Clinical course and duration of viremia in vertically transmitted hepatitis E virus infection in babies born to HEV-infected mothers

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1 Journal of Viral Hepatitis, 00 doi:./j x 0 Clinical course and duration of viremia in vertically transmitted hepatitis E virus infection in babies bn to HEV-infected mothers M. S. Khuroo, S. Kamili and M. S. Khuroo Digestive Diseases Centre, Dr KhurooÕs Medical Clinic, Srinagar, Kashmir, J&K, India; Experimental Pathology Section, Hepatitis Branch, Division of Viral and Rickettsial Diseases, US Centre f Disease Control and Prevention, Atlanta, GA, USA; and Department of Pathology, Sher-i-Kashmir Institute of Medical Sciences, Soura Srinagar, Kashmir, J&K, India Received October 00; accepted f publication November 00 SUMMARY. Infection with the hepatitis E virus (HEV) causes a self-limiting acute hepatitis. However, prolonged viremia and chronic hepatitis has been repted in gan transplant recipients. Vertically transmitted HEV infection is known to cause acute hepatitis in newbn babies. The clinical course and duration of viremia in vertically transmitted HEV infection in neonates in not known. We studied babies bn to HEV infected mothers. Babies were studied at birth and on a monthly basis to evaluate clinical profile, pattern of antibody response and duration of viremia in those infected with HEV. Fifteen (.%) babies had evidence of vertically transmitted HEV infection at birth (IgM anti-hev positive in and HEV RNA reactive in ) and three had sht-lasting IgG anti-hev positivity because of trans-placental antibody transmission. Seven HEV-infected babies had icteric hepatitis, five had anicteric hepatitis and three had high serum INTRODUCTION Hepatitis E was first recognized during an epidemic of hepatitis, which occurred in Kashmir valley in []. Very soon, hepatitis E was retrospectively traced to an epidemic of acute viral hepatitis, which had occurred in Delhi, India in []. The infection was successfully transmitted in into a human by self-ingestion of acute phase stool suspensions from a water-bne epidemic of non-a hepatitis in Central Asia []. Hepatitis E Virus (HEV) was cloned and sequenced in 0 []. HEV causes large scale water bne epidemics of acute hepatitis in developing countries []. HEV is the commonest cause of acute spadic viral hepatitis and fulminant hepatic failure (FHP) in endemic areas. HEV infections have been repted as impted disease in travellers and tourists from the developed countries to endemic Crespondence: Prof. Mohammad Sultan Khuroo, Direct, Digestive Diseases Centre, Dr KhurooÕs Medical Clinic, Srinagar, Kashmir, J&K, India. khuroo@yahoo.com bilirubin with nmal liver enzymes. Seven babies died in first week of birth (prematurity, icteric HEV, anicteric HEV and hyperbilirubinemia ). Nine babies survived and were followed up f clinical, biochemical, serological course and duration of viremia. Five of babies who survived were HEV RNA positive. HEV RNA was not detectable by weeks of birth in three babies, by weeks in one and by weeks in one. All surviving babies had self-limiting disease and none had prolonged viremia. Thus HEV infection is commonly transmitted from mother-to-foetus and causes high neonatal mtality. HEV infection in survivs is self-limiting with sht lasting viremia. Keywds: acute viral hepatitis, fulminant hepatic failure, hepatitis E virus, obstetric complications, vertical-transmission. areas. Recently HEV infections have been documented to occur in persons from developed countries, who have never travelled to endemic areas. Such infections may have zoonotic igin [,]. Hepatitis E Virus has unique epidemiologic features. The disease has high attack rate among adults years of age in developing countries. The overall mtality associated with HEV infection in these countries is similar to hepatitis A []. HEV infection has increased severity among pregnant women []. Pregnant women especially in the second and third trimester develop FHF. The mtality in the second trimester is around 0% and reaches up to % in the third trimester. The clinical course of non-e acute hepatitis in pregnant women is similar to that in nonpregnant women and men []. The cause of increased severity of HEV in pregnancy is not known. Hepatitis E Virus is commonly transmitted from infected mother to the foetus and causes serious foetal and neonatal infections with significant foetal loss and perinatal mbidity and mtality [,]. HEV infection is a self-limiting disease with sht lasting viremia. However, chronic HEV viremia Ó 00 The Auths J V H 0 B Dispatch:..0 Journal: JVH CE: Sittanand Journal Name Manuscript No. Auth Received: No. of pages: PE: Prasanna

