queen elizabeth hospital in blantyre , malawi , is a teaching hospital offering free services to a population of approximately 1 million . from 2006 until 2009 , 3 prospective adult ( age 16 years ) meningitis studies were conducted at the hospital ; a randomized controlled trial of adjunctive oral glycerol in bacterial meningitis ( n = 265 )   and descriptive studies of suspected meningitis of any cause ( n = 573 )   and suspected bacterial meningitis ( n = 161 ) ( unpublished ) .
each study was approved by the university of malawi , college of medicine research ethics committee ( p.04/05/363 , p.05/06/471 , p.09/08/700 ) and complied with all institutional guidelines .
patients were included in this study if they had an available csf sample , documented hiv status , and bacterial meningitis defined as a csf white cell count 100 cells / mm with polymorphs > 50% , or a positive csf gram stain , bacterial culture , or streptococcus pneumoniae polymerase chain reaction ( pcr ) test .
defining bacterial meningitis using the above csf white cell criteria identifies patients with the same clinical characteristics and outcomes as patients with microbiologically proven bacterial meningitis in this setting .
controls were patients from one of the descriptive studies who did not have meningitis based on a csf white cell count < 5 cells / mm and negative csf gram stain , cryptococcal antigen test , bacterial culture , and mycobacterial culture .
samples were stored at 70c until dna extraction from 200 l using a dna mini kit ( qiagen ) .
five microliters of extract was used for all assays except the cmv assay , which is optimized for use with 10 l .
real - time pcr was performed with an abi7300 machine using taqman master mix ( applied biosystems ) .
primers and probes targeting conserved regions of the dna polymerase gene were used for hsv1&2 ( forward - gacagcgaattcgagatgctg , reverse - atgttgtacccggtcacgaact , hsv1 probe ; 5-fam - catgacccttgtgaaaca - mgb-3-bhq1 , hsv2 probe ; 5-vic - tgaccttcgtcaagcag - mgb-3-bhq1 ) and vzv ( forward - gcgcggtagtaacagagaatttc , reverse - acgtgcatggaccggttaat , probe ; 5-fam - accatgtcatcgtttcaa - mgb-3-bhq1 ) . for vzv a selection of samples
the ebv assay targeted the bnrf1 gene , and the cmv assay targeted the ul123/ul55 genes [ 10 , 11 ] .
pcr conditions were activation of ung 50c for 2 minutes , activation of taq 95c for 15 minutes , amplification for 45 cycles at 95c for 15 seconds , and 60c for 1 minute .
each assay had a lower limit of detection of 1000 copies / ml assessed as the level at which the pcr reaction always gave a positive result .
positive controls consisted of extracted whole virus suspension ( national institute of biological standards and controls , united kingdom ) .
a positive control for the virus being tested and a negative control were tested in each run .
if virus was detected in 1 of 3 wells , the test was repeated and considered positive if it again yielded 1 of 3 wells positive .
samples positive for ebv were analyzed by quantitative pcr to determine ebv load using a plasmid - derived standard .
the standard was quantified by ultraviolet spectrophotometry , serially diluted to 1010 copies / ml , and optimized to 92% efficiency .
statistical analysis was performed using stata software , version 10 , with a level of significance of p < .05 .
associations were examined using fisher exact test , stratified cochran - mantel - haenszel tests , and logistic regression .
csf samples from 188 patients were analyzed . in total , 149 ( of whom 115 were hiv positive ) had bacterial meningitis and 39 ( of whom 24 were hiv positive ) had no meningitis ( table 1 ) .
cd4 testing was not routinely available in malawi during the studies ; however , where documented , there was no difference between samples with bacterial meningitis ( n = 45 ) and those without meningitis ( n = 23 ) ( median 188 cells / mm vs 157 cells / mm , respectively ; p = .43 ) . for patients with bacterial meningitis
the csf white cell count was lower in hiv - positive patients than in hiv - negative patients ( median 365 cells / mm vs 960 cells / mm ; p = .009 ) .
