encephalitis is one cause of epilepsy in the united states , as the central nervous system ( cns ) trauma ; however , there are 1.4 million cases of head injury in comparison with less than 50000 cases of cns infection per year .
the exact etiology of this disease is unknown and might be due to infective or noninfective causes , but viruses have been mostly implicated .
the most common viruses involved are human herpesviruses , flaviviruses , enteroviruses , and alphaviruses .
less common viral agents include influenza viruses , adenoviruses , rabies virus , lymphocytic choriomeningitis virus , measles , rubella and mumps .
vaccinations have dramatically decreased encephalitis because of childhood diseases such as mumps , measles and rubella . between herpesviruses , human herpesvirus-6 ( hhv-6 )
is found in cerebrospinal fluid ( csf ) of 10% of patients with suspected viral cns diseases .
viral encephalitis symptoms could be fever , headache , altered level of consciousness , and seizures ( 2 ) .
with production of new antiviral drugs , correct and timely diagnosis of the disease would be important for patients management ( 2 ) .  
it has been linked to different cns diseases such as meningitis , encephalitis , and multiple sclerosis , as well as psychological disorders .
hhv-6 primary infection manifests itself as roseola infantum with common symptoms of fever and rash in children between six months and two years old .
this disease itself is usually benign , but some major complications such as seizures , bulging of the anterior fontanel , and encephalitis have been reported after this disease ( 3 ) .
the question is that whether hhv-6 could cause encephalitis in the absence of roseola infantum .  
the diagnosis could be obtained by detection of viral nucleic acid as well as antibody or virus isolation from csf or brain tissue .
because of limitations in virus isolation and serological tests , polymerase chain reaction ( pcr ) has been widely used in clinical diagnosis of viruses .
even with this sensitive and specific technique , 30 - 60% of patients with encephalitis remain undiagnosed ( 4 ) .
the aim of this study was detection of hhv-6 dna in csf of patients with symptoms of possible acute encephalitis , using real - time pcr .
in this cross - sectional study , all children aged  2 years with encephalitis - like symptoms ( temperature > 38c , convulsion , and some with altered levels of consciousness ) , admitted to the emergency ward of children medical center of tehran university of medical sciences between march 2010 and march 2011 were enrolled .
special forms were filled to record the history and demographic information ( age and sex ) as well as clinical and laboratory findings ( symptoms , csf cell counts , and glucose and protein levels ) .
lumbar puncture was performed on the first day of admission and csf specimens were collected from all patients ; then , they were sent to the virology department , school of public health , tehran university of medical sciences , and stored at -70c .
dna was extracted from 140 l of each sample with high pure viral nucleic acid kit containing proteinase k ( roche , germany ) , according to the manufacturer s instruction .
when extracting the viral dna , proteinase k was added to the sample before adding to the column .
the csf specimens were tested for hhv-6 dna , using taq man real - time pcr .
the primers and the probe were directed against the u22 open reading frame of both hhv-6a and b ; forward primer : 5- tcg aaa taa gca tta ata ggc aca ct-3  , reverse primer : 5-cgg agt taa ggc att ggt tga -3  , and taq man probe : fam - cca agc agt tcc gtt tct ctg agc ca - tamra , which amplified a 99-bp fragment .
the probe was labeled at the 5 end with 6-carboxyfluorescein as the reporter dye and at the 3 end with 6-carboxytetramethylrhodamine as the quencher .
an adenine - to - guanine substitution at position 11 of the hhv6b sequence in the reverse primer and in the probe , as well as an ac - to - gt substitution at positions 13 and 14 , were accepted for designing the primer - probe ( 5 ) .   the dna extract ( 7 l )
was amplified by real - time pcr in a 25-l reaction mixture , containing 12.5 l of quanti fast probe pcr master mix ( qiagen , germany ) , 4 l of forward and reverse primers ( 10 pmol ) , 1 l of probe ( 10 pmol ) , and 0.5 l ddh2o in abi step one plus ( applied biosystems , u.s.a ) machine . the thermocycling process consisted of pcr initial heat activation for three minutes at 95c , followed by 40 cycles of denaturation at 95c for 15seconds , annealing at 56c for 30seconds , and extension at 60c for 30 seconds .
the concentration of hhv-6 control dna was 104 copies/l ; after dilution , we had 10 copies/l , which was positive in cycle threshold ( ct ) : 20 .
in this cross - sectional study , all children aged  2 years with encephalitis - like symptoms ( temperature > 38c , convulsion , and some with altered levels of consciousness ) , admitted to the emergency ward of children medical center of tehran university of medical sciences between march 2010 and march 2011 were enrolled .
