Research ArticleIdentification of Common Bacterial Pathogens CausingMeningitis in Culture-Negative Cerebrospinal Fluid SamplesUsing Real-Time Polymerase Chain Reaction
Walaa Shawky Khater and Safia Hamed Elabd
Medical Microbiology and Immunology Faculty of Medicine Ain Shams University Cairo 11566 Egypt
Correspondence should be addressed to Safia Hamed Elabd drsafia elabdmedasuedueg
Received 12 May 2016 Revised 18 June 2016 Accepted 4 July 2016
Academic Editor Giuseppe Comi
Copyright copy 2016 W S Khater and S H ElabdThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Background Meningitis is a serious communicable disease with high morbidity and mortality rates It is an endemic disease inEgypt caused mainly by Streptococcus pneumoniae Neisseria meningitidis and Haemophilus influenzae In some settings bacterialmeningitis is documented depending mainly on positive cerebrospinal fluid (CSF) culture results or CSF positive latexagglutination test missing the important role of prior antimicrobial intake which can yield negative culture and latex agglutinationtest results This study aimed to utilize molecular technology in order to diagnose bacterial meningitis in culture-negativeCSF samples Materials and Methods Forty culture-negative CSF samples from suspected cases of bacterial meningitis wereexamined by real-time polymerase chain reaction (real-time PCR) for the presence of lytA bexA and ctrA genes specific forStreptococcus pneumoniae Haemophilus influenzae and Neisseria meningitidis respectively Results Positive real-time PCR resultsfor Streptococcus pneumoniaewere detected in 36 (90) of culture-negative CSF samples while no positive results forHaemophilusinfluenzae or Neisseria meningitidis were detected Four (10) samples were negative by real-time PCR for all tested organismsConclusion The use of molecular techniques as real-time PCR can provide a valuable addition to the proportion of diagnosed casesof bacterial meningitis especially in settings with high rates of culture-negative results
1 Introduction
Bacterial meningitis is one of the serious communicablediseases associated with substantial morbidity and mortalityrates [1] About 10 to 20 of survivors develop disabling neu-rologic complications [2] mandating the prompt diagnosistreatment and prevention The incidence of meningitis isusually high in developing countries with poor-socioec-onomic status [3] and it is in fact reported endemic inEgypt [4] Streptococcus pneumoniae Neisseria meningitidisand Haemophilus influenzae type b account for most of theworldrsquos documented cases of community-acquired bacterialmeningitis [2ndash6] However over the past decade changes inthe epidemiology of the disease regarding the distribution ofthe causative agents and patientsrsquo age group have been noticedin different geographical areas owing to the implementationof different immunization strategies using conjugate polysac-charide vaccines [1 3 7 8]
Different methods exist for the diagnosis of bacterialmeningitis of which cerebrospinal fluid (CSF) culture isstill considered the ldquogold standardrdquo [9 10] However culturetechniques in some settings lack sensitivity particularly whenthe patient is pretreated with antimicrobials added to thatthe disadvantage of the turnaround time till results becomesavailable [9 11ndash13] Gram-stained smears of the CSF samplescan provide a rapid preliminary tool for diagnosis in 60ndash90of patientswhich correlateswith the concentration of bacteriain the CSF samples [9] However sensitivities of CSF Gramstaining vary considerably for different microorganisms [3]Some studies have also reported a low sensitivity of directantigen detection assays as latex agglutination test especiallyin pretreated patientswith antibiotics before lumbar puncture[14ndash16]
Accordingly molecular methods have been proposed tofill in these gaps as rapid and accurate methods especially inculture-negative situations [6 10 17ndash19]
Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 4197187 5 pageshttpdxdoiorg10115520164197187
2 International Journal of Microbiology
This study aimed to diagnose bacterial meningitis causedby Neisseria meningitidis Streptococcus pneumoniae andHaemophilus influenzae type b (Hib) in culture-negative CSFsamples by the aid of real-time PCR
2 Materials and Methods
This study was performed during the period from December2014 to March 2015 and was approved by the Ethics Com-mittee of Faculty of Medicine Ain Shams University CairoEgypt and in accordance with the ethical guidelines of theDeclaration of Helsinki 1975
The study comprised a total of 40 CSF samples recoveredfrom adult patients admitted to Abbasseya Fever Hospitalpresenting with clinical picture and abnormal CSF cellularand chemistry results suggestive of bacterial meningitis
Clinical criteria for inclusion of patients were fever head-ache vomiting photophobia and irritability (symptoms ofmeningeal irritation) along with neck rigidity Kernig signBrudzinski sign altered conscious level seizures and focalneurological signs (signs of meningeal irritation) [4]
The diagnostic laboratory criteria for bacterial meningi-tis included the following glucose concentration less than40mgL protein concentration more than 50mgdL a whitecell count more than 100 cells per mm3 and neutrophil per-centage more than 50 [20]
The collectedCSF sampleswere centrifuged at 10000 rpmfor 10min and the supernatant was examined for abnormal-ities in WBCs protein and glucose Ten microliters of thesedimentwas inoculated onto sheep blood agar and chocolateagar and the rest was aliquoted and stored at minus70∘C Bacterialgrowth was observed after overnight incubation of the agarplates at 37∘C in 5 CO
2atmosphere [21]
Data of patients with negative culture results wererecorded and their stored CSF samples were further exam-ined by real-time PCR
21 Real-Time PCR
211 Bacterial DNAExtraction Bacterial DNAwas extractedfrom CSF samples with the aid of QIAGEN DNA Mini kit(QIAGEN Inc California) as per themanufacturerrsquos protocolfor DNA purification The eluted DNA was stored at minus20∘Cuntil further processed
212 Real-Time PCR with SYBR Green I Three runs weresequentially performed for the detection of each organismseparatelyThe ctrA gene ofNeisseria meningitidis bexA geneof Haemophilus influenzae and lytA gene of Streptococcuspneumoniae [9] were used as species-specific targets Theprimers sequences are listed in Table 1 The mix for eachrun included 5 120583L of sample DNA 125 120583L of 2x QuantiTectSYBR Green PCR master mix (QIAGEN Inc Valencia CA)containing a buffer dNTP mix MgCl
2and HotStarTaq DNA
polymerase 1 120583L of the primer and RNase-free water fora final volume of 25 120583L DNA was amplified with the StepOne Real-Time PCR system (Applied Biosystems) by usingthe following temperature program an initial Hotstart Taqactivation step at 95∘C for 15min initial denaturing at 95∘C
for 15 seconds and 40 PCR cycles of denaturing at 95∘C for30 seconds annealing at 50∘C for 30 seconds and extensionat 72∘C for 30 seconds followed by melting curve stage of95∘C and 60∘C [22] Amplification data were analyzed byinstrument software (Step One Software v23) in terms ofmelting curve graphs of each sample (Figure 1) Positivecontrol strains and negative controls consisting of PCR gradewater instead of the target DNA were used in each run
22 Statistical Analysis Quantitative variables are presentedas mean and SD and intergroup differences are comparedusing the unpaired 119905-test Categorical variables are presentedas number and percentage Discrete and skewed continuousdata are presented as median and differences between groupsare compared using the Mann-Whitney test The statisticalprocedures were carried out using SPSS version 15 forWindows (SPSS Inc Chicago IL USA)
3 Results
The present study was conducted on 40 culture-negative CSFsamples withdrawn from 27 (675) males and 13 (325)females Age of patients ranged from 19 to 56 years (mean plusmnSD = 3833 plusmn 1044) Demographic data of the patients andbiochemical and cytological findings of the CSF samples arelisted in Table 2
CSF chemistry revealed elevated protein level(gt50mgdL) in 825 of the samples and decreasedglucose level (lt40mgdL) in 875 of them White blood cell(WBC) count gt1000 cellsmm3 was found in 575 of thesamples whereas 425 of the samples showed WBC countbetween 100 and 1000 cellsmm3 Predominant neutrophilicCSF (neutrophil gt 50) was found in 725 of the samples
Thirty-six samples (90) were positive for Streptococcuspneumoniae by real-timePCRwhereasNeisseriameningitidisand Haemophilus influenzae were not detected in any of thesamples (0) Four samples (10) were negative for all threeorganisms (Table 3)
No statistical significant difference was observed regard-ing CSF chemistry and cells between samples of CSF positiveand negative real-time PCR results (Table 4)
4 Discussion
