International Journal of Gynecology and Obstetrics 108 (2010) 207210

Contents lists available at ScienceDirect

International Journal of Gynecology and Obstetrics

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j g o

CLINICAL ARTICLE

Duration of hepatitis E viremia in pregnancy

Nargis Begum a,b,c,, Sunil Kumar Polipalli a,b, Syed Akhtar Husain b, Ashok Kumar c, Premashis Kar a
a
PCR Hepatitis Laboratory, Department of Medicine, Maulana Azad Medical College, University of Delhi, New Delhi, India
b
Department of Biosciences, Jamia Millia Islamia, New Delhi, India
c
Department of Obstetrics and Gynecology, Maulana Azad Medical College, University of Delhi, New Delhi, India

a r t i c l e i n f o a b s t r a c t

Article history: Objective: To investigate the duration of hepatitis E virus (HEV) infection in pregnant and non-pregnant
Received 11 July 2009 women with acute viral hepatitis (AVH) or fulminant hepatic failure (FHF). Methods: A prospective study
Received in revised form 2 September 2009 conducted with 20 pregnant women with AVH, 20 non-pregnant women with AVH, 10 pregnant women
Accepted 19 October 2009 with FHF, and 9 non-pregnant women with FHFall with HEV infection. The women were followed up on
day 7, 15, 30, 60, and 90 following recruitment for the presence of HEV-RNA. Results: Of the 59 patients with
Keywords:
HEV infection, only 2 (3.4%) revealed HEV viremia at day 30 and none of the patients was viremic at day 60
Acute viral hepatitis
Fulminant hepatic failure
and day 90. The proportion of HEV viremia in pregnant women with AVH and FHF at day 15 was signicantly
Hepatitis E virus higher than in non-pregnant women (88.3% vs 27.6%; P b 0.001). Similarly, HEV viremia among patients with
Pregnancy AVH was signicantly higher in pregnant women compared with non-pregnant women (100% vs 55%;
Viremia P b 0.001). Conclusion: HEV viremia may be prolonged in pregnancy.
2009 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Hepatitis E virus (HEV) is a single-stranded, spherical, non-
enveloped RNA virus that is classied under the new Hepevirus genus
(http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/ICTV8thReport%20Master%
20species%20list.htm). Hepatitis E is a worldwide public health problem
in locations where the availability of safe drinking water is limited. It is
an important cause of enterically-transmitted acute viral hepatitis
(AVH) in low-resource countries, including India, and is transmitted by
a fecaloral route [1]. HEV is the most common cause of acute sporadic
viral hepatitis and fulminant failure in endemic areas.
Hepatitis E virus has unique features. Its highest rate of attack is
in low-resource countries among adults aged 1540 years [2]. The
overall mortality associated with HEV infection in these countries is
similar to that of hepatitis A [2], while the greatest severity of HEV
infection is among pregnant women [3,4]. The incidence of HEV
infection during the second and third trimesters of pregnancy (19.4%
and 18.4%, respectively) was found to be much higher than in the rst
trimester (8.8%) or in non-pregnant women (2.1%) or men (2.8%) [4].
In Ghana, the prevalence of HEV was found to be higher in women in
the third trimester of pregnancy (30.3%) compared with women in
the second trimester of pregnancy (25.0%) [5]. However, the serologic
evidence for acute HEV infection was found to decline in Uzbekistan
from 71% to 12% from 1987 to 1992 [6]. A study from Bangladesh
reported an HEV prevalence of 58.3% among 144 patients who suf-
fered from AVH, 45.2% among 31 pregnant women, and 56.5% among
23 patients with fulminant hepatic failure (FHF). A mortality rate of
80% caused by HEV-induced FHF during the third trimester of
pregnancy has also been reported [7].
The fatality rate among pregnant women with FHF is reported to be
high in India at 22.2%, with the maximum severity occurring during the
third trimester (44.4%) [3,4,8]. Hepatitis E in pregnancy is also asso-
ciated with high rates of spontaneous abortion, intrauterine death, and
preterm labor [8]. However, there are conicting reports about the
severity related to pregnancy and HEV. The outcome of HEV-related
acute liver failure (ALF) is independent of the sex and pregnancy status
of patients (P = 0.103) [9]. The mortality in HEV-ALF and non-HEV-ALF
in pregnant women and girls has been reported as 51% (74/145) and
54.7% (52/95), respectively [9]. The outcome of pregnant patients with
ALF is also unrelated to the trimester of pregnancy [9]. The severity of
the HEV infection has been related to the hormonal and immunological
changes associated with pregnancy [10,11].
Current knowledge of the pathogenesis of HEV infection derives
from experimentally-infected nonhuman primates [12,13]. There is
sparse literature concerning the pathogenesis of HEV infection in
humans, and the duration of HEV viremia in the general population has
only recently been reported [1419]. The aim of the present prospective
study was to investigate the duration of hepatitis E viremia in pregnant
and non-pregnant women with acute AVH and FHF.

