1. Background
Hepatitis A and E virus (HAV and HEV) infections are worldwide public health problems, especially in developing countries. They are enterically transmitted, acute and self-limiting infections of liver which usually spread through oral-fecal route. Risk of catching hepatitis A and E virus increase with age. Although some differences distinguish them from each other, hepatitis E has higher mortality rate and lower prevalence compare to hepatitis A (1, 2). From the other point of view, patients with hemophilia (inherited bleeding disorder) and those with thalassemia (poly-transfused disease) are at higher risk of transfusion-transmissible viruses (HBV, HCV and HIV).
Due to the higher rate of HCV infection in these two groups, any superimposed infection with other viral hepatitis, especially A could lead to active liver failure(3). In Iran, there are significant number of HCV infected thalassemia and hemophilia patients (4, 5),those who are prone to complications of HCV infection, such as cirrhosis, liver failure, and hepatocellular carcinoma (6). Non-immune hemophilia and thalassemia patients against enterically transmitted viral hepatitis, in particular hepatitis A, are more susceptible to liver failure than healthy people. In Iran, epidemiological characteristics of HAV and HEV infections are located in a high endemic region; as a result there is not sufficient evidence in this field and we need to fill the ambiguity.
2. Objectives
The main aim of this study was to consider the seroprevalence of antibodies against hepatitis A and E in thalassemia and hemophilia patients infected with hepatitis C in Iran. Also, we aimed to determine epidemiological characteristics of HAV and HEV infections among these patients and to plan the most effective preventative strategies against these infections.
3. Patients and Methods
Patients were selected from those who had been registered in a national clinical trial project of hepatitis C treatment for Iranian thalassemia and hemophilia patients. All these patients were HCV RNA positive and were more than 12 years old.
In a cross-sectional study, sera of 219 thalassemia and hemophilia patients with HCV infection referred to Tehran Hepatitis Center (THC) between 2009 and 2010 from all provinces of Iran (12 to 76 years old). Sera of the patients were stored at -20°C to perform immunologic studies. Immunoglobulin G (IgG) antibodies against HAV and HEV were detected using Enzyme-linked immunosorbent assay (ELISA) (DIA.PRO, Diagnostic Bioprobes Srl, Italy) according to the manufacturer instructions. The cut-off value was defined using positive and negative control sera that were included in each assay. Samples with optical-density (OD) value above the cut-off value considered as positive. All positive samples were retested in duplicate with same enzyme immunoassay to confirm the initial results. The statistical analysis was performed using SPSS (version 18) software. Chi-square test, t-test, and independent sample test were done, and statistical significance was established at P values of < 0.05. The study was approved by the appropriate ethics committee.
4. Results
64 (29.2%) of HCV infected patients had thalassemia, and 155 of them (70.8%) had hemophilia. Generally, 32 (14.6%) of all patients diagnosed with liver cirrhosis. Thalassemia patients were about 5 years younger than hemophilia patients (25.08 ± 6.46 years old versus 30.63 ± 11.51 years old). IgG antibodies against HAV were observed in 164 (74.9%) of the samples. The antibodies were observed more frequently in thalassemia patients (60/64; 93.8%) than in hemophilia group (104/155; 67.1%, P < 0.001) (Table 1).
Table 1.
Status of Anti-HAV and HEV Antibodies among Thalassemia and Hemophilia Patients
| HAV Ab | HEV Ab | |||||
|---|---|---|---|---|---|---|
| Positive, No. (%) | negative, No. (%) | P value a | Positive, No. (%) | Negative, No. (%) | P value a | |
| Thalassemia | 60 (93.8) | 4 (6.3) | 0.000 | 1 (1.6) | 63 (98.4) | 0.674 |
| Hemophilia | 104 (67.1) | 51 (32.9) | 5 (3.2) | 150 (96.8) | ||
| Total | 164 (74.9) | 55 (25.1) | 6 (2.7) | 213 (97.3) | ||
aPvalue is calculated from Chi-square test and is significant < 0.05
The seroprevalence of anti-HAV antibody increased with age, ranging from 50% in patients below 20 years, 77.6% in20-40 years old patients, and up to 96% in patients above 40 years old. There was no significant difference in prevalence of anti-HAV antibody according to age groups (P = 0.000). The seroprevalence of anti-HEV IgG also increased with age, rising from 0% in patients below 20 years to 12% in above 40 years group. In addition, there was no significant difference in age groups for prevalence of anti-HEV IgG (P = 0.009) (Table 2).