special forms were filled to record the history and demographic information ( age and sex ) as well as clinical and laboratory findings ( symptoms , csf cell counts , and glucose and protein levels ) .
lumbar puncture was performed on the first day of admission and csf specimens were collected from all patients ; then , they were sent to the virology department , school of public health , tehran university of medical sciences , and stored at -70c .
dna was extracted from 140 l of each sample with high pure viral nucleic acid kit containing proteinase k ( roche , germany ) , according to the manufacturer s instruction . when extracting the viral dna , proteinase k was added to the sample before adding to the column .
the csf specimens were tested for hhv-6 dna , using taq man real - time pcr .
the primers and the probe were directed against the u22 open reading frame of both hhv-6a and b ; forward primer : 5- tcg aaa taa gca tta ata ggc aca ct-3  , reverse primer : 5-cgg agt taa ggc att ggt tga -3  , and taq man probe : fam - cca agc agt tcc gtt tct ctg agc ca - tamra , which amplified a 99-bp fragment .
the probe was labeled at the 5 end with 6-carboxyfluorescein as the reporter dye and at the 3 end with 6-carboxytetramethylrhodamine as the quencher .
an adenine - to - guanine substitution at position 11 of the hhv6b sequence in the reverse primer and in the probe , as well as an ac - to - gt substitution at positions 13 and 14 , were accepted for designing the primer - probe ( 5 ) .   the dna extract ( 7 l )
was amplified by real - time pcr in a 25-l reaction mixture , containing 12.5 l of quanti fast probe pcr master mix ( qiagen , germany ) , 4 l of forward and reverse primers ( 10 pmol ) , 1 l of probe ( 10 pmol ) , and 0.5 l ddh2o in abi step one plus ( applied biosystems , u.s.a ) machine . the thermocycling process
consisted of pcr initial heat activation for three minutes at 95c , followed by 40 cycles of denaturation at 95c for 15seconds , annealing at 56c for 30seconds , and extension at 60c for 30 seconds .
the concentration of hhv-6 control dna was 104 copies/l ; after dilution , we had 10 copies/l , which was positive in cycle threshold ( ct ) : 20 .
one hundred and fourteen children , two years old or younger , with symptoms of possible encephalitis , were enrolled in this study .
of the total patients , 68 were male and 46 were female , of which , 39 were older and 75 younger than 12 months old .
eleven patients had history of vaccination in the past two days , six of which were mmr , four were dpt , and one was influenza vaccine .
the disease incidence was 42.1% in autumn , 35.08% in winter , 17.54% in spring , and 5.26% in summer
.   in three of the children rash was identified , which was not specific for roseola infantum . in 10 ( 8.8% )
these children were younger than 13 months old ( the youngest one was two and the oldest 13 months old ) and 90% of them were boys .
their average temperature ( 38.9c ) at the time of admission was not significantly different from the temperature in the children with negative hhv-6 .
in hhv-6-positive patients , the incidence was 70% in winter , 20% in autumn , and 10% in summer .
two patients had fever two days before the convulsion and eight had fever at the day of convulsion .
of the 114 children , febrile convulsion ( fc ) was the first episode in 95 ( 83.33% ) and the second in 19 ( 16.66% ) .
dna was found in eight of 95 patients with first fc episode and two of 19 with second episode .
cell / mm and for the hhv-6-positive children it was 11986  8842 cell / mm .
all the 114 patients had fc , 10 had loss of consciousness , 16 coryza , 20 cough , 15 diarrhea , and eight vomiting . among hhv-6-positive patients ,
one had loss of consciousness , two coryza , two cough , and one diarrhea .
abbreviations : csf : cerebrospinal fluid ; esr : erythrocyte sedimentation rate ; hhv-6 : human herpesvirus 6 ; pmn : polymorphonuclear leukocytes ; rbc : red blood cell ; wbc : white blood cell .  
encephalitis is an acute inflammation of brain ; it is a crucial cause of acute symptomatic seizures and the subsequent epilepsy .
annually , 7.3 per 100000 people are hospitalized in the united states because of encephalitis ( 2 ) .
clinical syndromes of acute childhood encephalitis may range from mild illness including fever , convulsion and varying degree of consciousness , to a devastating disease resulting in death ( 4 ) .
having information about the disease etiology may be helpful in determining the therapeutic regimens as well as preventive advises .
certain virus families , especially herpes viruses such as hhv-6 , can cause encephalitis ( 5 ) .
after exclusion of noninfectious , bacterial , and fungal causes of encephalitis , viral diagnosis could be commonly achieved by pcr of csf samples .  
seroepidemiological surveys have shown that hhv-6 can infect almost all children up to two years of age .