2. Material and methods

Corresponding author. Department of Obstetrics and Gynecology, Maulana Azad
Medical College, New Delhi 110002, India. The study population included pregnant and non-pregnant
E-mail address: nargisbegum18@gmail.com (N. Begum). women with AVH and FHF. A total of 167 consecutive jaundiced

0020-7292/$ see front matter 2009 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijgo.2009.09.023
208 N. Begum et al. / International Journal of Gynecology and Obstetrics 108 (2010) 207210

patients with AVH or FHF who were 1835 years of age and registered
at the Lok Nayak Hospital, New Delhi, India, from December 2006 to
November 2007 were screened for the study. AVH was dened as an
acute self-limited disease with a serum aspartate aminotransferase
(AST) elevation of at least 5-fold above normal, clinical jaundice, or
both [20]. A diagnosis of FHF was made when, after a typical onset, the
patient became deeply jaundiced and went into hepatic encephalop-
athy within 4 weeks of the onset of disease and had no pre-existing
liver disease [21]. The study was reviewed and approved by the
Institutional Research Committee.
The demographic prole and details of the clinical examination
were recorded according to pre-designed and pre-tested proforma.
Blood samples were collected after obtaining consent from the pa-
tients, or their relatives in cases of FHF patients with altered
sensorium. Ten milliliters of blood was collected from each patient
to conduct a liver function test (LFT) and serological tests to detect
hepatitis A (IgM anti-HAV), hepatitis B (IgM anti-HBc and HBsAg),
hepatitis C (anti-HCV), and hepatitis E (IgM anti-HEV) (RADIM Diag-
nostic, Pomezia, Italy) infection. Serological tests were performed
using commercially available ELISA kits. Reverse transcriptase
polymerase chain reaction (RT-PCR) was used to detect the presence
of viremia on different follow-up days.
Those patients who exhibited hepatitis A, B, or C infection and co-
infection, or exposure to hepatotoxic drugs or chemicals were
excluded from the study since the duration of the virus and severity
of HEV-related disease may be altered by the presence of other
viruses.
RNA was extracted using a TRI reagent for serum (Sigma-Aldrich,
USA). Briey, 750 L of TRI reagent was added to 250 L of serum to
dissociate the nucleoprotein complexes. Then, 200 L of chloroform
was added to the nucleoprotein complexes and centrifuged at
12 000 g for 15 minutes at 4 C for separation into 3 phases. The
uppermost aqueous phase was removed and 500 L of isopropanol
was added and the mixture was kept at 20 C for 30 minutes to
precipitate the RNA. The RNA pellets were washed with 75% ethanol
and dried. The pellets were dissolved in 30 L of diethylpyrocarbo-
nate-treated water.
The nRT-PCR method involved the synthesis of cDNA by reverse
transcription. The primers used for amplication of the HEV RNA were
specic to the conserved region of ORF1 as reported earlier [22]. In
short, 5 L of RNA was added to 20 L of the RT master mix containing
2 L of 25 mM magnesium chloride, 1 L 10 mM (each) dNTPs,
20 pmol of reverse HEV outer primer, 10 U of RNase inhibitor, and
2.5 U of AMV reverse transcriptase (New England Biolabs, USA) and
then incubated at 42 C for 60 minutes.
For PCR, 20 L of the PCR master mix was prepared containing 1 L
of 25 mM magnesium chloride, 1 L of 10 mM (each) dNTPs, 20 pmol
of each HEV outer primers [22], 10 L of 10 x PCR buffer, 5 U of Taq
DNA polymerase (New England Biolabs, USA), and 5 L of cDNA. The
temperature prole used was 94 C for 4 minutes for the initial
denaturation followed by 35 cycles of 94 C for 1 minute, 50 C for
1 minute, and 72 C for 1 minute, for denaturation, annealing, and
extension respectively, and a nal extension at 72 C for 7 minutes.
Two L of the product from the rst round of PCR was added to 23 L
of the PCR master mixture II and subjected to a second round of PCR
for 35 cycles with the same temperature prole. The inner primers
were added to the master mixture II, in place of the outer primers.
Five L of the nal PCR product was subjected to electrophoresis in
ethidium bromide stained 2% agarose gel and observed under a UV
transilluminator to identify a 343 bp amplied product with a marker
of 100 bp. A sample of HEV isolate DEL 36 (DQ318854) was used as a
positive control. Saline water and monoinfection of hepatitis A virus
were used as the negative control to test the specicity of the primers.
Three randomly selected HEV RNA positive samples were sequenced
and found to be similar to that of HEV genotype 1.
The patients were followed-up on day 7, 15, 30, 60, and 90 fol-
lowing the day of recruitment. The sample withdrawn at 68 days
was labeled day 7. Samples drawn at 1416 days, 2832 days, 58
62 days, and 8892 days were labeled as day 15, 30, 60, and 90,
respectively. RT-PCR for HEV RNA was performed at each follow-up
visit.
Data were analyzed using EPI Info software version 3.5.1 (CDC,
Atlanta, GA, USA). The Fisher's exact test was used for univariate
analysis and the t test was used to compare continuous variables
between the 2 groups. P 0.05 was considered signicant.
3. Results
Hepatitis E infection (IgM anti-HEV positive) was identied in 39
pregnant patients (24 with AVH and 15 with FHF) and 31 non-
pregnant patients (21 with AVH and 10 with FHF) out of 167 eligible
patients. In the AVH group, 4 pregnant patients and 1 non-pregnant