Rapid and accurate laboratory diagnostics remain a crucialstep for the final diagnosis of bacterial meningitis specifyingthe treatment and implementing preventive measures forclose contacts when indicated Conventional culture meth-ods though the gold standard diagnostic technique cannotbe relied upon per se in certain situations owing to the delayin results availability and the relatively limited sensitivitythat had been repeatedly reported worldwide [12 13 23]and in conditions when prior antimicrobial therapy has beenreceived In Egypt a high percentage of culture-negativesamples was reported previously [24 25] These results wereexplained by the fact that most patients received antimicro-bial agents that are readily purchased as over-the-countermedications even prior to clinical evaluation alongside theoccasional delay in csf sampling For the above-mentioned
International Journal of Microbiology 3
200
175
150
125
100
75
50
25
0
ΔRn
Cycle4036322824201612840
4078621
(a)
600
500
400
300
200
100
Der
ivat
ive r
epor
ter (
minusRn
)
Tm 7678
Temperature (∘C)950850750650
(b)
Figure 1 (a) Real-time PCR amplification plot for lytA gene specific for Streptococcus pneumoniae Horizontal blue line represents thethreshold value of fluorescence (b) Melting curves of positive samples for Streptococcus pneumoniae
Data are presented as mean plusmn standard deviation for continuous variablesand as number (percentage) for categorical variablesWBCs white blood cells
reasons we employed a molecular method (real-time PCR)in this study to improve the diagnosis of bacterial meningitisin culture-negative purulent CSF samples
Demographic data analysis of patients revealed that theywere mainly males (675 versus 125 for females) Most ofthe cases belonged to the middle age group (mean = 383)This finding agreed with Fouad et al [4] who confirmedthat males were more significantly affected with bacterialmeningitis than females (61 versus 39 resp) though thedisease was distributed in all age groups with low rates ofoccurrence in the extremes of age (the neonates and above60 years)
Real-time PCR in this study was positive for Streptococcuspneumoniae in 36 culture-negative CSF samples (90) whileno positive results for Haemophilus influenzae or Neisseriameningitidis were detected The age group of the patients inthis study might have contributed to these results as the maincausative agents of bacterial meningitis in adults are gener-ally believed to be Streptococcus pneumoniae and Neisseriameningitidis [1 26 27] Streptococcus pneumoniae is foundto be the commonest etiology of bacterial meningitis in the
4 International Journal of Microbiology
Table 4 Comparison between real-time PCR positive and negative samples as regards CSF cells and chemistry results
Variables RT-PCR positive RT-PCR negative 119885lowast119905 p valueMedian
Mean plusmn SDNeutrophil percentage 75 plusmn 218 675 plusmn 185 lowast066 0513 (NS)Data are presented as median or mean plusmn standard deviationNS nonsignificant result (119901 value gt 005)119885 Mann-Whitney testlowast119905 unpaired 119905-test
WBCs white blood cells
United States and Europe accounting for 61 of total cases inthe United States [1 3 28] and inmost African countries withhigh human immune deficiency virus prevalence [29 30]Yet meningococcal meningitis is common in Sub-SaharanAfrica (the meningitis belt) but mostly apparent in theform of epidemics and outbreaks [31] In a laboratory-basedsurveillance study undergone in Egypt byAfifi et al [32] PCRwas performed on purulent culture-negative CSF specimenswithdrawn from patients who met the criteria for casedefinition of bacterial meningitis Streptococcus pneumoniaewas also reported as the most common etiology of bacterialmeningitis
Fouad et al [4] also documented Streptococcus pneu-moniae as the most frequent isolate (52) among bacteriacausing meningitisThe agreement between our findings andthose of the previously mentioned studies in Egypt consoli-dates the deduction of Shaban and Siam [33] in their reviewarticle that pneumococcal meningitis is currently the leadingcause of meningitis in Egypt as its incidence is constantlyrising at the expense of meningococcal meningitis whichmay be a reflection of the increased use of polysaccha-ride meningococcal vaccines
Wang et al [7] also identified bacterial meningitis in fivecases (9) by CSF cultures and 25 (45) by real-time PCRThey considered real-time PCR much more sensitive thanculture for the diagnosis of bacterial meningitis particularlyin their study where 68 of patients had received priorantimicrobial treatment and their CSF samples yielded neg-ative culture results A similar conclusion has been reachedby Wu et al [9] and Sacchi et al [19] who stated that real-time PCR increases diagnostic yield for bacterial meningitisand is ideal for incorporation into routine surveillance indeveloping countries
According to Brouwer et al [34] CSF culture is doc-umented to be positive only in 110th of the previouslyantibiotic treated patients in developing countries Sameresult was found by Afifi et al [32] who reported low ratesof culture positive CSF samples (8) of suspected cases withbacterial meningitis [32] This low yield in culture resultscould be attributed to the fact that antimicrobials are beingdispensed without prescriptions in Egypt
This study faces the limitation of the relatively lownumber ofCSF samples investigated and the lack of testing forother less commonly bacterial etiologies of adult meningitis
(eg Group B Streptococcus Listeria monocytogenes) Thismay provide an explanation for the negative results (10)obtained
According to our findings we conclude that the use ofmolecular technique in the diagnosis of bacterial meningitisshould be considered in suspected cases with negative cultureresults before reporting exclusion of the disease
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors gratefully acknowledge the support provided bythe physicians of the Microbiology Laboratory in AbbasseyaFever Hospital for supplying them with the CSF samplesincluded in this work
References
[1] D Van De Beek J De Gans L Spanjaard M Weisfelt J BReitsma and M Vermeulen ldquoClinical features and prognosticfactors in adults with bacterial meningitisrdquo New England Jour-nal of Medicine vol 351 no 18 pp 1849ndash1923 2004
[2] S A Namani R A Koci E Qehaja-Bucaj L Ajazaj-Berishaand M Mehmeti ldquoThe epidemiology of bacterial meningitis inKosovordquo Journal of Infection in Developing Countries vol 8 no7 pp 823ndash830 2014
[3] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
[4] R Fouad M Khairy W Fathalah T Gad B El-Kholy and AYosry ldquoRole of clinical presentations and routine CSF analysisin the rapid diagnosis of acute bacterial meningitis in cases ofnegative gram stained smearsrdquo Journal of TropicalMedicine vol2014 Article ID 213762 7 pages 2014
[5] C E Corless M Guiver R Borrow V Edwards-Jones A JFox and E B Kaczmarski ldquoSimultaneous detection ofNeisseriameningitidis Haemophilus influenzae and Streptococcus pneu-moniae in suspected cases of meningitis and septicemia usingreal-time PCRrdquo Journal of Clinical Microbiology vol 39 no 4pp 1553ndash1558 2001
[6] G M K Abdeldaim K Stralin J Korsgaard J Blomberg CWelinder-Olsson and B Herrmann ldquoMultiplex quantitative
International Journal of Microbiology 5
PCR for detection of lower respiratory tract infection andmeningitis caused by Streptococcus pneumoniaeHaemophilusinfluenzae and Neisseria meningitidisrdquo BMC Microbiology vol10 article 310 2010
[7] Y Wang G Guo H Wang et al ldquoComparative study ofbacteriological culture and real-time fluorescence quantitativePCR (RT-PCR) and multiplex PCR-based reverse line blot(mPCRRLB) hybridization assay in the diagnosis of bacterialneonatal meningitisrdquo BMC Pediatrics vol 14 no 1 article 2242014
[8] M W Bijlsma M C Brouwer E S Kasanmoentalib et alldquoCommunity-acquired bacterial meningitis in adults in theNetherlands 2006ndash2014 a prospective cohort studyrdquoTheLancetInfectious Diseases vol 16 no 3 pp 339ndash447 2016
[9] H M Wu S M Cordeiro B H Harcourt et al ldquoAccuracyof real-time PCR Gram stain and culture for StreptococcuspneumoniaeNeisseriameningitidis andHaemophilus influenzaemeningitis diagnosisrdquo BMC Infectious Diseases vol 13 no 1article 26 2013
[10] L D Saravolatz O Manzor N VanderVelde J Pawlak andB Belian ldquoBroad-range bacterial polymerase chain reactionfor early detection of bacterial meningitisrdquo Clinical InfectiousDiseases vol 36 no 1 pp 40ndash45 2003
[11] K Ahmadi A Akya B Numanpour A Salimi and A Veisi-Raigani ldquoFrequency of Streptococcus pneumoniae infection inpatients with suspected meningitis in Imam Reza Hospital ofKermanshah in the west of Iranrdquo Iranian Journal of Microbiol-ogy vol 7 no 1 pp 12ndash17 2015
[12] L E Nigrovic RMalley C GMacias et al ldquoEffect of antibioticpretreatment on cerebrospinal fluid profiles of children withbacterial meningitisrdquo Pediatrics vol 122 no 4 pp 726ndash7302008
[13] J P Bryan H Rodriques de Silva A Tavares H Rocha andW M Scheld ldquoEtiology and mortality of bacterial meningitisin northeastern Brazilrdquo Clinical Infectious Diseases vol 12 no1 pp 128ndash135 1990
