encephalitis is an inflammatory process of the brain parenchyma associated with clinical evidence of brain dysfunction due to infectious , immune - mediated or primary inflammatory causes . of all identifiable infectious agents , virus and bacteria account for 90% , with ameba , though rare , also being capable of causing infectious encephalitis . free - living amebas are widely distributed in nature , especially in fresh water and soil , but may occasionally invade and live as a parasite in the human tissue . several species of acanthamoeba , balamuthia mandrillaris , naegleria fowleri , and sappinia pedata are pathogenic and opportunistic free - living amebas known to cause diseases in humans . since the first case of amebic encephalitis was reported in 1965 , free - living amebas have increasingly been recognized as a pathogen causing central nervous system infections in humans . pathogenic free - living amebas cause 2 distinct clinical forms of encephalitis ; primary amebic meningoencephalitis ( pam ) and granulomatous amebic encephalitis ( gae ) . pam , caused by n. fowleri , is characterized by a fulminant course of disease with rapid progression to coma and death within a few days . on the other hand , gae , caused by acanthamoeba spp . or b. mandrillaris , usually runs a subacute to chronic course , and eventually progresses to death . due to its rarity , difficulty in diagnosis and lack of proven effective treatment , only 10 patients with balamuthia amebic encephalitis ( bae ) were reported to have survived thus far [ 6 - 15 ] . the purpose of this report was to demonstrate the clinical presentation , investigation , and treatment outcome of a child with bae , and to review the literature on cases with successful management . this report was informed and exempted by the institutional review board , faculty of medicine , chulalongkorn university , thailand . a previously healthy 4-year - old thai female patient initially presented with 1-month history of subacute intermittent bi - temporal headache with projectile vomiting , in the absence of fever or diarrhea . two weeks later , she developed unstable gait to the right side , and loss of distance approximation . there was no history of major illnesses , including sinusitis , pneumonia , chronic skin lesion , or any history of swimming in fresh water . she lived in buriram province , the northeastern part of thailand in rural settings , exposed to soil . abnormal neurological signs were head tilt to the right , jerky pursuit with nystagmus , hypometric succade , wide - based gait , and dysdiadochokinesia , which indicated right cerebellar hemispheric lesion . computer tomography ( ct ) scan of the brain at a local hospital showed an ill - defined 4.4 cm isodensity mass without calcification in the posterior fossa displaying heterogeneous enhancement on post - contrast study and obstructive hydrocephalus . magnetic resonance imaging ( mri ) of the brain at king chulalongkorn memorial hospital ( fig . 1 ) showed a 3.04.12.5 cm ill - defined infiltrating heterogeneously enhanced mass mainly occupying the right inferior cerebellar hemisphere with perilesional edema , causing right tonsillar herniation and mild obstructive hydrocephalus , highly suspicious for a malignant tumor such as medulloblastoma or high - grade glioma . total excision of the mass was performed on day 7 , and intraoperative finding revealed an infiltrative mass at the right cerebellum involving the hemisphere and tonsil with yellowish plaque and small cystic degeneration . the specimen was fixed in 10% neutral formalin , routinely processed , and embedded in paraffin wax . pathological examination demonstrated severely inflamed cerebellar tissue , with presence of scattered amebic trophozoites ( fig . dense lymphocyte infiltrates , aggregates of foamy histiocytes , few vague granulomata , and foci of tissue necrosis were found . the pathological diagnosis of amebic meningoencephalitis led to a prompt start of combined therapy of pentamidine ( 4 mg / kg / day ) , sulfasalazine ( 200 mg / kg / day ) , fluconazole ( 12 mg / kg / day ) , clarithromycin ( 14 mg / kg / day ) , and amphotericin b ( 1 mg / kg / day ) . on postoperative day 5 , cerebrospinal fluid ( csf ) obtained from lumbar puncture showed lymphocytic pleocytosis ( wbc 225 cell / mm , mononuclear cell 100% , rbc 157 cell / mm ) with increased protein of 148 mg / dl and decreased glucose levels of 27 mg / dl ( serum glucose level=126 mg / dl ) . no organisms were identified on gram and acid - fast bacillus stain and an indian ink preparation . these findings were consistent with amebic meningoencephalitis . in order to identify the specific causative agent , further molecular characterization was then performed , using the formalin - fixed brain tissue and fresh csf sample as detailed below . the dna was extracted from 1 mg