Table 1
Characteristic features of the patients at recruitment to the study.a

Characteristics Acute viral hepatitis Fulminant hepatic failure

Pregnant (n = 20) Non-pregnant (n = 20) P value Pregnant (n = 10) Non-pregnant (n = 9) P value

Age, y 25.1 4.6 23.8 9.3 0.58 23.7 3.4 21.7 2.9 0.19
(1935) (1745) (2032) (1722)
Gestational age, wk 30.5 7.9 N/A - 30.1 6.0 N/A -
(640) (1736)
b
Interval between onset of jaundice and recruitment, d 3.7 1.8 6.2 0.9 b0.001 1.7 0.5 2.8 0.8 0.002b
(28) (58) (12) (24)
Fever 9 (45.0) 3 (15.0) 0.08 7 (70.0) 4 (44.4) 0.37
Pruritus 7 (35.0) 4 (20.0) 0.48 2 (20.0) 4 (44.4) 0.35
Anorexia 12 (60.0) 1 (5.0) b0.001b 6 (60.0) 6 (66.7) 1.00
Pedal edema 11 (55.0) 0 (0) b0.001b 7 (70.0) 2 (22.2) 0.07
Prothrombin time, s 15.1 1.9 14 0.8 0.02b 29.8 12.3 17.1 3.4 0.008b
(1322) (1315) (2060) (1423)
Asparate aminotransferase, IU/mL 282.1 296.3 250.1 75.5 0.64 749.1 388.7 1077.1 1349 0.47
(181058) (156369) (1921506) (1353508)
Alanine aminotransferase, IU/mL 369.6 365.5 861.1 427.6 b0.001b 302.1 77.4 1268.7 1518 0.06
(361082) (2351715) (198416) (1403980)
Serum bilirubin, mg% 9.3 2.7 5.8 2.4 b0.001b 12.1 2.4 9.2 6.4 0.20
(3.212.5) (2.311.3) (9.716.2) (3.218.5)
Alkaline phosphate, KAU 151.2 133.1 191 124.8 0.33 247.5 262.3 296.3 322.3 0.72
(7521) (68512) (15826) (43985)
a
Values are given as mean SD (range) or number (percentage) unless otherwise indicated.
b
Signicant at b 0.05.
 N. Begum et al. / International Journal of Gynecology and Obstetrics 108 (2010) 207210
Fig. 1. Duration of HEV viremia in the study groups.
patient refused to give written consent and so were excluded from the
study. In the FHF group, 5 pregnant and 1 non-pregnant patient died
within 1 week of recruitment. Therefore, 20 pregnant patients (mean
gestational age 30.5 7.9 weeks) and 20 non-pregnant patients with
AVH, and 10 pregnant (mean gestational age 30.1 6.0 weeks) and 9
non-pregnant patients with FHF were followed up until the end of the
study. The characteristic features of the HEV IgM positive patients at
recruitment are presented in Table 1.
Of the 59 HEV positive patients who were followed-up until day
90, 2 (3.4%) patients were HEV RNA positive on day 30 of follow-
up and none of the patients were positive on day 60 and day 90.
Thirty-three (55.9%) patients were HEV RNA positive on the day 15,
50 (87.4%) patients were positive on day 7, and 56 (94.9%) patients
were positive only at recruitment (Fig. 1). In the pregnant group (AVH
and FHF), all of the women delivered within 7 days of recruitment,
except 3 in the AVH group who remained pregnant throughout the
full study period.
In the AVH non-pregnant group, 17 (85.0%) of 20 patients were
HEV RNA positive at recruitment and 11 (55.0%) patients had HEV
viremia on day 7 of follow-up. In the AVH pregnant group, 17 patients
delivered within 7 days of recruitment. Of these 17 patients, 15
(88.2%) had evidence of viremia for 15 days whereas the remaining 2
(11.8%) had viremia for 7 days. Three patients continued their preg-