patient characteristics and rates of detection of hsv 1&2 , vzv , ebv , and cmv values are median ( range ) unless otherwise stated .
abbreviations : csf , cerebrospinal fluid ; cmv , cytomegalovirus ; ebv , epstein - barr virus ; hiv , human immunodeficiency virus ; hsv 1 & 2 , herpes simplex virus types 1 and 2 ; na , not available ; pcr , polymerase chain reaction ; vzv , varicella zoster virus .
nine of the 90 positive pcr results were based on repeat testing following an initial result of 1 of 3 wells being positive ( 2 hsv1 , 1 cmv , 6 ebv ) .
hsv1 was detected in 2 of 39 patients without meningitis and 1 of 129 patients with bacterial meningitis ( table 1 ) .
cmv was detected in 11 of 115 ( 10% ) hiv - positive patients with bacterial meningitis ( median cd4 count , 121 cells / mm [ range , 1251 cells / mm ] ; n = 7 ) but was not detected in any hiv - negative patients with bacterial meningitis or 39 patients without meningitis .
ebv was found in the csf in 79 of 149 ( 53% ) patients with bacterial meningitis and was strongly associated with hiv seropositivity ( odds ratio 5.2 [ 95% confidence interval , 2.211.9 ] ; p < .001 ) .
in hiv - positive patients with bacterial meningitis , there was no correlation between csf ebv load and csf white cell count (  = 0.01 , p = .94 ) , csf lymphocyte count (  = 0.02 , p = .86 ) or csf red cell count (  = 0.01 , p = .98 ) . for those bacterial meningitis patients with cd4 count data available
there was no difference in the cd4 count between those with ( n = 33 ) and without ( n = 12 ) ebv in the csf ( median , 180 cells / mm vs 141 cells / mm , respectively ; p = .78 ) and csf ebv load did not correlate with the cd4 count (  = 0.01 , p = .97 ) . in hiv - positive patients without meningitis , ebv was present in 6 of 24 ( 25% ) patients , which was significantly less than in those with bacterial meningitis ( p = .002 ) .
ebv was not detected in any of the 15 hiv - negative patients without meningitis . in - hospital mortality from bacterial meningitis
was higher in hiv - positive ( 59% [ 64 of 108 ] ) than hiv - negative ( 35% [ 12 of 34 ] ) individuals ( p = .016 ) .
the presence of ebv in the csf of hiv - positive patients was not associated with outcome ( p = .32 ) ; however , when outcome was analyzed according to csf ebv load , adjusting for hiv status , there was a significant association with mortality ( p = .012 ) ( figure 1 ) . when this analysis was repeated excluding those who had no ebv in the csf ( n = 75 ) , the association remained ( p = .018 ) .
similarly , the association remained significant when other factors previously shown to be associated with poor outcome ( age 32 years , glasgow coma score < 12 , hemoglobin < 10 g / dl )   were included in the model ( p = .003 ) .
mortality from bacterial meningitis in adults infected with human immunodeficiency virus ( hiv ) according to epstein - barr virus ( ebv ) load in cerebrospinal fluid ( csf ) ( n = 108 ) and blood ( n = 83 ) . in total , 52 patients had no ebv in the csf , and 3 patients had no ebv in the blood .
csf ebv load quartile ranges are 5112186 copies / ml , 21876602 copies / ml , 709619  958 copies / ml , and 19  958223  323 copies / ml .
blood ebv load quartile ranges are 7519013 copies / ml , 964530  768 copies / ml , 31  251136  953 copies / ml , and 183  68135  658  200 copies / ml . ebv load in the blood was measured in 107 bacterial meningitis patients and detected in 80 of 83 ( 96% ) hiv - positive patients compared with 14 of 24 ( 58% ) hiv - negative patients ( p < .001 ) . unlike the csf , blood ebv load was not associated with outcome , adjusting for hiv status ( p = .37 ) ( figure 1 ) .
blood ebv load was higher than the corresponding csf load for all but 6 individuals , 3 of whom had no detectable ebv in the blood .