of brain tissue samples and 100 l ( confirm needed ) csf . the standard organic method ( phenol - chloroform extraction and alcohol precipitation ) was applied for the dna extraction . the resulting pellet was resuspended in a final volume of 30 l of deionized water . the gene encoding 18s rrna was amplified by pcr using specific primer sets . the reaction mixture consisted of 10 l of 2.5x mastermix ( eppendorf , hamburg , germany ) , 2 l of template , 0.5 m of each forward and reverse primer , and nuclease - free water to a final volume of 25 l . the reactions comprised of initial denaturation at 94 for 3 min , followed by 40 cycles of denaturation at 94 for 30 sec , annealing at 55 for 45 sec and extension at 72 for 60 sec , and concluded by a final extension step at 72 for 7 min . the pcr products were subjected to 2% agarose gel electrophoresis ( fmc bioproducts rockland , maine , usa ) and upon staining with ethidium bromide , visualized on a uv transilluminator . the band of the expected size was excised from the gel and purified using the agarose gel extraction mini kit ( 5prime , hamburg , germany ) according to the manufacturer 's specifications . the purified dna was sequenced by first - base laboratories sdnbhd ( selangor darul ehsan , malaysia ) . the nucleotide sequences were analyzed by basic local alignment search tool ( blast ) ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . direct sequencing of dna extracted from both brain tissue and csf showed 18s rdna of b. mandrillaris after blast analysis . the lasergene 6 package ( dnastar , madison , wisconsin , usa ) was used to assemble the nucleotide sequences . the positive sequences were compared with the reference sequences ( af019071 ) available at genbank . alignment of the 18s ribosomal dna sequences was performed by using clustal w applying the bioedit program ( version 7.0.4.1 ) . phylogenetic trees were constructed for sequence analysis applying the neighbor - joining ( nj ) method of the mega 4.0 program   and support tree topologies by boot - strapping applied with 1,000 replicates . according to fig . 3 , the phylogenetic tree also showed that this sequence is grouped with b. mandrillaris . an initial clinical response was noted for several days following surgical treatment and combined medical treatment . a follow - up ct scan ( fig . however , the patient gradually deteriorated and eventually succumbed to multiple organ failure and brain death ( declared on postoperative day 37 ) , approximately 3 months after her first clinical symptoms . the dna was extracted from 1 mg of brain tissue samples and 100 l ( confirm needed ) csf . the standard organic method ( phenol - chloroform extraction and alcohol precipitation ) was applied for the dna extraction . the resulting pellet was resuspended in a final volume of 30 l of deionized water . the reaction mixture consisted of 10 l of 2.5x mastermix ( eppendorf , hamburg , germany ) , 2 l of template , 0.5 m of each forward and reverse primer , and nuclease - free water to a final volume of 25 l . the reactions comprised of initial denaturation at 94 for 3 min , followed by 40 cycles of denaturation at 94 for 30 sec , annealing at 55 for 45 sec and extension at 72 for 60 sec , and concluded by a final extension step at 72 for 7 min . the pcr products were subjected to 2% agarose gel electrophoresis ( fmc bioproducts rockland , maine , usa ) and upon staining with ethidium bromide , visualized on a uv transilluminator . the band of the expected size was excised from the gel and purified using the agarose gel extraction mini kit ( 5prime , hamburg , germany ) according to the manufacturer 's specifications . the purified dna was sequenced by first - base laboratories sdnbhd ( selangor darul ehsan , malaysia ) . the nucleotide sequences were analyzed by basic local alignment search tool ( blast ) ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . direct sequencing of dna extracted from both brain tissue and csf showed 18s rdna of b. mandrillaris after blast analysis . the lasergene 6 package ( dnastar , madison , wisconsin , usa ) was used to assemble the nucleotide sequences . the positive sequences were compared with the reference sequences ( af019071 ) available at genbank . alignment of the 18s ribosomal dna sequences was performed by using clustal w applying the bioedit program ( version 7.0.4.1 ) . phylogenetic trees were constructed for sequence analysis applying the neighbor - joining ( nj ) method of the mega 4.0 program   and support tree topologies by boot - strapping applied with 1,000 replicates . according to fig . 