nancy after the completion of the study period. Two (66.7%) of these 3
patients had viremia for 30 days and 1 had it for 15 days.
In the FHF non-pregnant group, 8 (88.9%) of the 9 patients had
HEV RNA viremia for 15 days and 1 patient had viremia for 7 days. In
the FHF pregnant group, all 10 patients delivered within 3 days fol-
lowing admission. Of these, 7 (70%) patients had viremia for 15 days
and 3 (30%) had HEV viremia for 7 days.
When the proportion of HEV viremia in the pregnant (AVH and
FHF) group was compared with that for the non-pregnant (AVH and
FHF) group on day 15 of follow-up, the presence of viremia was
statistically higher among pregnant patients than non-pregnant
patients (25 [83.3%] vs 8 [27.6%]; P b 0.001). Similarly, when the
presence of HEV viremia among pregnant patients with AVH was
compared with non-pregnant on day 7 of follow-up, HEV viremia
was signicantly higher in pregnant patients with AVH (20 [100%] vs
11 [55%]; P b 0.001). However, when a comparison of the presence of
viremia in pregnant and non-pregnant patients with FHF was made
on day 15 of follow-up, the results for the 2 groups were comparable
(7 [70%] vs 8 [88.9%]; P N 0.05).
4. Discussion
Hepatitis E is an important public health problem in many tropical
and subtropical countries. The present study investigated the duration
209
of HEV viremia in pregnant and non-pregnant patients with AVH and
FHF.
The duration of HEV viremia was found to be longer in non-
pregnant patients with FHF than in non-pregnant patients with AVH
(15 vs 7 days; P b 0.001). This might be a result of the severe course of
the HEV infection. The duration of viremia in AVH cases was greater
in pregnant patients than non-pregnant patients (15 vs 7 days;
P b 0.001) suggesting that pregnancy may be a reason for longevity.
Similarly, patients whose pregnancy continued beyond 3 months had
a longer duration of the virus than those who delivered within
2 weeks of recruitment (30 vs 15 days; P b 0.01); this further suggests
that pregnancy might be a reason for extended viremia.
The durations of viremia for pregnant and non-pregnant patients
with FHF were similar. This can be explained because the pregnant
patients with FHF delivered within 1 week of recruitment. Therefore,
it is possible that the pregnant state may inuence the persistence of
the virus in HEV infection. Jilani et al. [10] suggest that diminished
cellular immunityindicated by CD4 decrease, CD8 cell count in-
crease, and lowered CD4/CD8 cell ratioand high levels of steroid
hormones inuencing viral replication/expression during pregnancy
are likely reasons for the severity of the disease. A higher HEV viral
load was also found in pregnant patients than in non-pregnant pa-
tients [23]. Thus, host factors and a higher viral load of pregnant
patients in the present study may have delayed the elimination of the
virus. The severe liver injury due to HEV infection during pregnancy
may be related to one of several possible host factors, such as dif-
ferences in immune and hormonal factors that occur during preg-
