lancefield group B Streptococcus) in clinical samples by real-time PCR. J Med Microbiol 61:1086–1090.  https://doi.org/10.1099/jmm.0.042879-0 CrossRefGoogle Scholar
  17. 17.
    Messmer TO, Black CM, Facklam RR (1995) Discrimination of Streptococcus pneumoniae from other upper respiratory tract streptococci by arbitrarily primed PCR. Clin Biochem 28:567–572.  https://doi.org/10.1016/0009-9120(95)00044-0 CrossRefGoogle Scholar
  18. 18.
    McHugh ML (2012) Interrater reliability: the kappa statistic. Biochem Med:276–282.  https://doi.org/10.11613/BM.2012.031
  19. 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
  20. 20.
    Liesman RM, Strasburg AP, Heitman AK, Theel ES, Patel R, Binnicker MJ (2018) Evaluation of a commercial multiplex molecular panel for diagnosis of infectious meningitis and encephalitis. J Clin Microbiol 56:e01927–e01917.  https://doi.org/10.1128/JCM.01927-17 CrossRefGoogle Scholar
  21. 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. 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. 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. 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. 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
  26. 26.
    Khazani NA, Noor NZNM, Yean C, Hasan H, Suraiya S, Mohamad S (2017) A thermostabilized, one-step PCR assay for simultaneous detection of Klebsiella pneumoniae and Haemophilus influenzae. J Trop Med 2017:1–8.  https://doi.org/10.1155/2017/7210849 CrossRefGoogle Scholar
  27. 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
  28. 28.
    Lindahl G, Stålhammar-Carlemalm M, Areschoug T (2005) Surface proteins of Streptococcus agalactiae and related proteins in other bacterial pathogens. Clin Microbiol Rev 18:102–127.  https://doi.org/10.1128/CMR.18.1.102-127.2005. CrossRefGoogle Scholar
  29. 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

Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Renata Chaves Albuquerque
    • 1
  • Ana Carolina Ramos Moreno
    • 1
    • 2
    Email author
  • Silvia Regina dos Santos
    • 3
  • Selma Lopes Betta Ragazzi
    • 3
  • Marina Baquerizo Martinez
    • 1
  1. 1.Faculdade de Ciências Farmacêuticas, Department of Clinical Chemistry and ToxicologyUniversidade de São Paulo (USP)São PauloBrazil
  2. 2.Biomedical Sciences Institute, Vaccine Development Laboratory, Department of MicrobiologyUSPSão PauloBrazil
  3. 3.Hospital Universitário, USPSão PauloBrazil

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