hepatitis a virus ( hav ) remains an important causative agent of acute viral hepatitis , causing large outbreak or severe cases of fulminant hepatitis with fatal outcomes .
acute hepatitis a can be complicated by extrahepatic manifestations , including hemolytic anemia , aplastic anemia , acute renal failure , and acute reactive arthritis.1,2 although hav displays only a single serotype , seven hav genotypes have been identified : four genotypes ( i , ii , iii and vii ) are of human origin , and other three ( iv , v , vi ) are of simian origin .
hav genotypes have unique geographic distributions ; however , co - circulation of multiple genotypes or subgenotypes has been reported in some regions of world.3 this report presents a peculiar coinfection hepatitis with the hav genotype ia and iiia , complicated by severe autoimmune hemolytic anemia and prolonged cholestasis , along with coexistence of igm anti - hepatitis e virus ( hev ) .
we performed immunohistochemical staining of hav antigen and hev antigen in the liver biopsy specimen , and molecular detection of hav rna and hev rna in the serial serum samples obtained from the patient .
a 37-year - old male visited the emergency room on december 28 , 2008 for treatment of fever ( 40 ) , chills , and jaundice lasting for 2 days .
he was a nonsmoking social drinker , and denied recent overseas travel , contact with hepatitis cases , or taking any medications .
his past medical history revealed an episode of pneumonia and bronchiectasis that occurred 20 years earlier .
his initial level of aspartate transaminase ( ast ) was 12,000 iu / l , alanine aminotransferase ( alt ) 8,129
iu / l , total bilirubin 4.5 mg / dl , prothrombin time 23.6 second ( international normalized ratio 2.06 ) and hemoglobin ( hb ) 14.3 g / dl .
igm anti - hav ( microparticle enzyme immunoassay , abbott , wiesbaden , germany ) and igg anti - hav ( radio - immunoassay kit , general biologicals corp . ,
hepatitis b virus surface antigen , igm anti - hbc , hepatitis c virus ( hcv ) rna , and anti - hcv were all negative .
igm anti - hev ( genelabs diganostics , singapore ) was negative . chest x ray and ct scan of the abdomen and chest showed no remarkable findings . under diagnosis of acute hepatitis a
, the patient received supportive care showing gradual recovery , and ast / alt levels decreased to 285/919 iu / l , with a stable hemoglobin level of 14.4 g / dl , while his total bilirubin level progressively increased up to 21.8 mg / dl at hospital discharge ( january 6 , 2009 ) suggesting a prolonged cholestatic feature . on january 12 , 2009 , he was readmitted for recurrent fever , general weakness , and aggravated jaundice . at this time , igm anti - hev was positive with persistently positive igm anti - hav .
total and direct bilirubin levels were 39 mg / dl and 29 mg / dl , respectively , with an alkaline phosphatase level of 374 iu / l , and hb level was 13.4 g / dl . until january 24
, total bilirubin level increased rapidly , up to 64.1 mg / dl ( direct bilirubin level of 41 mg / dl ) , and hb level fell rapidly to 5.5 g / dl , despite decreasing levels of transaminase .
haptoglobin level was 27.3 mg / dl and direct coombs ' test was positive ( anti - igg type ) with polychromasia and anisocytosis on a blood film ( fig .
oral prednisolone therapy ( 1 mg / kg ) was started on january 22 , however , his reticulocyte count remained low ( 0.13% ) , and he received 17 units of red blood cell transfusion for 11 days . to exclude the possibility of pure red cell aplasia
, bone marrow aspiration and biopsy were performed , and it showed increased erythropoiesis compatible with aiha without evidence of pure red cell aplasia ( fig .
the dosage of oral prednisolone therapy was incremented to 1.5 mg / kg / day , and after 8 days of the incremental dose therapy , the patient became gradually transfusion - independent with increase of hb level to 12.1 g / dl .
liver function tests showed ast of 22 iu / l , alt 136 iu / l , alkaline phosphatase 285 iu / l , gamma - glutamyl transferase ( ggt ) 897
iu / l , and total bilirubin of 5.4 mg / dl . to exclude the possibility of fibrosing cholestatic hepatitis associated with immunosuppressive therapy ,
percutaneous liver biopsy was undertaken on february 12 , which revealed hepatocellular and canalicular cholestasis , hepatocyte ballooning , and lobular spotty necrosis compatible with cholestatic hepatitis and extramedullary hematopoiesis ( fig .
2 ) . immunohistochemical staining was performed for detection of hav antigens and hev antigens using hav antibody ( purified hav , abcam , cambridge , uk ; mouse monoclonal , 1:50 ) and hev antibody ( abbiotec , ca , usa ; 1:200 ) , respectively .
strong cytoplasmic staining for hav was noted , whereas staining was absent in specimens used for negative control ( fig .