nancy and genetic and environmental factors that are found in certain
low-resource countries [11].
There are few reports of the duration of viremia among the general
population [1419]. HEV RNA was detected in 71% (15/21) of patients
at 03 days of illness, 91% (57/63) at 811 days, and 22% (2/9) at 27
41 days [15]. The study by Aggarwal et al. [16] based on 20 patients
reported that HEV viremia was present for a maximum duration of
45 days after the onset of symptoms. In an analysis of 4 patients,
protracted viremia lasted for 52112 days after the rst emergence of
symptoms [17]. However, in the present study of 59 patients, only 2
pregnant patients with AVH exhibited viremia on day 30 of follow-up.
The maximum HEV viremia duration of 51 days following the
onset of the symptoms was reported in 4 patients out of 32 and the
average duration of HEV RNA persistence in serial sera in 24 patients
was reported to be 20.6 days after the onset of illness [18]. Another
study of the duration of viremia in 5 neonates delivered to HEV
infected mothers showed no HEV viremia 4 weeks after birth in 3
neonates, no viremia 8 weeks after birth in 1, and no viremia
32 weeks after birth in the remaining neonate [19].
The main limitation of the present study is the small number of
patients. Statistical signicance is difcult to establish on the basis of
210 N. Begum et al. / International Journal of Gynecology and Obstetrics 108 (2010) 207210
this small prospective study. However, it suggests the longer duration
of HEV viremia in pregnant women from Northern India. It is possible
that a local and regional variation of host immune response to HEV is a
contributing factor. Further larger studies from other parts of the
world are required to consolidate the results.
To conclude, the duration of HEV viremia in pregnant patients with
AVH was signicantly longer than in non-pregnant patients with
AVH/FHF. Pregnancy appeared to be one of the risk factors for delayed
elimination of the virus.
Acknowledgments
The authors acknowledge the Indian Council of Medical Research,
New Delhi, for supporting the study.
Conict of interest
The authors have none to disclose.
References
[1] Krawczynski K. Hepatitis E. Hepatology 1993;17(5):93241.
[2] Khuroo MS. Study of an epidemic non-A, non-B hepatitis: possibility of an-
other human virus distinct from post-transfusion non-A, non-B type. Am J Med
1980;68(6):81824.
[3] Kumar A, Beniwal M, Kar P, Sharma JB, Murthy NS. Hepatitis E in pregnancy. Int J
Gynecol Obstet 2004;85(3):2404.
[4] Khuroo MS, Teli MR, Skidmore S, So MA, Khuroo MI. Incidence and severity of
viral hepatitis in pregnancy. Am J Med 1981;70(2):2525.
[5] Adjei AA, Tettey Y, Aviyase JT, Adu-Gyam C, Obed S, Mingle JA, et al. Hepatitis E
virus infection is highly prevalent among pregnant women in Accra, Ghana. Virol J
2009;6:108.
[6] Sharapov MB, Favorov MO, Yashina TL, Brown MS, Onischenko GG, Margolis HS, et
al. Acute viral hepatitis morbidity and mortality associated with hepatitis E virus
