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VOLUME 20 , ISSUE 2 ( July-December, 2018 ) > List of Articles

Original Article

Nested polymerase chain reaction targeting 16S rRNA gene in diagnosis of acute bacterial meningitis

Harshita Lal, Abhinav Agarwal, Ashish Kumar Singh, Rajniti Prasad, Anil Kumar Gulati, Pradyot Prakash

Keywords : Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae

Citation Information : Lal H, Agarwal A, Singh AK, Prasad R, Gulati AK, Prakash P. Nested polymerase chain reaction targeting 16S rRNA gene in diagnosis of acute bacterial meningitis. 2018; 20 (2):77-83.

DOI: 10.4103/jacm.jacm_11_18

License: CC BY-NC 4.0

Published Online: 17-08-2024

Copyright Statement:  Copyright © 2018; Wolters Kluwer India Pvt. Ltd.


Abstract

CONTEXT: Diagnosing acute bacterial meningitis (ABM) among children presenting to tertiary health care settings is often difficult because of prior administration of antimicrobials. AIMS: The present study was an attempt to diagnose cases of ABM in children with the help of nested polymerase chain reaction (PCR). SETTINGS AND DESIGN: It is a prospective observational study, in which a total of 84 clinically suspected cases and cerbrospinal fluid (CSF) biochemical parameters suggestive of ABM were included in the study. METHODS AND MATERIAL: CSF samples were subjected to Gram staining, bacterial culture and biochemical identification tests as well as panbacterial nested PCR targeting 16S rRNA gene sequence. Subsequently, a nested multiplex PCR for detection of the three fastidious organisms, viz. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae, was performed. STATISTICAL ANALYSIS USED: The sensitivity, specificity, PPV, NPV, LR+, and LR− of the tests were calculated. RESULTS: The sensitivity of Gram stain, bacterial culture, and nested PCR targeting 16SrRNA were observed to be 16.21%, 9.45%, and 97.29% respectively. Further, the NPV and LR− the PCR were found to be 83.33 and 0.02 respectively. Species specific nested multiplex PCR was able to detect S. pneumoniae (n = 7), N. meningitidis (n = 2) and H. influenzae (n = 1). CONCLUSIONS: The results indicates that nested PCR targeting 16S rRNA gene may be used in diagnosis of ABM. Further, nested multiplex PCR targeting the three important fastidious bacterial pathogens in ABM cases has showed their presence in our region.