Table 2.
Comparison of Anti-HAV and HEV Antibodies Among Age Groups
| HAV Ab | HEV Ab | |||||
|---|---|---|---|---|---|---|
| Positive, No. (%) | Negative, No. (%) | P value a | Positive, No. (%) | Negative, No. (%) | P value a | |
| Age | 0.000 | 0.009 | ||||
| < 20 | 19 (50.0) | 19 (50.0) | 0 (0) | 38 (100.0) | ||
| 20 - 40 | 121 (77.6) | 35 (22.4) | 3 (1.9) | 153 (98.1) | ||
| > 40 | 24 (96.0) | 1 (4.0) | 3 (12.0) | 22 (88.0) | ||
a P value is calculated from Chi-square test and is significant < 0.05
Among thalassemia patients, there was no significant association between HAV seropositivity and other variables like gender, marital status, region, province, level of education, and mean age (Table 3). But in hemophilia group, seropositive patients (mean ± SD age=33.88±11.3 years old) were significantly older than seronegative patients (mean ± SD age=24.00±8.69 years old) (P < 0.01). Single subjects in same group had higher prevalence of anti HAV Ab (P < 0.01) also, most seropositive hemophilia patients lived in urban area (P = 0.03). There was not any significant association between HAV seropositivity and history of icterus, addiction, and history of prison (P=not significant). The blood interval, history of splenectomy, and severity of liver disease were not related to HAV infection in thalassemia group (data not shown).
Table 3.
Demographic Distribution of HAV Seropositivity and Seronegativity Among Thalassemia and Hemophilia Patients Base on Marital Status, Region, Province, Level of Education, and Mean Age of Patients
| Thalassemia Anti HAV Posi- tive, No. (%) | Hemophilia Anti HAV Negative, No. (%) | P value a | Anti HAV Posi-tive, No. (%) | Anti HAV Negative, No. (%) | P value a | ||
|---|---|---|---|---|---|---|---|
| Gender | 1.00 | 0.75 | |||||
| Male | 30 (50) | 2 (50) | 95 (91.3) | 48 (94.1) | |||
| Female | 30 (50) | 2 (50) | 9 (8.7) | 3 (5.9) | |||
| Marital status | 0.42 | 0.000 | |||||
| Single | 53 (88.3) | 3 (75) | 42(40.4) | 42 (82.4) | |||
| Married | 7 (11.7) | 1 (25) | 62(59.6) | 9 (17.6) | |||
| Region | 1.00 | 0.03 | |||||
| Urban | 51 (85) | 4 (100) | 83 (79.8) | 47 (92.2) | |||
| Rural | 9 (15) | 0 (0) | 21 (20.2) | 4 (7.8) | |||
| Education | 0.62 | 0.87 | |||||
| Illiterate | 0 (0) | 0 (0) | 5 (4.8) | 2 (3.9) | |||
| Under diploma | 23 (38.3) | 1 (25) | 35 (33.7) | 19 (37.3) | |||
| Diploma or Bachelor of college | 31 (51.7) | 2 (50) | 51 (49) | 22 (43.1) | |||
| Bachelor of science (BS) and more | 6 (10) | 1 (25) | 13 (12.5) | 8 (15.7) | |||
| Iran’s provinces located in: | 0.93 | 0.39 | |||||
| Capital | 11(18.3) | 1(25) | 29 (27.9) | 16 (31.4) | |||
| North | 20 (33.3) | 1 (25) | 9 (8.7) | 9 (7.6) | |||
| South | 13 (21.7) | 1 (25) | 9 (8.7) | 6 (11.8) | |||
| East | 4 (6.7) | 0 (0) | 11 (10.6) | 5(9.8) | |||
| West | 5 (8.3) | 0 (0) | 23 (22.1) | 6 (11.8) | |||
| Center | 7 (11.7) | 1 (25) | 23 (22.1) | 9 (17.6) | |||
| Mean ± SD age | 25.15 ± 6.57 | 24.00 ± 5.22 | 0.73 b | 33.88 ± 11.3 | 24.00 ± 8.69 | 0.000 b | |
a P-value is calculated from Chi-square test and P < 0.05 was considered significant
b P-value is calculated from Independent Samples Test and is significant < 0.05
Six patients (2.7%) had seropositive HEV IgG antibody (one thalassemia, and 5 hemophilia patients) (Table 1). Neither gender, age, marital status nor province had significant association with HEV infection in both groups. Among hemophilia patients, HEV positive ones (mean ± SD age: 42.80 ± 19.94 years old) were significantly older than negative ones (mean±SD age: 30.22±11.00 years old) (P = 0.01) (Table 4).