infection: Uzbekistan surveillance data. BMC Infect Dis 2009;9:35.
[7] Mamun-Al-Mahtab, Rahman S, Khan M, Karim F. HEV infection as an aetiologic
factor for acute hepatitis: experience from a tertiary hospital in Bangladesh.
J Health Popul Nutr 2009;27(1):149.
[8] Dahiya M, Kumar A, Kar P, Gupta RK, Kumar A. Acute viral hepatitis in third
trimester of pregnancy. Indian J Gastroenterol 2005;24(3):1289.
[9] Bhatia V, Singhal A, Panda SK, Acharya SK. A 20-year single-center experience with
acute liver failure during pregnancy: is the prognosis really worse? Hepatology
2008;48(5):157785.
[10] Jilani N, Das BC, Husain SA, Baweja UK, Chattopadhya D, Gupta RK, et al. Hepatitis E
virus infection and fulminant hepatic failure during pregnancy. J Gastroenterol
Hepatol 2007;22(5):67682.
[11] Navaneethan U, Al Mohajer M, Shata MT. Hepatitis E and pregnancy: under-
standing the pathogenesis. Liver Int 2008;28(9):11909.
[12] Tsarev SA, Emerson SU, Reyes GR, Tsareva TS, Legters LJ, Malik IA, et al.
Characterization of a prototype strain of hepatitis E virus. Proc Natl Acad Sci USA
1992;89(2):55963.
[13] Li X, Kamili S, Krawczynski K. Quantitative detection of hepatitis E virus RNA and
dynamics of viral replication in experimental infection. J Viral Hepat 2006;13(12):
8359.
[14] Chauhan A, Jameel S, Dilawari JB, Chawla YK, Kaur U, Ganguly NK. Hepatitis E virus
transmission to a volunteer. Lancet 1993;341(8838):14950.
[15] Clayson ET, Myint KS, Snitbhan R, Vaughn DW, Innis BL, Chan L, et al. Viremia, fecal
shedding, and IgM and IgG responses in patients with hepatitis E. J Infect Dis
1995;172(4):92733.
[16] Aggarwal R, Kini D, Sofat S, Naik SR, Krawczynski K. Duration of viraemia and
faecal viral excretion in acute hepatitis E. Lancet 2000;356(9235):10812.
[17] Nanda SK, Ansari IH, Acharya SK, Jameel S, Panda SK. Protracted viremia during
acute sporadic hepatitis E virus infection. Gastroenterology 1995;108(1):22530.
[18] Zhao ZY, Ruan B, Shao H, Chen ZJ, Liu SL. Detection of hepatitis E virus RNA in sera
of patients with hepatitis E by polymerase chain reaction. Hepatobiliary Pancreat
Dis Int 2007;6(1):3842.
[19] Khuroo MS, Kamili S, Khuroo MS. Clinical course and duration of viremia in
vertically transmitted hepatitis E virus (HEV) infections in babies born to HEV-
infected mothers. J Viral Hepat 2009;16(7):51923.
[20] Smedile A, Farci P, Verme G, Caredda F, Cargnel A, Caporaso N, et al. Inuence of
delta infection on severity of hepatitis B. Lancet 1982;2(8305):9457.
[21] Trey C, Davidson CS. The management of fulminant hepatic failure. Prog Liver Dis
1970;3:28298.
[22] Madan K, Gopalkrishna V, Kar P, Sharma JK, Das UP, Das BC. Detection of hepatitis
C and E virus genomes in sera of patients with acute viral hepatitis and fulminant
hepatitis by their simultaneous amplication in PCR. J Gastroenterol Hepatol
1998;13(2):12530.
[23] Kar P, Jilani N, Husain SA, Pasha ST, Anand R, Rai A, et al. Does hepatitis E viral load
and genotypes inuence the nal outcome of acute liver failure during
pregnancy? Am J Gastroenterol 2008;103(10):2495501.