of these 6 patients csf white cell counts ranged from 120 to 5920 cells / mm , blood white cell counts ranged from 4.5 to 22.2 cells / l , and 3 patients died .
in total , 8 of 10 patients with cmv died ( outcome data were unavailable for 1 patient ) .
all 8 patients who died had dual infection with ebv , whereas the 2 survivors did not .
although the presence of cmv did not increase the risk of death ( p = .19 ) , ebv / cmv dual infection was associated with death ( p = .02 ) .
the cerebral injury that occurs in bacterial meningitis is largely due to a host - mediated inflammatory response .
our data suggest that this process may be amplified by ebv reactivation and possibly cmv either in the brain or periphery .
in contrast , we show that hsv1&2 and vzv are not commonly reactivated in adult bacterial meningitis in malawi .
we found a very high prevalence of ebv in the context of bacterial meningitis , particularly in hiv - positive patients .
ebv in the csf of hiv - positive patients is associated with cns lymphoma , although studies have detected ebv in other cns diseases [ 12 , 13 ] .
given that we found virus in patients with no white cells in the csf , and that replicative - cycle ebv messenger rna has been detected in csf from patients with other cns infections , it is likely that some ebv was replicating rather than latent .
alternatively , the inflammatory process may allow latently infected lymphocytes into the csf , leading to detection of latent virus .
immunosuppressed patients have increased levels of ebv in the blood due to a greater number of infected b lymphocytes .
if reactivation occurs in the circulation , then the disrupted blood - csf barrier in meningitis may allow free virus into the csf .
although most patients had higher ebv loads in the blood than in the csf , the finding of higher csf loads in some patients suggests that ebv can arise within the cns .
our data show that increasing csf ebv load is associated with an increased risk of death from bacterial meningitis .
ebv may simply be a marker of immune impairment , although this immune impairment appears to be poorly reflected in the cd4 count given the lack of correlation between cd4 count and ebv load .
alternatively , an immune threshold may exist at which ebv becomes detectable ; however , our data show is the ebv load that is associated with outcome rather than just the presence of virus .
ebv causes neurological damage by direct infection of brain cells or immune - mediated damage [ 14 , 15 ] .
ebv can also induce cytokines , which may allow further viral reactivation and impaired clearance of other infections .
the presence of ebv in 25% of hiv - positive patients with normal csf does show that ebv can occur in the csf without causing inflammation ; however , all of these patients had low ebv loads .
the absence of cmv in other patients indicates that detection is associated with inflammation in the csf in patients with underlying immunosuppression .
cmv neurological disease usually occurs at cd4 counts < 50 cells / mm ; however , in our study cmv - positive patients had a median cd4 count of 121 cells / mm .
a previous study showed that detection of cmv in the csf of hiv - positive individuals was almost always associated with autopsy findings of cmv neurological disease , even when other neurological diseases were likely to be the primary illness .
our study showed a significant association between dual ebv / cmv infection and mortality , suggesting that dual herpesvirus infections are associated with greater csf inflammation and worse outcome [ 5 , 13 ] .
our study supports the hypothesis that bacterial meningitis can trigger reactivation of herpesviruses , especially in hiv - positive patients .
this could be further supported by demonstrating that the virus is replicating and not latent .
we have shown that csf ebv load and dual ebv / cmv infection are associated with increased mortality from bacterial meningitis and propose that this reflects a causative role resulting from the effects of ebv and cmv on the inflammatory process responsible for poor outcome .
queen elizabeth hospital in blantyre , malawi , is a teaching hospital offering free services to a population of approximately 1 million . from 2006 until 2009 , 3 prospective adult ( age 16 years ) meningitis studies were conducted at the hospital ; a randomized controlled trial of adjunctive oral glycerol in bacterial meningitis ( n = 265 )   and descriptive studies of suspected meningitis of any cause ( n = 573 )   and suspected bacterial meningitis ( n = 161 ) ( unpublished ) .
each study was approved by the university of malawi , college of medicine research ethics committee ( p.04/05/363 , p.05/06/471 , p.09/08/700 ) and complied with all institutional guidelines .