Table 4.
Distribution of Demographic Data Between Groups Based on HEV Seropositive or Seronegative Patients
| Thalassemia HEV Ab Positive | Hemophilia HEV Ab Negative | P-value a | HEV Ab Posi- tive | HEV Ab Nega- tive | P value a | ||
|---|---|---|---|---|---|---|---|
| Gender | 1.00 | 1.00 | |||||
| Male | 0 (0) | 32 (50.8%) | 5 (100%) | 138 (92) | |||
| Female | 1 (100) | 31 (49.2%) | 0 (0%) | 12 (8) | |||
| Marital status | 0.12 | 0.13 | |||||
| Single | 0 (0) | 56 (88.9) | 1 (20) | 83 (55.3) | |||
| Married | 1 (100) | 7 (11.1) | 4 (80) | 67 (44.7) | |||
| Region | 1.00 | 1.00 | |||||
| Urban | 1.00 | 54 (85.7) | 5 (100) | 125 (83.3) | |||
| Rural | 0 (0) | 9 (14.3) | 0 (0) | 25 (16.7) | |||
| Education | 0.42 | 0.03 | |||||
| Illiterate | 0 (0) | 0 (0) | 1 (20) | 6 (4) | |||
| Under diploma | 1 (100) | 23 (36.5) | 4 (80) | 50 (33.3) | |||
| Diploma | 0 (0) | 33 (52.4) | 0 (0) | 73 (48.7) | |||
| BS and more | 0 (0) | 7 (11.1) | 0 (0) | 21 (14) | |||
| Iran’s prov- inces located in: | 0.83 | 0.21 | |||||
| Capital | 0 (0) | 12 (19) | 0 (0) | 45 (30) | |||
| North | 1 (100) | 20 (31.7) | 2 (40) | 16 (10.7) | |||
| South | 0 (0) | 14 (22.2) | 0 (0) | 15 (10) | |||
| East | 0 (0) | 4 (6.3) | 0 (0) | 16 (10.7) | |||
| West | 0 (0) | 5 (7.9) | 1 (20) | 28 (18.7) | |||
| Center | 0 (0) | 8 (12.7) | 2 (40) | 30 (20) | |||
| Mean±SD age | 36.00 | 24.90±6.36 | 0.08 b | 42.80 ± 19.94 | 30.22 ± 11.00 | 0.01 b |
aP value is calculated from Chi-square test and P < 0.05 was considered significant
bP value is calculated from Independent Samples Test and is significant < 0.05
5. Discussion
Thalassemia and hemophilia patients are at risk of viral hepatitis due to their needs to blood products. Most of them have chronic liver disease (CLD) such as chronic hepatitis C. The clinical courses of HAV and HEV infections are more severe in the patients with CLD (3, 7-9). Basically, patients with HCV infection are more exposed to HAV associated fulminant hepatic failure than those patients without CLD. Vento et al. found that 41.2% of patients with acute HAV super-infection had acute liver failure with a mortality rate of 35.3% (7).
In our study, the overall seroprevalence of IgG antibody against HAV and HEV in Iranian thalassemia and hemophilia patients were 74.9% and 2.7%, respectively. The seroprevalence of anti HAV antibody was significantly higher in thalassemia than hemophilia patients (93.8% and 67.1% respectively). Findings indicate that more thalassemia patients with HCV had already been exposed to HAV, and consequently had natural acquired immunity against it. In Iranian vaccination program, preventive immunization against HAV is not mandatory, however, it should be considered in non-immune hemophilia and thalassemia patients. Life of poly-transfused thalassemia patients depends on various factors including blood intervals, ferritin levels, and genetic characteristics (4).