this virus causes roseola infantum as the primary infection , but it can cause some neurological disorders too ( 6 ) . seizures , meningoencephalitis , bulging of the anterior fontanel , and encephalopathy are the most common manifestations . in one third of children younger than two years old with fcs , hhv-6 was detected .
the mechanism through which this virus causes cns complications is unclear , but it seems to be the result of direct invasion of cns .
its multiple manifestations as well as lack of routine diagnostic tests resulted in costly and lengthy evaluations and hospitalizations ; thus , understanding of the frequency and consequences of hhv-6 infection could be helpful in diagnosis and management of patients ( 7 ) .  
molecular methods of detection , especially pcr , have been established for diagnosis of cns infections .
even with the best and most specific methods , 30 - 60% of patients with suspected viral encephalitis will be undiagnosed ( 4 ) . in this research ,
hhv-6 was identified by real - time pcr in 10 of 114 csf specimens from children who showed symptoms of possible encephalitis ( incidence : 8.8%).encephalitis due to hhv-6 infection has been reported by several studies . in some of them , patients completely recovered , and in some others , neurologic sequela and death were reported .
the hhv-6 dna was identified in csf of several patients with pcr , revealing the direct viral invasion to cns ( 8 , 9 ) . in a study by yoshikawa et al . in 2009
( 10 ) , 86 cases of exanthema subitum with encephalopathy were evaluated , among which , the hhv-6 dna was detected in 21 patients with two deaths . in our research ,
all the patients survived and there was no confirmed encephalitis and death . in another study by suga
( 11 ) on 21 patients with exanthema subitum and cns complications , nine cases were hhv-6-positive for csf test , four had encephalitis , 17 had complete recovery , one case with encephalitis developed epilepsy , and one case died .
( 12 ) on csf samples of 150 children with meningoencephalitis in tehran showed that 9 ( 6% ) patients were positive for the hhv-6 dna .
encephalitis is important , both in the acute phase and the long term neurologic sequels .  
this is obviously an area of high clinical importance , requiring further investigations for diagnosis and treatment.hhv-6 is sensitive to ganciclovir and foscarnet and insensitive to acyclovir , same as cytomegalovirus ( 13 ) .
yoshida et al . ( 14 ) showed that hhv-6 was highly sensitive to cidofovir .
this lack of information might be due to the fact that studies on hhv-6 have been retrospective and there is not a routine laboratory test for identifying the virus in suspected patients .
this was a cross - sectional descriptive study on immunocompetent children , revealing the prevalence of hhv-6 in children less than two years old with possible encephalitis .
no significant differences could be found in clinical presentations and laboratory findings between the ten patients with and the 104 patients without hhv-6 infection .
all the patients had complete recovery without neurological deficit or death ; thus , we can conclude that all the patients were fc cases , some of which had post - ictal drowsiness .
further prospective studies on immunocompromised children are recommended for comparing the results between immunocompetent and immunocompromised children .
another issue is recognizing the common viruses such as herpes simplex virus , enteroviruses , etc . in follow up studies , to find the coinfection and other possible factors involved in this disease .
according to this study , evaluation of csf ( detecting the hhv-6 dna by pcr ) is recommended in children younger than 13 months old with possible encephalitis , for initiating the antiviral treatment .
we should follow up for identifying the type of viruses ( hhv-6 a or b ) as well as the prevalence of hhv-6 dna integration into the germline , which can permanently turn the individual into hhv-6-positive .
 background : encephalitis is swelling and inflammation of brain , usually due to viral infection . 
 viral encephalitis symptoms could be fever , headache , altered level of consciousness , and seizures.objectives:the aim of this study was detection of human herpesvirus-6 ( hhv-6 ) dna in cerebrospinal fluid ( csf ) of patients with symptoms of possible acute encephalitis and without typical signs or symptoms of roseola infantum , using real - time polymerase chain reaction ( pcr).patients and methods : we studied children two years old or younger , admitted to the pediatric emergency ward with encephalitis - like symptoms . 
 our evaluation included detection of hhv-6 in csf of these patients . 
 after dna extraction , real - time pcr was performed with primers and a probe specific for the u22 open reading frame of both hhv-6a and b.results:from a total of 114 patients , hhv-6 was detected in 10 ( 8.8% ) , 90% of which were boys with mean age 7.7 months and median of 7.5 months . 
 no significant differences were found in clinical presentations and laboratory findings between the patients positive and negative for hhv-6 . 
 all the children had complete recovery without neurological deficit or death.conclusions:according to this research and prevalence of hhv-6 in children , evaluation of csf ( detecting the hhv-6 dna by pcr ) is recommended in patients younger than 13 months with possible encephalitis . 