[14] A R Tunkel B J Hartman S L Kaplan et al ldquoPracticeguidelines for themanagement of bacterial meningitisrdquoClinicalInfectious Diseases vol 39 no 9 pp 1267ndash1284 2004
[15] L E Nigrovic N Kuppermann A J McAdam and R MalleyldquoCerebrospinal latex agglutination fails to contribute to themicrobiologic diagnosis of pretreated childrenwithmeningitisrdquoPediatric Infectious Disease Journal vol 23 no 8 pp 786ndash7882004
[16] K Tarafdar S Rao R A Recco and M M Zaman ldquoLackof sensitivity of the latex agglutination test to detect bacterialantigen in the cerebrospinal fluid of patients with culture-negative meningitisrdquo Clinical Infectious Diseases vol 33 no 3pp 406ndash408 2001
[17] E Van Gastel P Bruynseels W Verstrepen and A MertensldquoEvaluation of a real-time polymerase chain reaction assay forthe diagnosis of pneumococcal and meningococcal meningitisin a tertiary care hospitalrdquo European Journal of Clinical Micro-biology and Infectious Diseases vol 26 no 9 pp 651ndash653 2007
[18] M Tuyama R F Boente M C Rebelo R P Igreja and D EBarroso ldquoThe utility of the polymerase chain reaction assay foraetiologic definition of unspecified bacterial meningitis casesrdquoMemorias do Instituto Oswaldo Cruz vol 103 no 2 pp 138ndash1422008
[19] C T Sacchi L O Fukasawa M G Goncalves et al ldquoIncorpo-ration of real-time PCR into routine Public Health Surveillance
of Culture Negative Bacterial meningitis in Sao Paulo BrazilrdquoPLoS ONE vol 6 no 6 Article ID e20675 2011
[20] B Heydaria H Khalilib I Karimzadehc and H Emadi-Kochak ldquoClinical paraclinical and antimicrobial resistancefeatures of community-acquired acute bacterial meningitis at alarge infectious diseases ward in Tehran Iranrdquo Iranian Journalof Pharmaceutical Research vol 15 pp 347ndash354 2014
[21] M Cheesebrough ldquoBiochemical tests to identify bacteriardquo inDistrict Laboratory Practice in Tropical Countries M Chees-brough Ed pp 116ndash124 Cambridge University Press Cam-bridge UK 2nd edition 2007
[22] K Ohkusu K A Nash and C B Inderlied ldquoMolecular char-acterisation of Haemophilus influenzae type a and untypeablestrains isolated simultaneously from cerebrospinal fluid andblood novel use of quantitative real-time PCR based on the capcopy number to determine virulencerdquoClinicalMicrobiology andInfection vol 11 no 8 pp 637ndash643 2005
[23] L Ragunathan M Ramsay R Borrow M Guiver S Grayand E B Kaczmarski ldquoClinical features laboratory findingsand management of meningococcal meningitis in England andWales report of a 1997 surveyrdquo Journal of Infection vol 40 no1 pp 74ndash79 2000
[24] N Guirguis K Hafez M A El Kholy J B Robbins and EC Gotschlich ldquoBacterial meningitis in Egypt analysis of CSFisolates from hospital patients in Cairo 1977-1978rdquo Bulletin ofthe World Health Organization vol 61 no 3 pp 517ndash524 1983
[25] F G Youssef H El-Sakka A Azab et al ldquoEtiology antimi-crobial susceptibility profiles and mortality associated withbacterial meningitis among children in Egyptrdquo Annals of Epi-demiology vol 14 no 1 pp 44ndash48 2004
[26] M L Durand S B Calderwood D J Weber et al ldquoAcutebacterial meningitis in adults a review of 493 episodesrdquo TheNew England Journal of Medicine vol 328 no 1 pp 21ndash28 1993
[27] B Sigurdardottir O M Bjornsson K E Jonsdottir HErlendsdottir and S Gudmundsson ldquoAcute bacterial meningi-tis in adults a 20-year overviewrdquo Archives of Internal Medicinevol 157 no 4 pp 425ndash430 1997
[28] B Arda O R Sipahi S Atalay and S Ulusoy ldquoPooled analysisof 2408 cases of acute adult purulent meningitis from TurkeyrdquoMedical Principles and Practice vol 17 no 1 pp 76ndash79 2008
[29] M Scarborough S B Gordon C J MWhitty et al ldquoCorticos-teroids for bacterial meningitis in adults in sub-SaharanAfricardquoNewEngland Journal ofMedicine vol 357 no 24 pp 2441ndash24502007
[30] M Scarborough and G E Thwaites ldquoThe diagnosis andmanagement of acute bacterial meningitis in resource-poorsettingsrdquoThe Lancet Neurology vol 7 no 7 pp 637ndash648 2008
[31] G Campagne A Schuchat S Djibo AOusseini L Cisse and JP Chippaux ldquoEpidemiology of bacterial meningitis in NiameyNiger 1981ndash96rdquo Bulletin of the World Health Organization vol77 no 6 pp 499ndash508 1999
[32] S Afifi M O Wasfy M A Azab et al ldquoLaboratory-basedsurveillance of patients with bacterial meningitis in Egypt(1998ndash2004)rdquo European Journal of Clinical Microbiology andInfectious Diseases vol 26 no 5 pp 331ndash340 2007
[33] L Shaban andR Siam ldquoPrevalence and antimicrobial resistancepattern of bacterial meningitis in Egyptrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 8 article 26 2009
[34] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
This study aimed to diagnose bacterial meningitis causedby Neisseria meningitidis Streptococcus pneumoniae andHaemophilus influenzae type b (Hib) in culture-negative CSFsamples by the aid of real-time PCR
2 Materials and Methods
This study was performed during the period from December2014 to March 2015 and was approved by the Ethics Com-mittee of Faculty of Medicine Ain Shams University CairoEgypt and in accordance with the ethical guidelines of theDeclaration of Helsinki 1975
The study comprised a total of 40 CSF samples recoveredfrom adult patients admitted to Abbasseya Fever Hospitalpresenting with clinical picture and abnormal CSF cellularand chemistry results suggestive of bacterial meningitis
Clinical criteria for inclusion of patients were fever head-ache vomiting photophobia and irritability (symptoms ofmeningeal irritation) along with neck rigidity Kernig signBrudzinski sign altered conscious level seizures and focalneurological signs (signs of meningeal irritation) [4]
The diagnostic laboratory criteria for bacterial meningi-tis included the following glucose concentration less than40mgL protein concentration more than 50mgdL a whitecell count more than 100 cells per mm3 and neutrophil per-centage more than 50 [20]
The collectedCSF sampleswere centrifuged at 10000 rpmfor 10min and the supernatant was examined for abnormal-ities in WBCs protein and glucose Ten microliters of thesedimentwas inoculated onto sheep blood agar and chocolateagar and the rest was aliquoted and stored at minus70∘C Bacterialgrowth was observed after overnight incubation of the agarplates at 37∘C in 5 CO
2atmosphere [21]
Data of patients with negative culture results wererecorded and their stored CSF samples were further exam-ined by real-time PCR
21 Real-Time PCR
211 Bacterial DNAExtraction Bacterial DNAwas extractedfrom CSF samples with the aid of QIAGEN DNA Mini kit(QIAGEN Inc California) as per themanufacturerrsquos protocolfor DNA purification The eluted DNA was stored at minus20∘Cuntil further processed
212 Real-Time PCR with SYBR Green I Three runs weresequentially performed for the detection of each organismseparatelyThe ctrA gene ofNeisseria meningitidis bexA geneof Haemophilus influenzae and lytA gene of Streptococcuspneumoniae [9] were used as species-specific targets Theprimers sequences are listed in Table 1 The mix for eachrun included 5 120583L of sample DNA 125 120583L of 2x QuantiTectSYBR Green PCR master mix (QIAGEN Inc Valencia CA)containing a buffer dNTP mix MgCl
2and HotStarTaq DNA
polymerase 1 120583L of the primer and RNase-free water fora final volume of 25 120583L DNA was amplified with the StepOne Real-Time PCR system (Applied Biosystems) by usingthe following temperature program an initial Hotstart Taqactivation step at 95∘C for 15min initial denaturing at 95∘C
for 15 seconds and 40 PCR cycles of denaturing at 95∘C for30 seconds annealing at 50∘C for 30 seconds and extensionat 72∘C for 30 seconds followed by melting curve stage of95∘C and 60∘C [22] Amplification data were analyzed byinstrument software (Step One Software v23) in terms ofmelting curve graphs of each sample (Figure 1) Positivecontrol strains and negative controls consisting of PCR gradewater instead of the target DNA were used in each run
22 Statistical Analysis Quantitative variables are presentedas mean and SD and intergroup differences are comparedusing the unpaired 119905-test Categorical variables are presentedas number and percentage Discrete and skewed continuousdata are presented as median and differences between groupsare compared using the Mann-Whitney test The statisticalprocedures were carried out using SPSS version 15 forWindows (SPSS Inc Chicago IL USA)
3 Results
The present study was conducted on 40 culture-negative CSFsamples withdrawn from 27 (675) males and 13 (325)females Age of patients ranged from 19 to 56 years (mean plusmnSD = 3833 plusmn 1044) Demographic data of the patients andbiochemical and cytological findings of the CSF samples arelisted in Table 2
CSF chemistry revealed elevated protein level(gt50mgdL) in 825 of the samples and decreasedglucose level (lt40mgdL) in 875 of them White blood cell(WBC) count gt1000 cellsmm3 was found in 575 of thesamples whereas 425 of the samples showed WBC countbetween 100 and 1000 cellsmm3 Predominant neutrophilicCSF (neutrophil gt 50) was found in 725 of the samples