patients were included in this study if they had an available csf sample , documented hiv status , and bacterial meningitis defined as a csf white cell count 100 cells / mm with polymorphs > 50% , or a positive csf gram stain , bacterial culture , or streptococcus pneumoniae polymerase chain reaction ( pcr ) test .
defining bacterial meningitis using the above csf white cell criteria identifies patients with the same clinical characteristics and outcomes as patients with microbiologically proven bacterial meningitis in this setting .
controls were patients from one of the descriptive studies who did not have meningitis based on a csf white cell count < 5 cells / mm and negative csf gram stain , cryptococcal antigen test , bacterial culture , and mycobacterial culture .
samples were stored at 70c until dna extraction from 200 l using a dna mini kit ( qiagen ) .
five microliters of extract was used for all assays except the cmv assay , which is optimized for use with 10 l .
real - time pcr was performed with an abi7300 machine using taqman master mix ( applied biosystems ) .
primers and probes targeting conserved regions of the dna polymerase gene were used for hsv1&2 ( forward - gacagcgaattcgagatgctg , reverse - atgttgtacccggtcacgaact , hsv1 probe ; 5-fam - catgacccttgtgaaaca - mgb-3-bhq1 , hsv2 probe ; 5-vic - tgaccttcgtcaagcag - mgb-3-bhq1 ) and vzv ( forward - gcgcggtagtaacagagaatttc , reverse - acgtgcatggaccggttaat , probe ; 5-fam - accatgtcatcgtttcaa - mgb-3-bhq1 ) . for vzv a selection of samples
the ebv assay targeted the bnrf1 gene , and the cmv assay targeted the ul123/ul55 genes [ 10 , 11 ] .
pcr conditions were activation of ung 50c for 2 minutes , activation of taq 95c for 15 minutes , amplification for 45 cycles at 95c for 15 seconds , and 60c for 1 minute .
each assay had a lower limit of detection of 1000 copies / ml assessed as the level at which the pcr reaction always gave a positive result .
positive controls consisted of extracted whole virus suspension ( national institute of biological standards and controls , united kingdom ) .
a positive control for the virus being tested and a negative control were tested in each run .
if virus was detected in 1 of 3 wells , the test was repeated and considered positive if it again yielded 1 of 3 wells positive .
samples positive for ebv were analyzed by quantitative pcr to determine ebv load using a plasmid - derived standard .
the standard was quantified by ultraviolet spectrophotometry , serially diluted to 1010 copies / ml , and optimized to 92% efficiency .
statistical analysis was performed using stata software , version 10 , with a level of significance of p < .05 .
associations were examined using fisher exact test , stratified cochran - mantel - haenszel tests , and logistic regression .
csf samples from 188 patients were analyzed . in total , 149 ( of whom 115 were hiv positive ) had bacterial meningitis and 39 ( of whom 24 were hiv positive ) had no meningitis ( table 1 ) .
cd4 testing was not routinely available in malawi during the studies ; however , where documented , there was no difference between samples with bacterial meningitis ( n = 45 ) and those without meningitis ( n = 23 ) ( median 188 cells / mm vs 157 cells / mm , respectively ; p = .43 ) . for patients with bacterial meningitis
the csf white cell count was lower in hiv - positive patients than in hiv - negative patients ( median 365 cells / mm vs 960 cells / mm ; p = .009 ) .
patient characteristics and rates of detection of hsv 1&2 , vzv , ebv , and cmv values are median ( range ) unless otherwise stated .
abbreviations : csf , cerebrospinal fluid ; cmv , cytomegalovirus ; ebv , epstein - barr virus ; hiv , human immunodeficiency virus ; hsv 1 & 2 , herpes simplex virus types 1 and 2 ; na , not available ; pcr , polymerase chain reaction ; vzv , varicella zoster virus .
nine of the 90 positive pcr results were based on repeat testing following an initial result of 1 of 3 wells being positive ( 2 hsv1 , 1 cmv , 6 ebv ) . reclassifying these results as negative did not change the results of the statistical analyses .