2 0 M. S. Khuroo et al. and chronic hepatitis has been repted in gan transplant recipients [ ]. Prolonged viremia in this group of patients may be because of altered immune status of these patients. Foetal and neonatal periods are states of altered immunity and HBV infection in this age causes subclinical infections with high rate of chronicity []. The clinical course and duration of HEV viremia in vertically transmitted infections in neonates is not known. This study was carried out to assess clinical course of vertically transmitted HEV infection in newbn babies and to assess whether chronic viremia occurs in this age group. MATERIAL AND METHODS The study included pregnant women who had documented HEV infection. All patients were monited closely f maternal disease course, complications and outcome. Patients with FHF were admitted to intensive care unit and received standard supptive care with moniting of clinical, biochemical and hemodynamic parameters. None of the patients received a liver transplant artificial liver suppt. Foetal status was reviewed on a daily basis. A conservative and expectant approach was followed and no attempts were made to terminate pregnancy induce labour unless otherwise indicated on standard obstetric practices (foetal distress etc.). Baby was observed at birth and during the following weeks f any clinical signs of liver disease. Blood samples were collected from babies at birth and thereafter every weeks (in surviving babies) f liver tests, IgG anti- HEV, IgM anti-hev and HEV RNA. Samples were collected till the baby recovered from the clinical, biochemical and serological HEV infection (if infected) and became HEV RNA negative (if previous sample was positive). HEV genotyping was not carried out on these samples. Vertical HEV infection (from mother-to-foetus) in a pregnant woman with acute HEV infection was diagnosed when the blood collected from the baby had detectable IgM anti- HEV and/ HEV RNA persistent IgG anti-hev with elevated liver enzymes. Serology f hepatitis viruses was perfmed by enzyme immunosbant assays (ELISA) f markers of HAV (IgM anti-hav), HBV (HBsAg and IgM anti-hbc), HCV (anti-hcv nd generation) and HDV (IgG and IgM anti-hdv) using commercially available kits from Abbott Labaties (Nth Chicago, IL, USA). The assays were perfmed strictly accding to manufacturerõs instructions. Sera from all patients were also tested by ELISA f IgG and IgM antibodies to HEV by a kit using two recombinant HEV antigens cresponding to structural region (ORF) of the HEV (Diagnostic Biotechnology, Singape). All sera were tested by PCR f HEV RNA. The detection of HEV RNA was perfmed by reverse transcription nested PCR using primer sets / and HEV-/HEV-. The primer sequences and the conditions f PCR have been previously described []. To avoid bias, all sera were tested under code. Confirmed positive and negative controls were run with all PCR amplification reactions to ensure faithful amplifications. Strict application of containment measures was used to avoid false positives. Results of any PCR reactions were considered valid only if they were consistent in at least two independent experiments that included RNA extraction step as well. RESULTS The study included pregnant women (age, mean ± SD;. ±. years) with HEV infection. Serum bilirubin was. ±. (range..) mg/dl and serum ALT 0 ± (range 0) U/L. All patients were IgM anti-hev and IgG anti-hev positive. In addition, serum HEV RNA was positive in nine. One mother was in first trimester, five in second trimester and 0 in third trimester. Fifteen (.