Background/Aim: Hepatitis E virus (HEV) is a major public health problem in the developing countries. HEV infection in pregnant women is more common and fatal in the third trimester. The present study was designed to determine the seroprevalence of subclinical HEV infection in asymptomatic pregnant women. Materials and Methods: A total of 116 asymptomatic pregnant women divided into group 1, including 56 pregnant women with hepatitis C virus (HCV)-positive serology and group 2, including 60 pregnant women with negative HCV serology were included in this study. The prevalence of anti-HEV antibodies and anti-HCV was determined by an enzyme-linked immunosorbent assay (ELISA) kit. Results : The o verall prevalence of anti-HEV IgG was highly significant among pregnant women with chronic HCV infection [40/56 (71.42%)] than in pregnant women free from chronic HCV infection [28/60 (46.7%)] (P = 0.006). Chronic HCV infection in pregnant women appeared to be a risk factor associated with HEV IgG seropositivity (OR = 2.86, CI = 1.24-6.6 ) . The seropositivity of anti-HEV IgG was significantly high in rural areas than in urban areas (62.5% versus 37.5%) in group 1 and (78.58% versus 21.42%) in group 2 (P = 0.15) (OR = 2.2, CI = 0.65-7.7). A significant decrease in albumin (P = 0.047) and increase in bilirubin (P = 0.025), ALT (P = 0.032) and AST (P = 0.044) in pregnant women with positive HCV and IgG anti-HEV than in the second group with negative HCV serology was noted. Conclusions : The seroprevalence of anti-HEV IgG in pregnant women is high in Egypt, especially in the rural areas. With chronic HCV coinfection, marked increase in anti-HEV IgG seropositivity and significant worsening of the biochemical liver indices were noted. Increased public awareness about sound hygienic measures for a lower prevalence of HEV is strongly advised. The need for HEV vaccination for those at risk, especially pregnant ladies, should be considered.
Keywords: Chronic hepatitis C virus infection, Hepatitis E virus, pregnancy
Hepatitis E virus (HEV) is a major public health problem in the developing countries. The disease occurs either in the form of large epidemics, which are related to contamination of drinking water supplies, or in the form of sporadic cases in the absence of a conspicuous outbreak. , In countries with poor sanitation, HEV is endemic and typically causes explosive outbreaks of acute hepatitis, usually associated with fecal contamination of the water supply. The disease is generally mild, yet pregnant women suffer significant morbidity and mortality. ,, In contrast, in countries with high standards of sanitation, HEV occurs sporadically, initially identified as an imported disease in travelers from highly endemic regions, but subsequently diagnosed in patients with no travel history as well; this latter form has been named “hepatitis E indigenous to developed countries.” ,
HEV infection represents a significant public health and economic burden, particularly in countries where the absence of sanitation infrastructures, or their breakdown as a consequence of wars or natural disasters, brings the hygienic conditions below a safe level. ,,
Phylogenetic analysis of HEV genome from different isolates has led to the identification of our main genotypes, with genotypes 1 and 2 circulating in Africa and Asia, genotype 3 showing a broad distribution world wide and genotype 4 being restricted to Asia. Genotypes 3 and 4 are enzootic in a variety of wild and domestic animals, particularly pigs, , which gave rise to the question of whether human HEV infection is a zoonosis. Evidence from Japan ,, and China  now confirms that humans can acquire HEV infection from animals.
HEV infection can be diagnosed by either detection of viral particles in stool using electron microscopy or detection of anti-HEV antibodies in serum. Similar to hepatitis A virus, HEV occurs in high concentrations in stool in the weeks immediately prior to the onset of symptoms. Viral shedding in the stool usually continues about 2 weeks after the onset of jaundice, although in a few persons viral shedding has persisted as long as 4 weeks. Antibodies to HEV are detectable in nearly all infected patients upon presentation of their illness.  Enzyme immunoassays based on recombinant proteins of HEV have been used for most seroprevalence studies. The recombinant proteins contain immunodominant epitopes encoded by ORF2 and ORF3 of the HEV genome from different strains.  IgM antibody to HEV is used as an acute-phase marker of HEV infection, and HEV IgG is used to study the exposure to HEV in a given population. Evidence of exposure and/or positive IgG serology has been demonstrated in varying proportions (9.3-26%) in the healthy populations of developed countries. , This work was designed to study the seroprevalence of subclinical HEV in asymptomatic pregnant women with or without chronic HCV infection.
This study includes 116 asymptomatic pregnant women attending the antenatal clinic of Mansoura University Hospital for routine antenatal care during 2009. There were divided into:
A-group 1, which included 56 pregnant women with HCV-positive serology for more than 6 months as defined by positive viremia for hepatitis C virus (HCV) RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and had no other causes of acute or chronic liver diseases.
B-group 2, which included 60 pregnant women free from chronic HCV infection as defined by negative anti-HCV antibody and negative PCR.
All other causes of chronic liver diseases were excluded on the basis of analytical, clinical and epidemiological data, autoimmunity, metabolic and genetic disorders, non-alcoholic steatohepatitis, alcohol intake and drug toxicity, and all cases were negative for anti-HIV antibodies. Personal, family and socioeconomic history was recorded in detail. All females included in the study were subjected to full medical history and thorough clinical examination, including obstetric examinations. Laboratory investigations were performed, including liver function tests, using a Synchron autoanalyzer (Beckman Coulter, Fullerton, CA, USA). Informed consent was obtained from all patients.