Thirty-six samples (90) were positive for Streptococcuspneumoniae by real-timePCRwhereasNeisseriameningitidisand Haemophilus influenzae were not detected in any of thesamples (0) Four samples (10) were negative for all threeorganisms (Table 3)
No statistical significant difference was observed regard-ing CSF chemistry and cells between samples of CSF positiveand negative real-time PCR results (Table 4)
4 Discussion
Rapid and accurate laboratory diagnostics remain a crucialstep for the final diagnosis of bacterial meningitis specifyingthe treatment and implementing preventive measures forclose contacts when indicated Conventional culture meth-ods though the gold standard diagnostic technique cannotbe relied upon per se in certain situations owing to the delayin results availability and the relatively limited sensitivitythat had been repeatedly reported worldwide [12 13 23]and in conditions when prior antimicrobial therapy has beenreceived In Egypt a high percentage of culture-negativesamples was reported previously [24 25] These results wereexplained by the fact that most patients received antimicro-bial agents that are readily purchased as over-the-countermedications even prior to clinical evaluation alongside theoccasional delay in csf sampling For the above-mentioned
International Journal of Microbiology 3
200
175
150
125
100
75
50
25
0
ΔRn
Cycle4036322824201612840
4078621
(a)
600
500
400
300
200
100
Der
ivat
ive r
epor
ter (
minusRn
)
Tm 7678
Temperature (∘C)950850750650
(b)
Figure 1 (a) Real-time PCR amplification plot for lytA gene specific for Streptococcus pneumoniae Horizontal blue line represents thethreshold value of fluorescence (b) Melting curves of positive samples for Streptococcus pneumoniae
Data are presented as mean plusmn standard deviation for continuous variablesand as number (percentage) for categorical variablesWBCs white blood cells
reasons we employed a molecular method (real-time PCR)in this study to improve the diagnosis of bacterial meningitisin culture-negative purulent CSF samples
Demographic data analysis of patients revealed that theywere mainly males (675 versus 125 for females) Most ofthe cases belonged to the middle age group (mean = 383)This finding agreed with Fouad et al [4] who confirmedthat males were more significantly affected with bacterialmeningitis than females (61 versus 39 resp) though thedisease was distributed in all age groups with low rates ofoccurrence in the extremes of age (the neonates and above60 years)
Real-time PCR in this study was positive for Streptococcuspneumoniae in 36 culture-negative CSF samples (90) whileno positive results for Haemophilus influenzae or Neisseriameningitidis were detected The age group of the patients inthis study might have contributed to these results as the maincausative agents of bacterial meningitis in adults are gener-ally believed to be Streptococcus pneumoniae and Neisseriameningitidis [1 26 27] Streptococcus pneumoniae is foundto be the commonest etiology of bacterial meningitis in the
4 International Journal of Microbiology
Table 4 Comparison between real-time PCR positive and negative samples as regards CSF cells and chemistry results
Variables RT-PCR positive RT-PCR negative 119885lowast119905 p valueMedian
Mean plusmn SDNeutrophil percentage 75 plusmn 218 675 plusmn 185 lowast066 0513 (NS)Data are presented as median or mean plusmn standard deviationNS nonsignificant result (119901 value gt 005)119885 Mann-Whitney testlowast119905 unpaired 119905-test
WBCs white blood cells
United States and Europe accounting for 61 of total cases inthe United States [1 3 28] and inmost African countries withhigh human immune deficiency virus prevalence [29 30]Yet meningococcal meningitis is common in Sub-SaharanAfrica (the meningitis belt) but mostly apparent in theform of epidemics and outbreaks [31] In a laboratory-basedsurveillance study undergone in Egypt byAfifi et al [32] PCRwas performed on purulent culture-negative CSF specimenswithdrawn from patients who met the criteria for casedefinition of bacterial meningitis Streptococcus pneumoniaewas also reported as the most common etiology of bacterialmeningitis
Fouad et al [4] also documented Streptococcus pneu-moniae as the most frequent isolate (52) among bacteriacausing meningitisThe agreement between our findings andthose of the previously mentioned studies in Egypt consoli-dates the deduction of Shaban and Siam [33] in their reviewarticle that pneumococcal meningitis is currently the leadingcause of meningitis in Egypt as its incidence is constantlyrising at the expense of meningococcal meningitis whichmay be a reflection of the increased use of polysaccha-ride meningococcal vaccines
Wang et al [7] also identified bacterial meningitis in fivecases (9) by CSF cultures and 25 (45) by real-time PCRThey considered real-time PCR much more sensitive thanculture for the diagnosis of bacterial meningitis particularlyin their study where 68 of patients had received priorantimicrobial treatment and their CSF samples yielded neg-ative culture results A similar conclusion has been reachedby Wu et al [9] and Sacchi et al [19] who stated that real-time PCR increases diagnostic yield for bacterial meningitisand is ideal for incorporation into routine surveillance indeveloping countries
According to Brouwer et al [34] CSF culture is doc-umented to be positive only in 110th of the previouslyantibiotic treated patients in developing countries Sameresult was found by Afifi et al [32] who reported low ratesof culture positive CSF samples (8) of suspected cases withbacterial meningitis [32] This low yield in culture resultscould be attributed to the fact that antimicrobials are beingdispensed without prescriptions in Egypt
This study faces the limitation of the relatively lownumber ofCSF samples investigated and the lack of testing forother less commonly bacterial etiologies of adult meningitis
(eg Group B Streptococcus Listeria monocytogenes) Thismay provide an explanation for the negative results (10)obtained
According to our findings we conclude that the use ofmolecular technique in the diagnosis of bacterial meningitisshould be considered in suspected cases with negative cultureresults before reporting exclusion of the disease
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors gratefully acknowledge the support provided bythe physicians of the Microbiology Laboratory in AbbasseyaFever Hospital for supplying them with the CSF samplesincluded in this work
References
[1] D Van De Beek J De Gans L Spanjaard M Weisfelt J BReitsma and M Vermeulen ldquoClinical features and prognosticfactors in adults with bacterial meningitisrdquo New England Jour-nal of Medicine vol 351 no 18 pp 1849ndash1923 2004
[2] S A Namani R A Koci E Qehaja-Bucaj L Ajazaj-Berishaand M Mehmeti ldquoThe epidemiology of bacterial meningitis inKosovordquo Journal of Infection in Developing Countries vol 8 no7 pp 823ndash830 2014
[3] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
[4] R Fouad M Khairy W Fathalah T Gad B El-Kholy and AYosry ldquoRole of clinical presentations and routine CSF analysisin the rapid diagnosis of acute bacterial meningitis in cases ofnegative gram stained smearsrdquo Journal of TropicalMedicine vol2014 Article ID 213762 7 pages 2014
[5] C E Corless M Guiver R Borrow V Edwards-Jones A JFox and E B Kaczmarski ldquoSimultaneous detection ofNeisseriameningitidis Haemophilus influenzae and Streptococcus pneu-moniae in suspected cases of meningitis and septicemia usingreal-time PCRrdquo Journal of Clinical Microbiology vol 39 no 4pp 1553ndash1558 2001
[6] G M K Abdeldaim K Stralin J Korsgaard J Blomberg CWelinder-Olsson and B Herrmann ldquoMultiplex quantitative
International Journal of Microbiology 5
PCR for detection of lower respiratory tract infection andmeningitis caused by Streptococcus pneumoniaeHaemophilusinfluenzae and Neisseria meningitidisrdquo BMC Microbiology vol10 article 310 2010
[7] Y Wang G Guo H Wang et al ldquoComparative study ofbacteriological culture and real-time fluorescence quantitativePCR (RT-PCR) and multiplex PCR-based reverse line blot(mPCRRLB) hybridization assay in the diagnosis of bacterialneonatal meningitisrdquo BMC Pediatrics vol 14 no 1 article 2242014
[8] M W Bijlsma M C Brouwer E S Kasanmoentalib et alldquoCommunity-acquired bacterial meningitis in adults in theNetherlands 2006ndash2014 a prospective cohort studyrdquoTheLancetInfectious Diseases vol 16 no 3 pp 339ndash447 2016
[9] H M Wu S M Cordeiro B H Harcourt et al ldquoAccuracyof real-time PCR Gram stain and culture for StreptococcuspneumoniaeNeisseriameningitidis andHaemophilus influenzaemeningitis diagnosisrdquo BMC Infectious Diseases vol 13 no 1article 26 2013
[10] L D Saravolatz O Manzor N VanderVelde J Pawlak andB Belian ldquoBroad-range bacterial polymerase chain reactionfor early detection of bacterial meningitisrdquo Clinical InfectiousDiseases vol 36 no 1 pp 40ndash45 2003