%) mothers had developed FHF. Of these, presented with clinical features of FHF and another three developed FHF during the hospital course. Nine (0.0%) of the mothers with FHF died and (.0%) survived. All mothers with nonfulminant course of illness had clinical, biochemical and serologic recovery in. ±. weeks (range weeks). Five mothers with FHF died with babies undelivered. Two (FHF and nonfulminant AVH ) mothers abted, (FHF and nonfulminant AVH ) mothers delivered premature babies and mothers (FHF and nonfulminant AVH ) delivered full-term babies. Nineteen live bne babies (full term and premature delivery ) were evaluated f clinical course and duration of HEV RNA (Table ). All babies were negative f evidence of neonatal infections such as toxoplasmosis, rubella virus, cytomegalovirus and herpes simplex virus (TORCH complex). Fifteen (.%) babies at birth revealed evidence of HEV infection. Markers of HEV in these babies were as follows: (i) HEV RNA, IgM anti-hev and IgG anti-hev reactive ; (ii) HEV RNA and IgG anti-hev reactive ; (iii) IgM anti-hev and IgG anti- HEV reactive ; (iv) IgG anti-hev reactive babies. The diagnosis of HEV infection in two babies with IgG anti-hev positive alone was based on associated elevated liver tests and persistence of antibody response f weeks (as against Table Obstetric outcome of pregnant women studied Fulminant hepatic failure Mother died undelivered Mother delivered full term baby Mother delivered premature baby Mother abted Acute viral hepatitis Mother delivered full term baby Mother delivered premature baby Mother abted Ó 00 The Auths

3 Hepatitis E virus infection in babies bn to HEV-infected mothers 0 trans-placental IgG anti-hev transmission). Three babies were reactive f IgG anti-hev alone with nmal liver tests. The antibody disappeared on serial samples within weeks in two and at weeks in one baby. This antibody response was suggestive of trans-placental antibody transmission (Table ). Nine babies were icteric at birth and one baby developed icterus in the first week of birth. On biochemical evaluation, seven babies had icteric hepatitis, five had anicteric hepatitis and three babies had elevated bilirubin (mixed type) with nmal liver enzymes. Seven babies died in the first week of birth. The cause of death was prematurity in one baby and HEV infection in six. These six babies (icteric hepatitis, anicteric hepatitis and elevated bilirubin group ) developed a clinical state of unresponsiveness, hypoglycemia and hypothermia. Post-mtem needle biopsy of one baby with anicteric hepatitis revealed massive hepatic necrosis. HEV RNA was detected on liver biopsy sample by PCR. Nine HEV infected babies survived and had serial clinical, biochemical, serological and virological evaluation. Liver tests settled to nmal levels in all by weeks. HEV RNA was reactive at birth in five babies. HEV RNA was not detectable by weeks of birth in three babies, by weeks in one and by weeks in one. IgM anti-hev was reactive at birth in seven babies and of the six with repeat samples the antibody persisted f weeks in three and f weeks in two babies. All babies had IgG anti-hev reactive and this antibody persisted in samples taken at weeks (n = ), weeks (n = ) and weeks (n = ). None of the HEV infected baby lost IgG anti-hev over the observation period. DISCUSSION In this study, we followed a coht of pregnant women with HEV infection. Fifteen women either presented with FHF developed FHF during hospital course. Increased severity of viral hepatitis in pregnant women had been repted from the Indian subcontinent, Iran and many other developing countries [ ]. These data were looked upon with scepticism as selection bias could not be ruled out in the hospital data which fmed the basis of these repts. Data collected from an outbreak of hepatitis E in Kashmir () showed that the frequency of hepatitis was greater in pregnant (.%) that in nonpregnant (.%) women and in men (.%) []. The frequency of the disease in the first, second and third trimesters were.%,.% and.% respectively. Severe disease in the fm of FHP developed me often in pregnant women (.%) that in nonpregnant women, none of whom had this complication, and in men (.%). Fulminant hepatitis developed exclusively in the third trimester. The overall mtality in pregnant women was found to be 0%. The nutritional status of women in the area affected by the outbreak those women who developed FHP was assessed as good. Since then, a number of repts have identified Ó 00 The Auths pregnancy especially second and third trimester as a high risk f development of FHF during epidemic as well as spadic HEV infections [,0]. Vertical HEV infection (mother-to-foetus) was diagnosed in of the live births in this study. The clinical profile of HEV infected babies varied from elevated liver enzymes alone, elevated bilirubin alone and elevated bilirubin with elevated liver enzymes. Elevated bilirubin (unconjugated) could be because of physiological jaundice which occurs in neonates. However, pattern of serum bilirubin (mixed pattern) in three babies in this study was against it and suggested it was likely because of the HEV infection. The liver disease in the babies bn with vertically transmitted HEV infection was severe and (0.