IgG anti-HEV enzyme-linked immunosorbent assay (ELISA)
All serum samples from pregnant women were tested with IgG anti-HEV ELISA kits (Genelabs Diagnostics, Singapore). Fusion proteins M 3-2, B 6-1-4 and M 4-2, corresponding to the immunodominant epitopes found in ORF2 and ORF3, were used to coat the solid phase of the ELISA to detect IgG anti-HEV. The ELISA was performed according to the protocols provided by the manufacturer.
The statistical analysis of data was performed using Excel program and SPSS program (statistical package for social science) version 10. The description of the data performed was presented in the form of mean±SD for quantitative data and frequency and proportion for qualitative data. The analysis of the data was carried out to test the statistical significant difference between groups. For quantitative data, Student’s t-test was used to compare between the groups and Paired Sample t-test was used to compare each group at different measurements. Significant data in univarate analysis were entered in a multivariate logistic regression to detect the predictable data. A P-value <0.05 is considered to be significant.
A total of 116 pregnant women were enrolled in this study. They were divided into two groups: group 1 included 56 pregnant women with chronic HCV infection with a mean age of 32.5 ± 12.3 years and group 2 included 60 pregnant women free from such infection with a mean age of 33.6 ± 7.8 years. [Table 1] shows that 40/56 patients (71.42%) were tested positive for IgG anti-HEV antibodies among the pregnant women with chronic HCV, while 28/60 patients (46.7%) tested positive for IgG anti-HEV antibodies among the pregnant women free from chronic HCV infection (OR 2.86; CI 1.24-6.6).
The seropositivity of IgG anti-HEV when compared with the place of residence was found to be significantly higher in rural areas than in urban areas (62.5% versus 37.5% in group 1 and 78.58% versus 21.42% in group 2 [P = 0.15]) (OR 2.2; CI 0.65-7.7). The majority of pregnant women in our study were primipara, 80.35% in pregnant women with chronic HCV infection and 66.67% in pregnant women free from chronic HCV infection. Most pregnant women in both groups were above 30 years of age, 67.84% in group 1 and 53.3% in group 2.
[Table 2] shows a significant decrease in albumin (P = 0.047) and increase in bilirubin (P = 0.025), ALT (P = 0.032) and AST (P = 0.044) in patients with positive HCV serology and IgG anti-HEV than in patients with negative serology for HCV with positive anti-HEV IgG.
Studies reported very high levels of anti-HEV prevalence among healthy adults and pregnant females in rural areas in Egypt (67.7% and 84.3%, respectively). , The authors hypothesize that both zoonotic and anthroponotic transmission of a virulent (possibly genotype-3) HEV is occurring extensively in these rural villages, and that the rate of seropositivity increases with age.
In our study, we found that the seroprevalence of anti-HEV IgG was 71.42% among pregnant females with chronic HCV infection and 46.7% among pregnant females free from chronic HCV infection. Compared with other studies, , the better results in our study may be explained by the continuous official efforts that resulted in remarkable improvement of sewage disposal and more sanitary water supply in both urban and rural areas during the last few years.
On the other hand, when comparing our findings with international studies, pregnant women in our study showed a higher prevalence of anti-HEV IgG than that in other countries (2-13%). ,, This finding could indicate that the journey is still at its beginning and more mutual efforts among the people and the health authorities are required.
In the present work, the seroprevalence of anti-HEV IgG among pregnant females with chronic HCV infection is 71.42%. A striking association between HEV and HCV infection was reported from southern Italy  and from Greece  (27.0% and 10.7%, respectively). This association may be explained by the fact that transmission of HEV occurs predominantly by the fecal-oral route, which is an easier route for transmission in areas endemic for both viruses and, doubtfully, sanitary health conditions. However, the parenteral route has also been implicated. 
In this study, when our patients with positive anti-HEV IgG were compared, we found a significant increase in AST, ALT and bilirubin and a significant decrease in the albumin level in patients with chronic HCV infection than in those with a negative HCV serology. This result may be explained by the superadded hepatic necroinflammation induced by chronic HCV infection. 
In our study, the seroprevalence of anti-HEV IgG was high in rural areas than in urban areas (62.5% versus 37.5%) in group 1 and 78.58% versus 21.42% in group 2; this result is in agreement with those of Begum et al. (2009), who found that the lowered socioeconomic status appeared to be the major risk factor for the increased prevalence HEV infection among pregnant women. Health measures such as health directions to improve the personal as well as the public hygiene are known to be the most-effective available measures for controlling the spread of HEV infection. 
The seroprevalence of anti-HEV IgG in pregnant women is high in Egypt, especially in rural areas. With chronic HCV coinfection, a marked increase in anti-HEV IgG seropositivity and a significant worsening of the biochemical liver indices were noted. Increased public awareness about sound hygienic measures for a lower prevalence of HEV is strongly advised. The need for HEV vaccination for those at risk, especially pregnant ladies, should be considered.
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]