[11] K Ahmadi A Akya B Numanpour A Salimi and A Veisi-Raigani ldquoFrequency of Streptococcus pneumoniae infection inpatients with suspected meningitis in Imam Reza Hospital ofKermanshah in the west of Iranrdquo Iranian Journal of Microbiol-ogy vol 7 no 1 pp 12ndash17 2015
[12] L E Nigrovic RMalley C GMacias et al ldquoEffect of antibioticpretreatment on cerebrospinal fluid profiles of children withbacterial meningitisrdquo Pediatrics vol 122 no 4 pp 726ndash7302008
[13] J P Bryan H Rodriques de Silva A Tavares H Rocha andW M Scheld ldquoEtiology and mortality of bacterial meningitisin northeastern Brazilrdquo Clinical Infectious Diseases vol 12 no1 pp 128ndash135 1990
[14] A R Tunkel B J Hartman S L Kaplan et al ldquoPracticeguidelines for themanagement of bacterial meningitisrdquoClinicalInfectious Diseases vol 39 no 9 pp 1267ndash1284 2004
[15] L E Nigrovic N Kuppermann A J McAdam and R MalleyldquoCerebrospinal latex agglutination fails to contribute to themicrobiologic diagnosis of pretreated childrenwithmeningitisrdquoPediatric Infectious Disease Journal vol 23 no 8 pp 786ndash7882004
[16] K Tarafdar S Rao R A Recco and M M Zaman ldquoLackof sensitivity of the latex agglutination test to detect bacterialantigen in the cerebrospinal fluid of patients with culture-negative meningitisrdquo Clinical Infectious Diseases vol 33 no 3pp 406ndash408 2001
[17] E Van Gastel P Bruynseels W Verstrepen and A MertensldquoEvaluation of a real-time polymerase chain reaction assay forthe diagnosis of pneumococcal and meningococcal meningitisin a tertiary care hospitalrdquo European Journal of Clinical Micro-biology and Infectious Diseases vol 26 no 9 pp 651ndash653 2007
[18] M Tuyama R F Boente M C Rebelo R P Igreja and D EBarroso ldquoThe utility of the polymerase chain reaction assay foraetiologic definition of unspecified bacterial meningitis casesrdquoMemorias do Instituto Oswaldo Cruz vol 103 no 2 pp 138ndash1422008
[19] C T Sacchi L O Fukasawa M G Goncalves et al ldquoIncorpo-ration of real-time PCR into routine Public Health Surveillance
of Culture Negative Bacterial meningitis in Sao Paulo BrazilrdquoPLoS ONE vol 6 no 6 Article ID e20675 2011
[20] B Heydaria H Khalilib I Karimzadehc and H Emadi-Kochak ldquoClinical paraclinical and antimicrobial resistancefeatures of community-acquired acute bacterial meningitis at alarge infectious diseases ward in Tehran Iranrdquo Iranian Journalof Pharmaceutical Research vol 15 pp 347ndash354 2014
[21] M Cheesebrough ldquoBiochemical tests to identify bacteriardquo inDistrict Laboratory Practice in Tropical Countries M Chees-brough Ed pp 116ndash124 Cambridge University Press Cam-bridge UK 2nd edition 2007
[22] K Ohkusu K A Nash and C B Inderlied ldquoMolecular char-acterisation of Haemophilus influenzae type a and untypeablestrains isolated simultaneously from cerebrospinal fluid andblood novel use of quantitative real-time PCR based on the capcopy number to determine virulencerdquoClinicalMicrobiology andInfection vol 11 no 8 pp 637ndash643 2005
[23] L Ragunathan M Ramsay R Borrow M Guiver S Grayand E B Kaczmarski ldquoClinical features laboratory findingsand management of meningococcal meningitis in England andWales report of a 1997 surveyrdquo Journal of Infection vol 40 no1 pp 74ndash79 2000
[24] N Guirguis K Hafez M A El Kholy J B Robbins and EC Gotschlich ldquoBacterial meningitis in Egypt analysis of CSFisolates from hospital patients in Cairo 1977-1978rdquo Bulletin ofthe World Health Organization vol 61 no 3 pp 517ndash524 1983
[25] F G Youssef H El-Sakka A Azab et al ldquoEtiology antimi-crobial susceptibility profiles and mortality associated withbacterial meningitis among children in Egyptrdquo Annals of Epi-demiology vol 14 no 1 pp 44ndash48 2004
[26] M L Durand S B Calderwood D J Weber et al ldquoAcutebacterial meningitis in adults a review of 493 episodesrdquo TheNew England Journal of Medicine vol 328 no 1 pp 21ndash28 1993
[27] B Sigurdardottir O M Bjornsson K E Jonsdottir HErlendsdottir and S Gudmundsson ldquoAcute bacterial meningi-tis in adults a 20-year overviewrdquo Archives of Internal Medicinevol 157 no 4 pp 425ndash430 1997
[28] B Arda O R Sipahi S Atalay and S Ulusoy ldquoPooled analysisof 2408 cases of acute adult purulent meningitis from TurkeyrdquoMedical Principles and Practice vol 17 no 1 pp 76ndash79 2008
[29] M Scarborough S B Gordon C J MWhitty et al ldquoCorticos-teroids for bacterial meningitis in adults in sub-SaharanAfricardquoNewEngland Journal ofMedicine vol 357 no 24 pp 2441ndash24502007
[30] M Scarborough and G E Thwaites ldquoThe diagnosis andmanagement of acute bacterial meningitis in resource-poorsettingsrdquoThe Lancet Neurology vol 7 no 7 pp 637ndash648 2008
[31] G Campagne A Schuchat S Djibo AOusseini L Cisse and JP Chippaux ldquoEpidemiology of bacterial meningitis in NiameyNiger 1981ndash96rdquo Bulletin of the World Health Organization vol77 no 6 pp 499ndash508 1999
[32] S Afifi M O Wasfy M A Azab et al ldquoLaboratory-basedsurveillance of patients with bacterial meningitis in Egypt(1998ndash2004)rdquo European Journal of Clinical Microbiology andInfectious Diseases vol 26 no 5 pp 331ndash340 2007
[33] L Shaban andR Siam ldquoPrevalence and antimicrobial resistancepattern of bacterial meningitis in Egyptrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 8 article 26 2009
[34] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
Figure 1 (a) Real-time PCR amplification plot for lytA gene specific for Streptococcus pneumoniae Horizontal blue line represents thethreshold value of fluorescence (b) Melting curves of positive samples for Streptococcus pneumoniae
Data are presented as mean plusmn standard deviation for continuous variablesand as number (percentage) for categorical variablesWBCs white blood cells
reasons we employed a molecular method (real-time PCR)in this study to improve the diagnosis of bacterial meningitisin culture-negative purulent CSF samples
Demographic data analysis of patients revealed that theywere mainly males (675 versus 125 for females) Most ofthe cases belonged to the middle age group (mean = 383)This finding agreed with Fouad et al [4] who confirmedthat males were more significantly affected with bacterialmeningitis than females (61 versus 39 resp) though thedisease was distributed in all age groups with low rates ofoccurrence in the extremes of age (the neonates and above60 years)
Real-time PCR in this study was positive for Streptococcuspneumoniae in 36 culture-negative CSF samples (90) whileno positive results for Haemophilus influenzae or Neisseriameningitidis were detected The age group of the patients inthis study might have contributed to these results as the maincausative agents of bacterial meningitis in adults are gener-ally believed to be Streptococcus pneumoniae and Neisseriameningitidis [1 26 27] Streptococcus pneumoniae is foundto be the commonest etiology of bacterial meningitis in the
4 International Journal of Microbiology
Table 4 Comparison between real-time PCR positive and negative samples as regards CSF cells and chemistry results
Variables RT-PCR positive RT-PCR negative 119885lowast119905 p valueMedian
Mean plusmn SDNeutrophil percentage 75 plusmn 218 675 plusmn 185 lowast066 0513 (NS)Data are presented as median or mean plusmn standard deviationNS nonsignificant result (119901 value gt 005)119885 Mann-Whitney testlowast119905 unpaired 119905-test
WBCs white blood cells
United States and Europe accounting for 61 of total cases inthe United States [1 3 28] and inmost African countries withhigh human immune deficiency virus prevalence [29 30]Yet meningococcal meningitis is common in Sub-SaharanAfrica (the meningitis belt) but mostly apparent in theform of epidemics and outbreaks [31] In a laboratory-basedsurveillance study undergone in Egypt byAfifi et al [32] PCRwas performed on purulent culture-negative CSF specimenswithdrawn from patients who met the criteria for casedefinition of bacterial meningitis Streptococcus pneumoniaewas also reported as the most common etiology of bacterialmeningitis
Fouad et al [4] also documented Streptococcus pneu-moniae as the most frequent isolate (52) among bacteriacausing meningitisThe agreement between our findings andthose of the previously mentioned studies in Egypt consoli-dates the deduction of Shaban and Siam [33] in their reviewarticle that pneumococcal meningitis is currently the leadingcause of meningitis in Egypt as its incidence is constantlyrising at the expense of meningococcal meningitis whichmay be a reflection of the increased use of polysaccha-ride meningococcal vaccines
Wang et al [7] also identified bacterial meningitis in fivecases (9) by CSF cultures and 25 (45) by real-time PCRThey considered real-time PCR much more sensitive thanculture for the diagnosis of bacterial meningitis particularlyin their study where 68 of patients had received priorantimicrobial treatment and their CSF samples yielded neg-ative culture results A similar conclusion has been reachedby Wu et al [9] and Sacchi et al [19] who stated that real-time PCR increases diagnostic yield for bacterial meningitisand is ideal for incorporation into routine surveillance indeveloping countries