%) babies infected developed a syndrome of fatal FHF. This syndrome in neonates presented with unresponsiveness, hypoglycemia and hypothermia, as repted earlier by us and others [,]. Of significance was the finding that such a syndrome occurred in few babies even with anicteric hepatitis. In fact one such baby with anicteric hepatitis and syndrome of FHP had massive hepatic necrosis in post mtem liver biopsy. In an earlier rept we studied eight babies bn to mothers infected with HEV infection in third trimester. Six infants had evidence of vertically transmitted HEV infection []. The clinical, biochemical, serological and virological profile of babies who survived showed that HEV infection (mother-to-foetus transmission) in neonates is a self-limiting disease and does not cause chronic viremia prolonged clinical course. This is in contrast to clinical course of HBV infection acquired by neonates by peri-natal transmission. This is possibly related to the different ways these two viruses cause hepatic injury. HBV is a not a cyto-pathogenic virus and liver injury is caused by immune response of the host against the pathogen []. In contrast liver injury in HEV is likely related direct cyto-pathogenic changes in liver cells []. The data regarding epidemiology of HEV pertain to genotype as all HEV infections from humans in Kashmir and the Indian subcontinent are related to genotype []. HEV genotype infections in men and nonpregnant women are known to cause a self limiting acute hepatitis with low rates of FHP in developing countries []. However, the disease presents high incidence and severity in pregnant women especially in second and third trimester [,]. In this study we showed that HEV infection in foetus and babies causes severe liver disease with high rates of FHP and deaths. These data cannot be generalized to HEV infections in the West. Autochthonous HEV infections in the West and Japan are caused by genotype. HEV genotype infections are less severe and most of the infections may run a subclinical course []. There have been no repts of HEV genotype infections in pregnant women and data in this direction shall be watched with interest. It is of interest that HEV infection should run a severe course in pregnant women as well as foetus/newbn. This

4 M. S. Khuroo et al. 0 Table Serial clinical, biochemical, serological and virological data of live bn babies Baby number Mother disease Del Age newbn Bil ALT RNA IgM IgG Clinical status Proven HEV infection in newbn babies FHF FTD Birth AIH, recovery W W W 0. ) ) + AVH FTD Birth AIH, recovery W 0. 0 ) + + W 0. ) ) + W 0. ) ) + FHF FTD Birth 0. + ) + AIH, hypothermia, hypoglycemia, expired AVH FTD Birth IH, recovery W W. ) ) + FHF FTD Birth AIH, hypothermia, expired, MHN AVH FTD Birth. ) ) + IH, recovery W. ) ) + W. ) ) + AVH FTD Birth IH, recovery W ) + + W 0. ) ) + AVH FTD Birth HB, recovery W. ) + + W 0. ) + + FHF FTD Birth. ) + + IH, recovery AVH PMD Birth. ) + + IH, hypothermia, expired AVH FTD Birth 0. ) ) + AIH, recovery W 0. ) ) + W 0. ) ) + FHF PMD Birth IH, expired FHF PMD Birth HB, expired AVH FTD Birth. ) + + HB, recovery FHF FTD Birth IH, hypothermia, expired Noninfected newbn babies FHF FTD Birth 0. ) ) + TPAT W 0. ) ) + W 0. ) ) + W 0. 0 ) ) ) AVH FTD Birth 0. ) ) + TPAT W 0. ) ) + W 0. ) ) ) AVH FTD Birth 0. ) ) + TPAT W 0. ) ) + W 0. ) ) ) FHF PMD Birth 0. 0 ) ) ) Expired because of prematurity FHF, fulminant hepatic failure; AVH, acute viral hepatitis; FTD, full term delivery; PMD, premature delivery; AIH, anicteric hepatitis; IH, icteric hepatitis; TPAT, trans-placental IgG transmission; HB, hyperbilirubinemia; MHN, massive hepatic necrosis. Ó 00 The Auths