hsv1 was detected in 2 of 39 patients without meningitis and 1 of 129 patients with bacterial meningitis ( table 1 ) .
cmv was detected in 11 of 115 ( 10% ) hiv - positive patients with bacterial meningitis ( median cd4 count , 121 cells / mm [ range , 1251 cells / mm ] ; n = 7 ) but was not detected in any hiv - negative patients with bacterial meningitis or 39 patients without meningitis .
ebv was found in the csf in 79 of 149 ( 53% ) patients with bacterial meningitis and was strongly associated with hiv seropositivity ( odds ratio 5.2 [ 95% confidence interval , 2.211.9 ] ; p < .001 ) .
in hiv - positive patients with bacterial meningitis , there was no correlation between csf ebv load and csf white cell count (  = 0.01 , p = .94 ) , csf lymphocyte count (  = 0.02 , p = .86 ) or csf red cell count (  = 0.01 , p = .98 ) .
for those bacterial meningitis patients with cd4 count data available there was no difference in the cd4 count between those with ( n = 33 ) and without ( n = 12 ) ebv in the csf ( median , 180 cells / mm vs 141 cells / mm , respectively ; p = .78 ) and csf ebv load did not correlate with the cd4 count (  = 0.01 , p = .97 ) . in hiv - positive patients without meningitis , ebv was present in 6 of 24 ( 25% ) patients , which was significantly less than in those with bacterial meningitis ( p = .002 ) .
ebv was not detected in any of the 15 hiv - negative patients without meningitis . in - hospital
mortality from bacterial meningitis was higher in hiv - positive ( 59% [ 64 of 108 ] ) than hiv - negative ( 35% [ 12 of 34 ] ) individuals ( p = .016 ) .
the presence of ebv in the csf of hiv - positive patients was not associated with outcome ( p = .32 ) ; however , when outcome was analyzed according to csf ebv load , adjusting for hiv status , there was a significant association with mortality ( p = .012 ) ( figure 1 ) . when this analysis was repeated excluding those who had no ebv in the csf ( n = 75 ) , the association remained ( p = .018 ) .
similarly , the association remained significant when other factors previously shown to be associated with poor outcome ( age 32 years , glasgow coma score < 12 , hemoglobin < 10 g / dl )   were included in the model ( p = .003 ) .
mortality from bacterial meningitis in adults infected with human immunodeficiency virus ( hiv ) according to epstein - barr virus ( ebv ) load in cerebrospinal fluid ( csf ) ( n = 108 ) and blood ( n = 83 ) . in total , 52 patients had no ebv in the csf , and 3 patients had no ebv in the blood .
csf ebv load quartile ranges are 5112186 copies / ml , 21876602 copies / ml , 709619  958 copies / ml , and 19  958223  323 copies / ml .
blood ebv load quartile ranges are 7519013 copies / ml , 964530  768 copies / ml , 31  251136  953 copies / ml , and 183  68135  658  200 copies / ml . ebv load in the blood was measured in 107 bacterial meningitis patients and detected in 80 of 83 ( 96% ) hiv - positive patients compared with 14 of 24 ( 58% ) hiv - negative patients ( p < .001 ) . unlike the csf , blood ebv load was not associated with outcome , adjusting for hiv status ( p = .37 ) ( figure 1 ) .
blood ebv load was higher than the corresponding csf load for all but 6 individuals , 3 of whom had no detectable ebv in the blood .
of these 6 patients csf white cell counts ranged from 120 to 5920 cells / mm , blood white cell counts ranged from 4.5 to 22.2 cells / l , and 3 patients died .
in total , 8 of 10 patients with cmv died ( outcome data were unavailable for 1 patient ) .
all 8 patients who died had dual infection with ebv , whereas the 2 survivors did not .
although the presence of cmv did not increase the risk of death ( p = .19 ) , ebv / cmv dual infection was associated with death ( p = .02 ) .
the cerebral injury that occurs in bacterial meningitis is largely due to a host - mediated inflammatory response .