3 , the phylogenetic tree also showed that this sequence is grouped with b. mandrillaris . an initial clinical response was noted for several days following surgical treatment and combined medical treatment . a follow - up ct scan ( fig . however , the patient gradually deteriorated and eventually succumbed to multiple organ failure and brain death ( declared on postoperative day 37 ) , approximately 3 months after her first clinical symptoms . b. mandrillaris , formerly known as leptomyxoid ameba ( leptomyxidae ) , was first identified in the brain of a pregnant mandrill baboon having died of encephalitis in 1986 . in humans , it can cause diseases in both immunocompromised or immunocompetent individuals , especially young children and the elderly . recently , it has also been reported in solid organ transplant recipients . to date , approximately 150 cases of bae have been reported worldwide , mostly in the warm regions of the american continent , asia , australia , and europe . the first case of bae in thailand and southeast asia has been reported by intalapaporn et al . . the case involved a 23-year - old healthy man with prior history of motorcycle accident and upon falling into a swamp 6 months before , he developed a chronic nasal ulcer and had progressive central nervous system ( cns ) deterioration leading to death in spite of receiving full empirical treatment . at autopsy , his brain section was sent for fluorescent immunohistochemical analysis to confirm b. mandrillaris infection . however , our case was the first thai child with a premortal diagnosis of bae . like other amebas , b. mandrillaris can be isolated from soil samples and fresh water . b. mandrillaris can invade through skin wounds or be inhaled through nose or mouth , causing draining ulcers , chronic sinusitis or pneumonia before spreading hematogeneously to the cns . the skin lesion is typically a painless plaque or ulcer in the center of the face or extremity ( especially the knee ) . correct identification of the skin lesion is of great importance , allowing early diagnosis , weeks or months prior to cns manifestation . nevertheless , most patients , including the present case , presented with neurological involvement from the start , without skin manifestation . cns symptoms consist of fever , personality changes , headache , focal seizures , nonspecific cranial nerve dysfunction , or localized motor deficit . signs of increased intracranial pressure and alteration of consciousness may develop later as the disease progresses . due to the indolent clinical course , bae is difficult to diagnose and mostly recognized postmortem . csf usually reveals lymphocyte pleocytosis , with mild to severe elevation of protein levels ( > 1,000 mg / dl ) and normal to low glucose levels which are nonspecific . as b. mandrillaris trophozoites are rarely found in the csf , a large number of patients were initially misdiagnosed for other organisms . ct scan and mri typically show multifocally enhanced mass - like lesions with ring enhancement , edema , and hydrocephalus mimicking brain abscess , brain metastasis , or intracerebral hematoma . a small number of solitary mass lesion was also reported [ 8 - 10,25,26 ] which mimicked a brain tumor with no obvious sign of encephalitis , similar to our case . interestingly , a single mass in most cases situated in the anterior cranial fossa . to the best of our knowledge , this was the first case of bae manifesting with a solitary cerebellar mass lesion . diagnosis of amebic encephalitis can be confirmed by microscopic identification of characteristic amebic trophozoites in the skin or brain tissue or in csf . skin and brain pathology typically show granulomatous inflammation formed by aggregation of foamy macrophages , multinucleated giant cells , and lymphocytes . other findings may include presence of neutrophils , eosinophils , and small foci of vasculitis producing large areas of hemorrhage , infarction , and necrosis . 3 ) , it is not possible to morphologically distinguish between these 2 amebas . moreover , gae caused by acanthamoeba spp . or b. mandrillaris are similar in terms of clinical course , neuroimaging , and pathological findings . therefore , the diagnosis must be confirmed with indirect immunofluorescence ( iif ) techniques , immunohistochemistry ( ihc ) , or pcr . since iif and ihc techniques for these amebas are readily available in only few specialized centers such as the centers for disease control and prevention ( cdc ; atlanta , georgia , usa ) , pcr technique , as performed on our patient , may be a more easily accessible tool leading to an earlier diagnosis and treatment of bae before mortality . further to our own case , 10 patients who had survived bae were searched for under " balamuthia " in medline ( pubmed ) . the 10 cases were summarized ( table 1 ) and reviewed in order to demonstrate common factors or antimicrobial agents that may contribute to a better chance of survival . the 10 survivors were distributed equally into 3 age groups , 4 children ( < 12 years ) , 3 middle - aged patients ( 13 - 60 years ) , and 3 elderly patients ( > 60 years ) . according to the data collected from all bae cases , the proportion of elderly patients who survived appeared to be the highest , approximately 25% survival compared to less than 10% survival in the other groups . concerning gender , male individuals were more susceptible to bae , with male : female ratio of 2.5:1 , since they tend to be exposed to outdoor activities more than in females . thus , the proportion of female survivors outnumbered that of males , with approximately 15% survival in females and 5% survival in males . hence , female gender may play some role in increasing the chances of survival of these patients . skin lesions are the first clinical manifestation preceding neurological symptoms in approximately 1/4 of all patients with bae . therefore , skin manifestation may allow for early recognition and diagnosis of bae leading to prompt treatment which potentially increase the patient 's chances of survival . in terms of treatment , apart from surgical approach , multi - drug regimens based on in vitro data and the clinical experience in successfully treated cases were also administered . the novel antimicrobial drug needs to have amebicidal activity , good penetration through the blood - brain barrier with minimal toxicity . pentamidine , propamidine isethionates , polymyxin b , gramicidin s , and miltefosine were found to have strong inhibitory effects in vitro against b. mandrillaris . unfortunately , some of these amebicidal agents are of limited use because they are not suitable for parenteral use , except for miltefosine in 1 survivor and pentamidine in 4 survivors , including our patient . other antimicrobial agents with little or no in vitro effects included azithromycin , clarithromycin , the azole compounds , trimethoprim , sulfamethoxazole , and amphotericin b . of the 9 survivors with documented specific antimicrobial treatment regimens , almost all have shown similar treatment regimens consisting of azoles ( fluconazole , itraconazole ) in 8 of 9 survivors , macrolides ( clarithromycin , azithromycin ) in 7 of 9 survivors . in contrast with in vitro study results , azoles and macrolides may represent good choices as potential antimicrobial agents for treating bae . the optimal duration of drug therapy for bae has not yet been ascertained due to the complete absence of any validated biochemical markers of response to treatment , but most survivors had been treated for a few months up to more than 5 years . however , long - term use of multiple drugs might be limited by some undesirable side effects , such as hyperglycemia due to pentamidine or hyponatremia due to thioridazine , resulting in discontinuation of medications . currently , patients recently diagnosed with this disease are being treated according to the reported successful regimens ; however , case fatality of this disease is still high . due to the limited number of survivors reported , the prognostic factors and effective treatment regimens have so far remained elusive and require more detailed further research . although it has become more recognizable among physicians , more case reports and additional research will still be required for more information and a better insight into this disease . a high index of suspicion of clinicians , immunostaining or molecular techniques to promote early diagnosis , multidisciplinary team approach , and prompt initiation of antimicrobial therapy are key factors which might improve survival of the patients .
balamuthia mandrillaris is one of the 4 amebas in fresh water and soil that cause diseases in humans . 
 granulomatous amebic encephalitis ( gae ) , caused by b. mandrillaris , is a rare but life - threatening condition . a 4-year - old , previously healthy , thai girl presented with progressive headache and ataxia for over a month . 
 neuroimaging studies showed an infiltrative mass at the right cerebellar hemisphere mimicking a malignant cerebellar tumor . 
 the pathological finding after total mass removal revealed severe necrotizing inflammation , with presence of scattered amebic trophozoites . 
 cerebrospinal fluid ( csf ) obtained from lumbar puncture showed evidence of non - specific inflammation without identifiable organisms . 
 a combination of pentamidine , sulfasalazine , fluconazole , and clarithromycin had been initiated promptly before pcr confirmed the diagnosis of balamuthia amebic encephalitis ( bae ) . 
 the patient showed initial improvement after the surgery and combined medical treatment , but gradually deteriorated and died of multiple organ failure within 46 days upon admission despite early diagnosis and treatment . 
 in addition to the case , 10 survivors of bae reported in the pubmed database were briefly reviewed in an attempt to identify the possible factors leading to survival of the patients diagnosed with this rare disease .