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  1. World Health Organization, Centers for Disease Control and Prevention. Laboratory methods for the diagnosis of meningitis caused by neisseria meningitidis, streptococcus pneumoniae, and haemophilus influenzae: WHO manual. Available from: https://www.cdc.gov/meningitis/lab-manual/full-manual.pdf. [Last accessed on 2018 Oct 05].
  2. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev 2010;23:467-92.
  3. Roos KL, Tyler KL. Meningitis, encephalitis, brain abscess and empyema. In: Harrison's Textbook of Internal Medicine. 19th ed. Kasper SL, Fauci AS, Hauser SL, Longo DL, Jameson JL, Loscalzo J, editors. New Delhi: McGraw Hills Education; 2015. p. 883-906.
  4. Das BK, Gurubacharya RL, Mohapatra TM, Mishra OP. Bacterial antigen detection test in meningitis. Indian J Pediatr 2003;70:799-801.
  5. Mani R, Pradhan S, Nagarathna S, Wasiulla R, Chandramuki A. Bacteriological profile of community acquired acute bacterial meningitis: A ten-year retrospective study in a tertiary neurocare centre in South India. Indian J Med Microbiol 2007;25:108-14.
  6. Abro AH, Abdou AH, Ali H, Ustadi AM, Hasab AAH. Cerebrospinal fluid analysis acute bacterial versus viral meningitis. Pak J Med Sci 2008;24:645-50.
  7. Alamgir F, Miah RA, Saleh AA. Bacterial antigen detection and CRP estimation. Bangladesh Med J 2008;13:17-20.
  8. Dalton HP, Allison MJ. Modification of laboratory results by partial treatment of bacterial meningitis. Am J Clin Pathol 1968;49:410-3.
  9. Perkins MD, Mirrett S, Reller LB. Rapid bacterial antigen detection is not clinically useful. J Clin Microbiol 1995;33:1486-91.
  10. Welinder-Olsson C, Dotevall L, Hogevik H, Jungnelius R, Trollfors B, Wahl M, et al. Comparison of broad-range bacterial PCR and culture of cerebrospinal fluid for diagnosis of community-acquired bacterial meningitis. Clin Microbiol Infect 2007;13:879-86.
  11. Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM, et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis 2004;39:1267-84.
  12. Okhravi N, Adamson P, Matheson MM, Towler HM, Lightman S. PCR-RFLP-mediated detection and speciation of bacterial species causing endophthalmitis. Invest Ophthalmol Vis Sci 2000;41:1438-47.
  13. de Filippis I, do Nascimento CR, Clementino MB, Sereno AB, Rebelo C, Souza NN, et al. Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene. Diagn Microbiol Infect Dis 2005;51:85-90.
  14. Toikka P, Nikkari S, Ruuskanen O, Leinonen M, Mertsola J. Pneumolysin PCR-based diagnosis of invasive pneumococcal infection in children. J Clin Microbiol 1999;37:633-7.
  15. Richardson DC, Louie L, Louie M, Simor AE. Evaluation of a rapid PCR assay for diagnosis of meningococcal meningitis. J Clin Microbiol 2003;41:3851-3.
  16. Greenberg RS, Daniels SR, Flanders WD, Eley JW, Boring JR, editors. Diagnostic testing. In: Medical Epidemiology. 3rd ed. New York: McGraw-Hill; 1996. p. 77-88.
  17. Schlech WF 3rd, Ward JI, Band JD, Hightower A, Fraser DW, Broome CV, et al. Bacterial meningitis in the united states, 1978 through 1981. The national bacterial meningitis surveillance study. JAMA 1985;253:1749-54.
  18. Dery MA, Hasbun R. Changing epidemiology of bacterial meningitis. Curr Infect Dis Rep 2007;9:301-7.
  19. Basu RN. Meningococcal meningitis in Delhi and other areas. Community Dis Bull 1985;2:1-10.
  20. Kalghatgi AT, Praharaj AK, Sahni AK, Pradhan D, Kumaravelu S, Prasad PL, et al. Detection of bacterial pathogens in cerebrospinal fluid using restriction fragment length polymorphism. Med J Armed Forces India 2008;64:29-32.
  21. Chakrabarti P, Das BK, Kapil A. Application of 16S rDNA based seminested PCR for diagnosis of acute bacterial meningitis. Indian J Med Res 2009;129:182-8.
  22. Rådström P, Bäckman A, Qian N, Kragsbjerg P, Påhlson C, Olcén P, et al. Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy. J Clin Microbiol 1994;32:2738-44.
  23. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB, et al. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 2001;39:1553-8.
  24. Tzanakaki G, Tsopanomichalou M, Kesanopoulos K, Matzourani R, Sioumala M, Tabaki A, et al. Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae. Clin Microbiol Infect 2005;11:386-90.
  25. Diggle MA, Clarke SC. Detection and genotyping of meningococci using a nested PCR approach. J Med Microbiol 2003;52:51-7.
  26. du Plessis M, Smith AM, Klugman KP. Rapid detection of penicillin-resistant Streptococcus pneumoniae in cerebrospinal fluid by a seminested-PCR strategy. J Clin Microbiol 1998;36:453-7.
  27. Khan F, Rizvi M, Fatima N, Shukla I, Malik A, Khatoon R. Bacterial meningitis in North India: Trends over a period of eight years. Neurol Asia 2011;16:47-56.
  28. Nigrovic LE, Malley R, Macias CG, Kanegaye JT, Moro-Sutherland DM, Schremmer RD, et al. Effect of antibiotic pretreatment on cerebrospinal fluid profiles of children with bacterial meningitis. Pediatrics 2008;122:726-30.
  29. Talan DA, Hoffman JR, Yoshikawa TT, Overturf GD. Role of empiric parenteral antibiotics prior to lumbar puncture in suspected bacterial meningitis: State of the art. Rev Infect Dis 1988;10:365-76.
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