our data suggest that this process may be amplified by ebv reactivation and possibly cmv either in the brain or periphery .
in contrast , we show that hsv1&2 and vzv are not commonly reactivated in adult bacterial meningitis in malawi .
we found a very high prevalence of ebv in the context of bacterial meningitis , particularly in hiv - positive patients .
ebv in the csf of hiv - positive patients is associated with cns lymphoma , although studies have detected ebv in other cns diseases [ 12 , 13 ] .
given that we found virus in patients with no white cells in the csf , and that replicative - cycle ebv messenger rna has been detected in csf from patients with other cns infections , it is likely that some ebv was replicating rather than latent .
alternatively , the inflammatory process may allow latently infected lymphocytes into the csf , leading to detection of latent virus .
immunosuppressed patients have increased levels of ebv in the blood due to a greater number of infected b lymphocytes .
if reactivation occurs in the circulation , then the disrupted blood - csf barrier in meningitis may allow free virus into the csf .
although most patients had higher ebv loads in the blood than in the csf , the finding of higher csf loads in some patients suggests that ebv can arise within the cns .
our data show that increasing csf ebv load is associated with an increased risk of death from bacterial meningitis .
ebv may simply be a marker of immune impairment , although this immune impairment appears to be poorly reflected in the cd4 count given the lack of correlation between cd4 count and ebv load .
alternatively , an immune threshold may exist at which ebv becomes detectable ; however , our data show is the ebv load that is associated with outcome rather than just the presence of virus .
ebv causes neurological damage by direct infection of brain cells or immune - mediated damage [ 14 , 15 ] .
ebv can also induce cytokines , which may allow further viral reactivation and impaired clearance of other infections .
the presence of ebv in 25% of hiv - positive patients with normal csf does show that ebv can occur in the csf without causing inflammation ; however , all of these patients had low ebv loads .
the absence of cmv in other patients indicates that detection is associated with inflammation in the csf in patients with underlying immunosuppression .
cmv neurological disease usually occurs at cd4 counts < 50 cells / mm ; however , in our study cmv - positive patients had a median cd4 count of 121 cells / mm .
a previous study showed that detection of cmv in the csf of hiv - positive individuals was almost always associated with autopsy findings of cmv neurological disease , even when other neurological diseases were likely to be the primary illness .
our study showed a significant association between dual ebv / cmv infection and mortality , suggesting that dual herpesvirus infections are associated with greater csf inflammation and worse outcome [ 5 , 13 ] .
our study supports the hypothesis that bacterial meningitis can trigger reactivation of herpesviruses , especially in hiv - positive patients .
this could be further supported by demonstrating that the virus is replicating and not latent .
we have shown that csf ebv load and dual ebv / cmv infection are associated with increased mortality from bacterial meningitis and propose that this reflects a causative role resulting from the effects of ebv and cmv on the inflammatory process responsible for poor outcome .
this work was supported by grants from the meningitis research foundation ( 0905.0 ) and the wellcome trust ( 084679/z/08/z ) , united kingdom .
conflicts that the editors consider relevant to the content of the manuscript have been disclosed .
 mortality from adult bacterial meningitis exceeds 50% in sub - saharan africa . 
 we postulated that  particularly in individuals infected with human immunodeficiency virus ( hiv)herpes simplex virus , varicella zoster virus , epstein - barr virus ( ebv ) , and cytomegalovirus ( cmv ) in the cerebrospinal fluid ( csf ) contribute to poor outcome . 
 csf from 149 malawian adults with bacterial meningitis and 39 controls were analyzed using polymerase chain reaction . 
 ebv was detected in 79 of 149 bacterial meningitis patients . 
 mortality ( 54% ) was associated with higher csf ebv load when adjusted for hiv ( p = .01 ) . 
 cmv was detected in 11 of 115 hiv - infected patients , 8 of whom died . 
 the mechanisms by which ebv and cmv contribute to poor outcome require further investigation . 
None
Methods
Results
Discussion
Acknowledgments.
Financial support.
Potential conflicts of interest.