Immune deficiency attributed to multiple transfusions and iron overload (10) which could predispose thalassemia patients to HAV infection. No data exists that immune deficiency predispose to HAV infection (11); this seems to be a probable reason for the fact that multi-transfusion influences on getting HAV infection. Another possibility is that in thalassemia patients anti HAV IgG might have been transferred passively by means of transfusions and not the expression of actively acquired immunity (11). In addition, high concentration of antibodies in plasma preparations of multiple donors assists the passive transmission of antibodies in hemophilia patients (11). In overall, potential transfusion of HAV-specific antibodies to the recipient of multiple blood units, and rising seroprevalence to HAV with age significantly reduce risk of post-transfusion hepatitis A.
Most of our statistical populations were hemophilia patients (70.8%). As this disease is x-linked, 79.9% of all patients were male who could superimpose our results (76.2% of seropositive cases were male) in opposite of some related studies (12, 13) but similar to this study in Thailand (14). In addition, thalassemia patients were 5 years younger than hemophilia patients with higher prevalence of HAV infection. As risk of HAV infection increases with age (15), it is suggested to think about vaccination against HAV in childhood. Geographic distribution was not related to seroprevalence of HAV; main location of the project was in Tehran thus we could not determine the exact role of residency area in the study.
In different countries, anti-HAV IgG antibody seroprevalence reported in the range of 40-70% among patients with hemophilia (16-19). In Iran, it is not known how many of these patients are anti-HAV IgG antibody seroprevalence. However, in healthy populations was determined to be different across regions of Iran. Recently published article reported that anti-HAV IgG antibody seroprevalence in Tehran is 90% (20), in northern part of the country is ranging from 19.20% in Savadkoh (21), 38.9% in Sari (12) to 98.6% in Golestan(22), in Fars 88.2% (23), and among HCV patients in Isfahan is 94.9% (24).
In addition, some outbreaks of acute hepatitis A observed in Europe, USA, and South Africa among these patients in the early nineties. The patients receiving clotting factor concentrate, particularly products inactivated by solvent-detergent which was ineffective against non-lipid enveloped viruses such as HAV (25-30). In developed countries, hepatitis A vaccine is recommended for HAV seronegative people with clotting disorders, and those with CLD (31, 32). On the other hand, some effective examinations such as blood-donor screening, viral inactivation of plasma-derived products, and development of recombinant clotting factors are used to reduce viral exposure (31). Several studies that were done in Japan reported high prevalence of HAV and HEV infection among hemophilia patients compare to healthy individual (control group) (33, 34).
As Iran is located in the endemic area of HEV, (35) a few suspected outbreaks of HEV infection were observed in Lordegan (Southeast of Iran) in 1992. Unsanitary sewage disposal with secondary contamination of drinking water was reason for outbreak of HEV infection (36). Like hepatitis A, the seroprevalence of anti-HEV IgG in healthy Iranian populations was demonstrated to be different across regions of Iran, ranging from 2.3% in northern region (37), 3.8% in Isfahan (38), 7.8% in Tabriz (39), 8.5%-11.5% in southern region(40, 41) and 9.3%in Nahavand (42). Interestingly HEV seroprevalence was reported 30.8% in kidney transplant recipients (43).
Our results were similar to seroprevalence of Iranian general population, this indicates that blood products cannot be a transmission way for Iranian thalassemia and hemophilia. Contradictory with our data, researchers in a study in Saudi Arabia proposed higher prevalence of HEV among thalassemia children (10.7%) (44). Toyoda et al. reported 16.3% HEV seropositivity among hemophilia patients. They also suggested that the parenteral transmission of HEV may have occurred in Japanese patients with hemophilia via non-virus-inactivated coagulation factors (34). Other studies in different countries showed lower HEV seropositivity among hemophilia patients (45-48) than Japan.
In conclusion, vaccination of non-immune individuals against HAV infection in high risk groups, especially hemophilia and thalassemia patients are recommended. About seroprevalence of HEV, our results did not show any differences with Iranian general population.