3 ) . in this case , there was no staining for hev antigen ( data not shown ) .
after a 2 month - period of gradual tapering of prednisolone , complete recovery of liver function was achieved at 5 months after the patient 's initial admission . however , his igm anti - hav was positive until may 29 , while both igm anti - hev and igg anti - hev ( genelabs diganostics , singapore ) were negative . during the course of his illness ,
serial serum samples were available for detection of hav rna and hev rna , according to the method described in previous studies.4 all hav rna - positive samples were sequenced using the bigdye terminator ( abi prism 377 automatic sequencer , toroed , norway ) .
interestingly , initial hav detected on january 14 , 2009 was genotype ia , which was subsequently replaced by hav genotype iiia during the later phase of the illness ( april 22 , 2009 ) , while hev rna was not detected by okamoto 's method , as previously reported.5,6
this report described a case of acute viral hepatitis coinfected by hav genotype ia and genotype iiia complicated with severe autoimmune hemolytic anemia , prolonged cholestasis , and coexistence of igm anti - hev , which was successfully treated by high dose ( 1.5 mg / kg / day ) prednisolone therapy .
there have been a few reports of severe coombs - positive aiha with or without pure red cell aplasia , complicating acute viral hepatitis.7 however , coinfection of hav genotype ia and iiia complicated with severe aiha has not been reported .
genotype 1a hav was a major genotype in korea , however , during recent nationwide outbreak of hepatitis a , a rapid genotypic shift from genotype ia to iiia has occurred.4,8 therefore , at least two genotypes of hav co - circulate in korea .
coinfection of the hav genotype ia and iiia was a peculiar finding in this case . from onset of symptoms to development of aiha ,
subsequently , during prolonged follow - up , his hav genotype was replaced from genotype ia to iiia .
this replacement can be explained by initially mixed infection of hav genotype ia and iiia with a dominant population of hav genotype ia and reciprocal inhibition of genotype iiia . during his initial hepatitis course , vigorous immune responses
may have controlled hav genotype ia up to an undetectable level , and subsequent strong immunosuppression for the treatment of hemolytic anemia may support replication of hav genotype iiia , a minor population . to our knowledge , this is the first case of coinfection of hepatitis a genotype ia and iiia documented .
coppola et al9 reported a case of acute hepatitis a complicated with prolonged cholestasis and coinfection of genotype ia and ib , and suggested that coinfection of two hav subgenotypes may be a reason for prolonged cholestasis and viremia due to impairment of antibody production or neutralizing activity on the basis of the simultaneous antigenic stimulatory effect of the two hav isolates .
however , hav has only one common serotype , and this finding was not supported by experimental data .
the interesting finding of this case was that igm anti - hev at the first manifestation was negative , which turned out to be positive at the initiation of hemolytic anemia .
afterwards , the transiently positive result for igm anti - hev turned out to be negative , along with a negative result for igg anti - hev during the follow - up period .
both hepatitis a virus ( hav ) and hepatitis e virus ( hev ) infection can be transmitted by the fecal - oral route with similar incubation periods and clinical presentations . among patients with sporadic acute viral hepatitis ,
cases showing simultaneously positive results for immunoglobulin m ( igm ) anti - hav and igm anti - hev have been reported , and were regarded as coinfection of hav and hev.10,11 however , hev rna was not detected in serum and stool samples , while hav rna was detected and genotyped . in immunohistochemical staining of liver biopsy specimens ,
hav antigens showed strongly positive staining , in contrast to no staining for hev antigens , although we were not able to obtain adequate positive control specimens .
all of these results suggested the false positive reaction of initially positive igm anti - hev , rather than a true coinfection of hev along with hav.12 severe cell necrosis associated with hav infection may induce autoimmune reaction such as hemolytic anemia or autoimmune hepatitis.13,14 nonspecific induction of igm anti - hev during massive hepatic necrosis death in the hepatitis phase of this case may be a possibility . in conclusion
, severe autoimmune hemolytic anemia and prolonged cholestasis can be complicated with acute hepatitis co - infected by hav genotype ia and iiia , which was successfully treated with high - dose prednisolone therapy .
the coexistence of igm anti - hev in the setting of hepatitis a seems to be a false positive reaction rather than a true coinfection .
 a 37-year - old male presented with fever and jaundice was diagnosed as hepatitis a complicated with progressive cholestasis and severe autoimmune hemolytic anemia . 
 he was treated with high - dose prednisolone ( 1.5 mg / kg ) , and eventually recovered . 
 his initial serum contained genotype ia hepatitis a virus ( hav ) , which was subsequently replaced by genotype iiia hav . 
 moreover , at the time of development of hemolytic anemia , he became positive for immunoglobulin m ( igm ) anti - hepatitis e virus ( hev ) . 
 we detected hav antigens in the liver biopsy specimen , while we detected neither hev antigen in the liver nor hev rna in his serum . 
 this is the first report of hepatitis a coinfected with two different genotypes manifesting with autoimmune hemolytic anemia , prolonged cholestasis , and false - positive igm anti - hev . 
INTRODUCTION
CASE REPORT
DISCUSSION