According to Brouwer et al [34] CSF culture is doc-umented to be positive only in 110th of the previouslyantibiotic treated patients in developing countries Sameresult was found by Afifi et al [32] who reported low ratesof culture positive CSF samples (8) of suspected cases withbacterial meningitis [32] This low yield in culture resultscould be attributed to the fact that antimicrobials are beingdispensed without prescriptions in Egypt
This study faces the limitation of the relatively lownumber ofCSF samples investigated and the lack of testing forother less commonly bacterial etiologies of adult meningitis
(eg Group B Streptococcus Listeria monocytogenes) Thismay provide an explanation for the negative results (10)obtained
According to our findings we conclude that the use ofmolecular technique in the diagnosis of bacterial meningitisshould be considered in suspected cases with negative cultureresults before reporting exclusion of the disease
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors gratefully acknowledge the support provided bythe physicians of the Microbiology Laboratory in AbbasseyaFever Hospital for supplying them with the CSF samplesincluded in this work
References
[1] D Van De Beek J De Gans L Spanjaard M Weisfelt J BReitsma and M Vermeulen ldquoClinical features and prognosticfactors in adults with bacterial meningitisrdquo New England Jour-nal of Medicine vol 351 no 18 pp 1849ndash1923 2004
[2] S A Namani R A Koci E Qehaja-Bucaj L Ajazaj-Berishaand M Mehmeti ldquoThe epidemiology of bacterial meningitis inKosovordquo Journal of Infection in Developing Countries vol 8 no7 pp 823ndash830 2014
[3] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
[4] R Fouad M Khairy W Fathalah T Gad B El-Kholy and AYosry ldquoRole of clinical presentations and routine CSF analysisin the rapid diagnosis of acute bacterial meningitis in cases ofnegative gram stained smearsrdquo Journal of TropicalMedicine vol2014 Article ID 213762 7 pages 2014
[5] C E Corless M Guiver R Borrow V Edwards-Jones A JFox and E B Kaczmarski ldquoSimultaneous detection ofNeisseriameningitidis Haemophilus influenzae and Streptococcus pneu-moniae in suspected cases of meningitis and septicemia usingreal-time PCRrdquo Journal of Clinical Microbiology vol 39 no 4pp 1553ndash1558 2001
[6] G M K Abdeldaim K Stralin J Korsgaard J Blomberg CWelinder-Olsson and B Herrmann ldquoMultiplex quantitative
International Journal of Microbiology 5
PCR for detection of lower respiratory tract infection andmeningitis caused by Streptococcus pneumoniaeHaemophilusinfluenzae and Neisseria meningitidisrdquo BMC Microbiology vol10 article 310 2010
[7] Y Wang G Guo H Wang et al ldquoComparative study ofbacteriological culture and real-time fluorescence quantitativePCR (RT-PCR) and multiplex PCR-based reverse line blot(mPCRRLB) hybridization assay in the diagnosis of bacterialneonatal meningitisrdquo BMC Pediatrics vol 14 no 1 article 2242014
[8] M W Bijlsma M C Brouwer E S Kasanmoentalib et alldquoCommunity-acquired bacterial meningitis in adults in theNetherlands 2006ndash2014 a prospective cohort studyrdquoTheLancetInfectious Diseases vol 16 no 3 pp 339ndash447 2016
[9] H M Wu S M Cordeiro B H Harcourt et al ldquoAccuracyof real-time PCR Gram stain and culture for StreptococcuspneumoniaeNeisseriameningitidis andHaemophilus influenzaemeningitis diagnosisrdquo BMC Infectious Diseases vol 13 no 1article 26 2013
[10] L D Saravolatz O Manzor N VanderVelde J Pawlak andB Belian ldquoBroad-range bacterial polymerase chain reactionfor early detection of bacterial meningitisrdquo Clinical InfectiousDiseases vol 36 no 1 pp 40ndash45 2003
[11] K Ahmadi A Akya B Numanpour A Salimi and A Veisi-Raigani ldquoFrequency of Streptococcus pneumoniae infection inpatients with suspected meningitis in Imam Reza Hospital ofKermanshah in the west of Iranrdquo Iranian Journal of Microbiol-ogy vol 7 no 1 pp 12ndash17 2015
[12] L E Nigrovic RMalley C GMacias et al ldquoEffect of antibioticpretreatment on cerebrospinal fluid profiles of children withbacterial meningitisrdquo Pediatrics vol 122 no 4 pp 726ndash7302008
[13] J P Bryan H Rodriques de Silva A Tavares H Rocha andW M Scheld ldquoEtiology and mortality of bacterial meningitisin northeastern Brazilrdquo Clinical Infectious Diseases vol 12 no1 pp 128ndash135 1990
[14] A R Tunkel B J Hartman S L Kaplan et al ldquoPracticeguidelines for themanagement of bacterial meningitisrdquoClinicalInfectious Diseases vol 39 no 9 pp 1267ndash1284 2004
[15] L E Nigrovic N Kuppermann A J McAdam and R MalleyldquoCerebrospinal latex agglutination fails to contribute to themicrobiologic diagnosis of pretreated childrenwithmeningitisrdquoPediatric Infectious Disease Journal vol 23 no 8 pp 786ndash7882004
[16] K Tarafdar S Rao R A Recco and M M Zaman ldquoLackof sensitivity of the latex agglutination test to detect bacterialantigen in the cerebrospinal fluid of patients with culture-negative meningitisrdquo Clinical Infectious Diseases vol 33 no 3pp 406ndash408 2001
[17] E Van Gastel P Bruynseels W Verstrepen and A MertensldquoEvaluation of a real-time polymerase chain reaction assay forthe diagnosis of pneumococcal and meningococcal meningitisin a tertiary care hospitalrdquo European Journal of Clinical Micro-biology and Infectious Diseases vol 26 no 9 pp 651ndash653 2007
[18] M Tuyama R F Boente M C Rebelo R P Igreja and D EBarroso ldquoThe utility of the polymerase chain reaction assay foraetiologic definition of unspecified bacterial meningitis casesrdquoMemorias do Instituto Oswaldo Cruz vol 103 no 2 pp 138ndash1422008
[19] C T Sacchi L O Fukasawa M G Goncalves et al ldquoIncorpo-ration of real-time PCR into routine Public Health Surveillance
of Culture Negative Bacterial meningitis in Sao Paulo BrazilrdquoPLoS ONE vol 6 no 6 Article ID e20675 2011
[20] B Heydaria H Khalilib I Karimzadehc and H Emadi-Kochak ldquoClinical paraclinical and antimicrobial resistancefeatures of community-acquired acute bacterial meningitis at alarge infectious diseases ward in Tehran Iranrdquo Iranian Journalof Pharmaceutical Research vol 15 pp 347ndash354 2014
[21] M Cheesebrough ldquoBiochemical tests to identify bacteriardquo inDistrict Laboratory Practice in Tropical Countries M Chees-brough Ed pp 116ndash124 Cambridge University Press Cam-bridge UK 2nd edition 2007
[22] K Ohkusu K A Nash and C B Inderlied ldquoMolecular char-acterisation of Haemophilus influenzae type a and untypeablestrains isolated simultaneously from cerebrospinal fluid andblood novel use of quantitative real-time PCR based on the capcopy number to determine virulencerdquoClinicalMicrobiology andInfection vol 11 no 8 pp 637ndash643 2005
[23] L Ragunathan M Ramsay R Borrow M Guiver S Grayand E B Kaczmarski ldquoClinical features laboratory findingsand management of meningococcal meningitis in England andWales report of a 1997 surveyrdquo Journal of Infection vol 40 no1 pp 74ndash79 2000
[24] N Guirguis K Hafez M A El Kholy J B Robbins and EC Gotschlich ldquoBacterial meningitis in Egypt analysis of CSFisolates from hospital patients in Cairo 1977-1978rdquo Bulletin ofthe World Health Organization vol 61 no 3 pp 517ndash524 1983
[25] F G Youssef H El-Sakka A Azab et al ldquoEtiology antimi-crobial susceptibility profiles and mortality associated withbacterial meningitis among children in Egyptrdquo Annals of Epi-demiology vol 14 no 1 pp 44ndash48 2004
[26] M L Durand S B Calderwood D J Weber et al ldquoAcutebacterial meningitis in adults a review of 493 episodesrdquo TheNew England Journal of Medicine vol 328 no 1 pp 21ndash28 1993
[27] B Sigurdardottir O M Bjornsson K E Jonsdottir HErlendsdottir and S Gudmundsson ldquoAcute bacterial meningi-tis in adults a 20-year overviewrdquo Archives of Internal Medicinevol 157 no 4 pp 425ndash430 1997
[28] B Arda O R Sipahi S Atalay and S Ulusoy ldquoPooled analysisof 2408 cases of acute adult purulent meningitis from TurkeyrdquoMedical Principles and Practice vol 17 no 1 pp 76ndash79 2008
[29] M Scarborough S B Gordon C J MWhitty et al ldquoCorticos-teroids for bacterial meningitis in adults in sub-SaharanAfricardquoNewEngland Journal ofMedicine vol 357 no 24 pp 2441ndash24502007
[30] M Scarborough and G E Thwaites ldquoThe diagnosis andmanagement of acute bacterial meningitis in resource-poorsettingsrdquoThe Lancet Neurology vol 7 no 7 pp 637ndash648 2008
[31] G Campagne A Schuchat S Djibo AOusseini L Cisse and JP Chippaux ldquoEpidemiology of bacterial meningitis in NiameyNiger 1981ndash96rdquo Bulletin of the World Health Organization vol77 no 6 pp 499ndash508 1999
[32] S Afifi M O Wasfy M A Azab et al ldquoLaboratory-basedsurveillance of patients with bacterial meningitis in Egypt(1998ndash2004)rdquo European Journal of Clinical Microbiology andInfectious Diseases vol 26 no 5 pp 331ndash340 2007