5 Hepatitis E virus infection in babies bn to HEV-infected mothers 0 may be related to a common link which increases the pathogenecity of HEV infection in both mother and foetus. What that link could be is obscure and to find it out is a matter of great urgency. Also, the severity of HEV infection in mother and baby may be related to each other. Data from another study has shown definite relationship between FHF of the infants and mother [,]. These data suggested that foetal disease influenced the course of maternal HEV infection. The possible mechanism of increased severity of liver disease in the mother may be production of toxic metabolite in the foetus with FHF, which adversely affected the clinical course of HEV infection in the mother. REFERENCES Khuroo MS. Study of an epidemic of non-a, non-b hepatitis: possibility of another human hepatitis virus distinct from post-transfusion non-a, non-b type. Am J Med 0; :. Wong DC, Purcell RH, Sreenivasn MA, Prasad SR, Pavri KM. Epidemic and endemic hepatitis in India: evidence f a non-a, non-b hepatitis etiology. Lancet 0; :. Balyan MS., Andzjaparridze AG, Savinskaya SS et al. Evidence of a virus in non-a non-b hepatitis transmitted via the fecal-al route. Intervirology?????; 0:. Reyes GR, Purdy MA, Kim J et al. Isolation of a cdna from the virus responsible f enterically transmitted non- A, non-b hepatitis. Science?????; :. Khuroo MS, Khuroo MS. Hepatitis E Virus. Curr Opinion Infect Dis 00; :. Péron JM, Bureau C, Poirson H et al. Fulminant liver failure from acute autochthonous hepatitis E in France: description of seven patients with acute hepatitis E and encephalopathy. J Viral Hepat 00; ():. Yazaki Y, Mizuo H, Takhashi??? et al. Spadic acute fulminant hepatitis E in Hokkaido, Japan, may be foodbne, as suggested by the presence of HEV in pig liver as food. J Gen Virol 00; :. Khuroo MS, Teli MR, Skidme S, Sofi MA, Khuroo MI. Incidence and severity of viral hepatitis in pregnancy. Am J Med ; 0:. Knox TA, Olans LB. Liver disease in pregnancy. N Engl J Med ; :. Khuroo MS, Kamili S, Jameel S. Vertical transmission of hepatitis E virus. Lancet ; :. Kumar RM, Uduman S, Rana S, Kochiyil JK, Usmani A, Thomas L. Sero-prevalence and mother-to-infant transmission of hepatitis E virus among pregnant women in the United Arab Emirates. Eur J Obstet Gynaecol Reprod Biol 00; 0:. Kamar N, Selves J, Mansuy JM et al. Hepatitis E virus and chronic hepatitis in gan-transplant recipients. N Eng J Med 00; ():. Schildgen O, Müller A, Simon A. Chronic hepatitis E and gan transplants. N Engl J Med 00; ():. Haagsma EB, van den Berg AP, Pte RJ et al. Chronic hepatitis E virus infection in liver transplant recipients. Liver Transpl 00; ():. Lee WM. Hepatitis B virus infection. N Eng J Med ; :. Vishwanathan R. Infectious hepatitis in Delhi ( ): a critical study: epidemiology. Ind J Med Res ; (Suppl):. Bhanmanesh F, Haghigi P, Hekmat K, Rezaizadeh K, Ghavani A. Viral hepatitis during pregnancy: severity and effect on gestation. Gastroenterology ; :. Hammerli UP. Jaundice during pregnancy. Acta Med Scand ; (Suppl.):. Khuroo MS, Kamili S. Etiology and prognostic facts of acute liver failure in India. J Viral Hepat 00; :. 0 Patra S, Kumar A, Trivedi SS et al. Maternal and fetal outcomes in pregnant women with acute hepatitis E virus infection. Ann Intern Med 00; ():. Merle P, Zoulin F, Trepo C. Hepatitis B virus infection. In: Knawy BA, Shiffman ML, Weisner RH, eds. Hepatology A Practical Approach.?????????: Elsevier Academic press, 00:. Kamili S. Immunity to hepatitis E virus: new insights and future challenges. Ind J Gastroenterol 00; :. Kamili S. Hepatitis E: Studies on Transmission, Etiological Agent and Seroepidemiology. Thesis submitted to the University of Kashmir f award of the degree of Doct of Philosophy in Biochemistry, December????????:?????, :????. Khuroo MS, Rustgi VK, Dawson GJ et al. Spectrum of Hepatitis E virus infection in India. J Med Virol ; :. Shata MT, Navaneethan U. The mystery of hepatitis E seroprevalence in developed countries: is there subclinical infection due to hepatitis E virus? Clin Infect Dis 00; :. Khuroo MS, Kamili S. Association of severity of hepatitis E virus infection in the mother and vertically transmitted infection in the fetus. JK Pract 00; : 0. Khuroo MS, Khuroo M. Association of severity of HEV infection in the mother and vertically transmitted infection in fetus. Ann Intern Med 00; :. (letter to Edit, rapid response). Ó 00 The Auths

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