[33] L Shaban andR Siam ldquoPrevalence and antimicrobial resistancepattern of bacterial meningitis in Egyptrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 8 article 26 2009
[34] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
Mean plusmn SDNeutrophil percentage 75 plusmn 218 675 plusmn 185 lowast066 0513 (NS)Data are presented as median or mean plusmn standard deviationNS nonsignificant result (119901 value gt 005)119885 Mann-Whitney testlowast119905 unpaired 119905-test
WBCs white blood cells
United States and Europe accounting for 61 of total cases inthe United States [1 3 28] and inmost African countries withhigh human immune deficiency virus prevalence [29 30]Yet meningococcal meningitis is common in Sub-SaharanAfrica (the meningitis belt) but mostly apparent in theform of epidemics and outbreaks [31] In a laboratory-basedsurveillance study undergone in Egypt byAfifi et al [32] PCRwas performed on purulent culture-negative CSF specimenswithdrawn from patients who met the criteria for casedefinition of bacterial meningitis Streptococcus pneumoniaewas also reported as the most common etiology of bacterialmeningitis
Fouad et al [4] also documented Streptococcus pneu-moniae as the most frequent isolate (52) among bacteriacausing meningitisThe agreement between our findings andthose of the previously mentioned studies in Egypt consoli-dates the deduction of Shaban and Siam [33] in their reviewarticle that pneumococcal meningitis is currently the leadingcause of meningitis in Egypt as its incidence is constantlyrising at the expense of meningococcal meningitis whichmay be a reflection of the increased use of polysaccha-ride meningococcal vaccines
Wang et al [7] also identified bacterial meningitis in fivecases (9) by CSF cultures and 25 (45) by real-time PCRThey considered real-time PCR much more sensitive thanculture for the diagnosis of bacterial meningitis particularlyin their study where 68 of patients had received priorantimicrobial treatment and their CSF samples yielded neg-ative culture results A similar conclusion has been reachedby Wu et al [9] and Sacchi et al [19] who stated that real-time PCR increases diagnostic yield for bacterial meningitisand is ideal for incorporation into routine surveillance indeveloping countries
According to Brouwer et al [34] CSF culture is doc-umented to be positive only in 110th of the previouslyantibiotic treated patients in developing countries Sameresult was found by Afifi et al [32] who reported low ratesof culture positive CSF samples (8) of suspected cases withbacterial meningitis [32] This low yield in culture resultscould be attributed to the fact that antimicrobials are beingdispensed without prescriptions in Egypt
This study faces the limitation of the relatively lownumber ofCSF samples investigated and the lack of testing forother less commonly bacterial etiologies of adult meningitis
(eg Group B Streptococcus Listeria monocytogenes) Thismay provide an explanation for the negative results (10)obtained
According to our findings we conclude that the use ofmolecular technique in the diagnosis of bacterial meningitisshould be considered in suspected cases with negative cultureresults before reporting exclusion of the disease
Competing Interests
The authors declare that they have no competing interests
Acknowledgments
The authors gratefully acknowledge the support provided bythe physicians of the Microbiology Laboratory in AbbasseyaFever Hospital for supplying them with the CSF samplesincluded in this work
References
[1] D Van De Beek J De Gans L Spanjaard M Weisfelt J BReitsma and M Vermeulen ldquoClinical features and prognosticfactors in adults with bacterial meningitisrdquo New England Jour-nal of Medicine vol 351 no 18 pp 1849ndash1923 2004
[2] S A Namani R A Koci E Qehaja-Bucaj L Ajazaj-Berishaand M Mehmeti ldquoThe epidemiology of bacterial meningitis inKosovordquo Journal of Infection in Developing Countries vol 8 no7 pp 823ndash830 2014
[3] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
[4] R Fouad M Khairy W Fathalah T Gad B El-Kholy and AYosry ldquoRole of clinical presentations and routine CSF analysisin the rapid diagnosis of acute bacterial meningitis in cases ofnegative gram stained smearsrdquo Journal of TropicalMedicine vol2014 Article ID 213762 7 pages 2014
[5] C E Corless M Guiver R Borrow V Edwards-Jones A JFox and E B Kaczmarski ldquoSimultaneous detection ofNeisseriameningitidis Haemophilus influenzae and Streptococcus pneu-moniae in suspected cases of meningitis and septicemia usingreal-time PCRrdquo Journal of Clinical Microbiology vol 39 no 4pp 1553ndash1558 2001
[6] G M K Abdeldaim K Stralin J Korsgaard J Blomberg CWelinder-Olsson and B Herrmann ldquoMultiplex quantitative
International Journal of Microbiology 5
PCR for detection of lower respiratory tract infection andmeningitis caused by Streptococcus pneumoniaeHaemophilusinfluenzae and Neisseria meningitidisrdquo BMC Microbiology vol10 article 310 2010
[7] Y Wang G Guo H Wang et al ldquoComparative study ofbacteriological culture and real-time fluorescence quantitativePCR (RT-PCR) and multiplex PCR-based reverse line blot(mPCRRLB) hybridization assay in the diagnosis of bacterialneonatal meningitisrdquo BMC Pediatrics vol 14 no 1 article 2242014
[8] M W Bijlsma M C Brouwer E S Kasanmoentalib et alldquoCommunity-acquired bacterial meningitis in adults in theNetherlands 2006ndash2014 a prospective cohort studyrdquoTheLancetInfectious Diseases vol 16 no 3 pp 339ndash447 2016
[9] H M Wu S M Cordeiro B H Harcourt et al ldquoAccuracyof real-time PCR Gram stain and culture for StreptococcuspneumoniaeNeisseriameningitidis andHaemophilus influenzaemeningitis diagnosisrdquo BMC Infectious Diseases vol 13 no 1article 26 2013
[10] L D Saravolatz O Manzor N VanderVelde J Pawlak andB Belian ldquoBroad-range bacterial polymerase chain reactionfor early detection of bacterial meningitisrdquo Clinical InfectiousDiseases vol 36 no 1 pp 40ndash45 2003
[11] K Ahmadi A Akya B Numanpour A Salimi and A Veisi-Raigani ldquoFrequency of Streptococcus pneumoniae infection inpatients with suspected meningitis in Imam Reza Hospital ofKermanshah in the west of Iranrdquo Iranian Journal of Microbiol-ogy vol 7 no 1 pp 12ndash17 2015
[12] L E Nigrovic RMalley C GMacias et al ldquoEffect of antibioticpretreatment on cerebrospinal fluid profiles of children withbacterial meningitisrdquo Pediatrics vol 122 no 4 pp 726ndash7302008
[13] J P Bryan H Rodriques de Silva A Tavares H Rocha andW M Scheld ldquoEtiology and mortality of bacterial meningitisin northeastern Brazilrdquo Clinical Infectious Diseases vol 12 no1 pp 128ndash135 1990
[14] A R Tunkel B J Hartman S L Kaplan et al ldquoPracticeguidelines for themanagement of bacterial meningitisrdquoClinicalInfectious Diseases vol 39 no 9 pp 1267ndash1284 2004
[15] L E Nigrovic N Kuppermann A J McAdam and R MalleyldquoCerebrospinal latex agglutination fails to contribute to themicrobiologic diagnosis of pretreated childrenwithmeningitisrdquoPediatric Infectious Disease Journal vol 23 no 8 pp 786ndash7882004
[16] K Tarafdar S Rao R A Recco and M M Zaman ldquoLackof sensitivity of the latex agglutination test to detect bacterialantigen in the cerebrospinal fluid of patients with culture-negative meningitisrdquo Clinical Infectious Diseases vol 33 no 3pp 406ndash408 2001
[17] E Van Gastel P Bruynseels W Verstrepen and A MertensldquoEvaluation of a real-time polymerase chain reaction assay forthe diagnosis of pneumococcal and meningococcal meningitisin a tertiary care hospitalrdquo European Journal of Clinical Micro-biology and Infectious Diseases vol 26 no 9 pp 651ndash653 2007
[18] M Tuyama R F Boente M C Rebelo R P Igreja and D EBarroso ldquoThe utility of the polymerase chain reaction assay foraetiologic definition of unspecified bacterial meningitis casesrdquoMemorias do Instituto Oswaldo Cruz vol 103 no 2 pp 138ndash1422008
[19] C T Sacchi L O Fukasawa M G Goncalves et al ldquoIncorpo-ration of real-time PCR into routine Public Health Surveillance
of Culture Negative Bacterial meningitis in Sao Paulo BrazilrdquoPLoS ONE vol 6 no 6 Article ID e20675 2011
[20] B Heydaria H Khalilib I Karimzadehc and H Emadi-Kochak ldquoClinical paraclinical and antimicrobial resistancefeatures of community-acquired acute bacterial meningitis at alarge infectious diseases ward in Tehran Iranrdquo Iranian Journalof Pharmaceutical Research vol 15 pp 347ndash354 2014
[21] M Cheesebrough ldquoBiochemical tests to identify bacteriardquo inDistrict Laboratory Practice in Tropical Countries M Chees-brough Ed pp 116ndash124 Cambridge University Press Cam-bridge UK 2nd edition 2007
[22] K Ohkusu K A Nash and C B Inderlied ldquoMolecular char-acterisation of Haemophilus influenzae type a and untypeablestrains isolated simultaneously from cerebrospinal fluid andblood novel use of quantitative real-time PCR based on the capcopy number to determine virulencerdquoClinicalMicrobiology andInfection vol 11 no 8 pp 637ndash643 2005
[23] L Ragunathan M Ramsay R Borrow M Guiver S Grayand E B Kaczmarski ldquoClinical features laboratory findingsand management of meningococcal meningitis in England andWales report of a 1997 surveyrdquo Journal of Infection vol 40 no1 pp 74ndash79 2000
[24] N Guirguis K Hafez M A El Kholy J B Robbins and EC Gotschlich ldquoBacterial meningitis in Egypt analysis of CSFisolates from hospital patients in Cairo 1977-1978rdquo Bulletin ofthe World Health Organization vol 61 no 3 pp 517ndash524 1983
[25] F G Youssef H El-Sakka A Azab et al ldquoEtiology antimi-crobial susceptibility profiles and mortality associated withbacterial meningitis among children in Egyptrdquo Annals of Epi-demiology vol 14 no 1 pp 44ndash48 2004
[26] M L Durand S B Calderwood D J Weber et al ldquoAcutebacterial meningitis in adults a review of 493 episodesrdquo TheNew England Journal of Medicine vol 328 no 1 pp 21ndash28 1993
[27] B Sigurdardottir O M Bjornsson K E Jonsdottir HErlendsdottir and S Gudmundsson ldquoAcute bacterial meningi-tis in adults a 20-year overviewrdquo Archives of Internal Medicinevol 157 no 4 pp 425ndash430 1997
[28] B Arda O R Sipahi S Atalay and S Ulusoy ldquoPooled analysisof 2408 cases of acute adult purulent meningitis from TurkeyrdquoMedical Principles and Practice vol 17 no 1 pp 76ndash79 2008
[29] M Scarborough S B Gordon C J MWhitty et al ldquoCorticos-teroids for bacterial meningitis in adults in sub-SaharanAfricardquoNewEngland Journal ofMedicine vol 357 no 24 pp 2441ndash24502007
[30] M Scarborough and G E Thwaites ldquoThe diagnosis andmanagement of acute bacterial meningitis in resource-poorsettingsrdquoThe Lancet Neurology vol 7 no 7 pp 637ndash648 2008
[31] G Campagne A Schuchat S Djibo AOusseini L Cisse and JP Chippaux ldquoEpidemiology of bacterial meningitis in NiameyNiger 1981ndash96rdquo Bulletin of the World Health Organization vol77 no 6 pp 499ndash508 1999
[32] S Afifi M O Wasfy M A Azab et al ldquoLaboratory-basedsurveillance of patients with bacterial meningitis in Egypt(1998ndash2004)rdquo European Journal of Clinical Microbiology andInfectious Diseases vol 26 no 5 pp 331ndash340 2007
[33] L Shaban andR Siam ldquoPrevalence and antimicrobial resistancepattern of bacterial meningitis in Egyptrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 8 article 26 2009
[34] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
PCR for detection of lower respiratory tract infection andmeningitis caused by Streptococcus pneumoniaeHaemophilusinfluenzae and Neisseria meningitidisrdquo BMC Microbiology vol10 article 310 2010
[7] Y Wang G Guo H Wang et al ldquoComparative study ofbacteriological culture and real-time fluorescence quantitativePCR (RT-PCR) and multiplex PCR-based reverse line blot(mPCRRLB) hybridization assay in the diagnosis of bacterialneonatal meningitisrdquo BMC Pediatrics vol 14 no 1 article 2242014
[8] M W Bijlsma M C Brouwer E S Kasanmoentalib et alldquoCommunity-acquired bacterial meningitis in adults in theNetherlands 2006ndash2014 a prospective cohort studyrdquoTheLancetInfectious Diseases vol 16 no 3 pp 339ndash447 2016
[9] H M Wu S M Cordeiro B H Harcourt et al ldquoAccuracyof real-time PCR Gram stain and culture for StreptococcuspneumoniaeNeisseriameningitidis andHaemophilus influenzaemeningitis diagnosisrdquo BMC Infectious Diseases vol 13 no 1article 26 2013
[10] L D Saravolatz O Manzor N VanderVelde J Pawlak andB Belian ldquoBroad-range bacterial polymerase chain reactionfor early detection of bacterial meningitisrdquo Clinical InfectiousDiseases vol 36 no 1 pp 40ndash45 2003
[11] K Ahmadi A Akya B Numanpour A Salimi and A Veisi-Raigani ldquoFrequency of Streptococcus pneumoniae infection inpatients with suspected meningitis in Imam Reza Hospital ofKermanshah in the west of Iranrdquo Iranian Journal of Microbiol-ogy vol 7 no 1 pp 12ndash17 2015
[12] L E Nigrovic RMalley C GMacias et al ldquoEffect of antibioticpretreatment on cerebrospinal fluid profiles of children withbacterial meningitisrdquo Pediatrics vol 122 no 4 pp 726ndash7302008
[13] J P Bryan H Rodriques de Silva A Tavares H Rocha andW M Scheld ldquoEtiology and mortality of bacterial meningitisin northeastern Brazilrdquo Clinical Infectious Diseases vol 12 no1 pp 128ndash135 1990
[14] A R Tunkel B J Hartman S L Kaplan et al ldquoPracticeguidelines for themanagement of bacterial meningitisrdquoClinicalInfectious Diseases vol 39 no 9 pp 1267ndash1284 2004
[15] L E Nigrovic N Kuppermann A J McAdam and R MalleyldquoCerebrospinal latex agglutination fails to contribute to themicrobiologic diagnosis of pretreated childrenwithmeningitisrdquoPediatric Infectious Disease Journal vol 23 no 8 pp 786ndash7882004
[16] K Tarafdar S Rao R A Recco and M M Zaman ldquoLackof sensitivity of the latex agglutination test to detect bacterialantigen in the cerebrospinal fluid of patients with culture-negative meningitisrdquo Clinical Infectious Diseases vol 33 no 3pp 406ndash408 2001
[17] E Van Gastel P Bruynseels W Verstrepen and A MertensldquoEvaluation of a real-time polymerase chain reaction assay forthe diagnosis of pneumococcal and meningococcal meningitisin a tertiary care hospitalrdquo European Journal of Clinical Micro-biology and Infectious Diseases vol 26 no 9 pp 651ndash653 2007
[18] M Tuyama R F Boente M C Rebelo R P Igreja and D EBarroso ldquoThe utility of the polymerase chain reaction assay foraetiologic definition of unspecified bacterial meningitis casesrdquoMemorias do Instituto Oswaldo Cruz vol 103 no 2 pp 138ndash1422008
[19] C T Sacchi L O Fukasawa M G Goncalves et al ldquoIncorpo-ration of real-time PCR into routine Public Health Surveillance
of Culture Negative Bacterial meningitis in Sao Paulo BrazilrdquoPLoS ONE vol 6 no 6 Article ID e20675 2011
[20] B Heydaria H Khalilib I Karimzadehc and H Emadi-Kochak ldquoClinical paraclinical and antimicrobial resistancefeatures of community-acquired acute bacterial meningitis at alarge infectious diseases ward in Tehran Iranrdquo Iranian Journalof Pharmaceutical Research vol 15 pp 347ndash354 2014
[21] M Cheesebrough ldquoBiochemical tests to identify bacteriardquo inDistrict Laboratory Practice in Tropical Countries M Chees-brough Ed pp 116ndash124 Cambridge University Press Cam-bridge UK 2nd edition 2007
[22] K Ohkusu K A Nash and C B Inderlied ldquoMolecular char-acterisation of Haemophilus influenzae type a and untypeablestrains isolated simultaneously from cerebrospinal fluid andblood novel use of quantitative real-time PCR based on the capcopy number to determine virulencerdquoClinicalMicrobiology andInfection vol 11 no 8 pp 637ndash643 2005
[23] L Ragunathan M Ramsay R Borrow M Guiver S Grayand E B Kaczmarski ldquoClinical features laboratory findingsand management of meningococcal meningitis in England andWales report of a 1997 surveyrdquo Journal of Infection vol 40 no1 pp 74ndash79 2000
[24] N Guirguis K Hafez M A El Kholy J B Robbins and EC Gotschlich ldquoBacterial meningitis in Egypt analysis of CSFisolates from hospital patients in Cairo 1977-1978rdquo Bulletin ofthe World Health Organization vol 61 no 3 pp 517ndash524 1983
[25] F G Youssef H El-Sakka A Azab et al ldquoEtiology antimi-crobial susceptibility profiles and mortality associated withbacterial meningitis among children in Egyptrdquo Annals of Epi-demiology vol 14 no 1 pp 44ndash48 2004
[26] M L Durand S B Calderwood D J Weber et al ldquoAcutebacterial meningitis in adults a review of 493 episodesrdquo TheNew England Journal of Medicine vol 328 no 1 pp 21ndash28 1993
[27] B Sigurdardottir O M Bjornsson K E Jonsdottir HErlendsdottir and S Gudmundsson ldquoAcute bacterial meningi-tis in adults a 20-year overviewrdquo Archives of Internal Medicinevol 157 no 4 pp 425ndash430 1997
[28] B Arda O R Sipahi S Atalay and S Ulusoy ldquoPooled analysisof 2408 cases of acute adult purulent meningitis from TurkeyrdquoMedical Principles and Practice vol 17 no 1 pp 76ndash79 2008
[29] M Scarborough S B Gordon C J MWhitty et al ldquoCorticos-teroids for bacterial meningitis in adults in sub-SaharanAfricardquoNewEngland Journal ofMedicine vol 357 no 24 pp 2441ndash24502007
[30] M Scarborough and G E Thwaites ldquoThe diagnosis andmanagement of acute bacterial meningitis in resource-poorsettingsrdquoThe Lancet Neurology vol 7 no 7 pp 637ndash648 2008
[31] G Campagne A Schuchat S Djibo AOusseini L Cisse and JP Chippaux ldquoEpidemiology of bacterial meningitis in NiameyNiger 1981ndash96rdquo Bulletin of the World Health Organization vol77 no 6 pp 499ndash508 1999
[32] S Afifi M O Wasfy M A Azab et al ldquoLaboratory-basedsurveillance of patients with bacterial meningitis in Egypt(1998ndash2004)rdquo European Journal of Clinical Microbiology andInfectious Diseases vol 26 no 5 pp 331ndash340 2007
[33] L Shaban andR Siam ldquoPrevalence and antimicrobial resistancepattern of bacterial meningitis in Egyptrdquo Annals of ClinicalMicrobiology and Antimicrobials vol 8 article 26 2009
[34] M C Brouwer A R Tunkel and D Van De Beek ldquoEpidemiol-ogy diagnosis and antimicrobial treatment of acute bacterialmeningitisrdquo Clinical Microbiology Reviews vol 23 no 3 pp467ndash492 2010
