Hepatitis E virus prevalence in Egyptian children with transfusion-dependent thalassemia

ABSTRACT

Hepatitis E virus (HEV) infection is one of the major public health problems in developing countries. HEV can cause chronic infections in immunocompromised individuals e.g. thalassemic patients with increased risk of morbidity and mortality. In addition there is possibility of HEV transmission through blood transfusion. Therefore, the present study aimed to investigate the seroprevalence and risk factors of HEV infection in β-thalassemic children.

Methods:

This cross-sectional study was conducted on 140 Egyptian children suffering from β-thalassemia, attending the hematology outpatient clinic from April to October 2016. Serum samples from patients were collected and anti-HEV antibodies; Immunoglobulin G (IgG) and Immunoglobulin M (IgM)were measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The seroprevalence of HEV in β-thalassemic chidren was relatively high (27.15%). Anti-HEV IgG prevalence was 24.29% while that of IgM was 2.86%. There was significant association between HEV infection and age, residence, liver enzymes and amount of blood transfusion per year.

Conclusions:

Thalasemic patients are vulnerable to chronicity and increased risk of morbidity and mortality from HEV infection. Frequent assessment of liver enzymes in thalassemic patients to monitor subclinical HEV is recommended. Close monitoring and HEV screening of blood donations should be taken in consideration. Public awareness about HEV endemicity, modes of transmission, and risk hazards especially in high risk group should be done to reduce the disease burden.

Keywords
Hepatitis E virus (HEV); Prevalence; Thalassemia; Transfusion

Introduction

Hepatitis E virus infection is endemic in many developing countries while sporadic HEV infections have also been reported in some developed countries. ¹ Although HEV infection is usually associated with acute self-limited hepatitis, fulminant hepatic failure with morbidity and mortality may occur especially in immunocompromised hosts. ² High prevalence of HEV infection was reported in Africa, Central and Southeast Asia, and Mexico. ³ In France, the seroprevalence of HEV (anti-HEV immunoglobulin G (IgG) and IgM) varied from 21.9% to 71.3%, according to geographic distribution, with average 39.1%. ⁴ People with low educational level may be at a higher risk of exposure and infection than those with higher educational level. In Egypt, low educational level and socioeconomic status resulted in lack of knowledge about avoidable possible risk factors associated with HEV infection. Thus HEV was highly endemic in Egyptian rural communities with genotype 1 subtype 3 most circulating.^5,6 There are four documented routes of transmission of HEV including fecal-oral route, vehicle-borne including water-borne and zoonotic foodborne, bloodborne transmission through parenteral blood transfusion, and perinatal via vertical transmission from mother-to-infant. ^7,8 Chronic HEV infection was acquired in 1.4% of patients following liver transplantion which subsequently resulted in persistent damage of the liver graft. ⁹ Thalassemia is one of the most common genetic diseases worldwide. It is a major public health problem, causing much morbidity, early mortality, and great financial and emotional burden for a family. Thalassemia is the most common form of inherited anemia around the world.¹⁰ β-thalassemia frequencies are common in almost all Arab countries. ¹¹ In Egypt, β-thalassemia is the most common type with a carrier rate ranging from 5.3% to ≥9%. It was estimated that 1000/1.5 million live births/year suffer from thalassemia disease in Egypt. ¹² Many thalassemia patients develop chronic liver disease (CLD) like chronic hepatitis C. HAV and HEV infections show more severe clinical courses in the CLD patients. ¹³ Some authors documented HEV seropositivity among hemodialysis and thalassemia patients. ^14,15 It was reported that HEV RNA detected in blood donations through studies by blood banks in several European countries.¹⁶ Therefore, some countries began HEV RNA screening of blood donations and others selectively applied blood screening for high risk patients. ¹⁷ A previous study conducted on patients with acute viral hepatitis in Egypt reported HEV infection in about 12-42% of all cases. ¹⁸ Another study carried out on blood samples collected from 134 jaundiced patients in Egypt, revealed that 51 (38.1%) patients were positive for anti-HEV IgG. ¹⁹ Also, study on children with acute viral hepatitis in Assiut, Egypt, detected 30.9% HEV infection in children. ²⁰ The present study aimed to detect the seroprevalence of HEV infection in thalassemic children to assess the role of blood transfusion as a risk factor for HEV infection.

Patients and methods

Patients

This cross-sectional study was conducted on 140 Egyptian children suffering from β-thalassemia, attending the hematology outpatient clinic in Mansoura University Children's Hospital (MUCH) from April 2016 through October 2016. Patients included in this study were patients with beta-thalassemia aged up to 18 years who frequently received blood transfusion. However, patients with hemolytic anemia other than thalassemia, patients with alpha-thalassemia, and patients with less than 10 blood transfusions were excluded from the study. This study was approved by the Medical Research Ethics Committee, Mansoura University (IRB no. MS/16/03/82).

Data collected from patients

Data collected from patients included age, sex, residence, amount of blood transfused per year, abdominal examination of liver and spleen, serum ferritin, liver function tests (ALT and AST), and viral hepatitis markers (HBs Ag and HCV Abs).

Sample collection and processing

Two milliliters of venous blood were obtained from each selected patient and collected in a sterile tube, centrifuged immediately for serum separation. Serum samples were stored at −20 °C. Presence of anti-HEV antibodies (anti-HEV IgG indicating past infection and anti-HEV IgM indicating recent infection) in the patients' sera were measured by enzyme-linked immunosorbent assay; HEV-IGM Elisa (Immunospec, USA, BL147710) and HEV-IGG Elisa (Immunospec, USA, BL147709).

Statistical analysis

The collected data was coded, processed and analyzed using SPSS (Statistical package for social science) version 23 for windows. Descriptive statistics were calculated by frequency (number-percent). Chi square test was used for inter-group comparison of categorical data. p-Value less than 0.05 (5%) was considered statistically significant. Significant variables on univariate analysis were entered into a logistic regression model to determine which of these factors were independently associated with HEV infection, followed by multivariate analysis.

Results

The seroprevalence of HEV antibodies in β-thalassemic patients was 27.15% (38/140 patients); 34 (24.29%) patients were positive for anti-HEV IgG, and four (2.86%) patients were positive for anti-HEV IgM. As shown in Table 1, there was significant association between age and anti-HEV IgG and IgM seropositivity (p = 0.004, and 0.044, respectively). HEV seropositivity increased with age. There was significant association between rural residence and anti-HEV IgG (p < 0.001). Regarding clinical data, there was significant association between liver enzymes and anti-HEV IgG. No patients in our study had HBV infection. Splenectomy and HCV infection were not significantly associated with HEV seropositivity (Table 2). Table 3. In Univariate analysis, there was significant association between Age, residence, liver enzymes, and amount of blood transfusions per with HEV seropositivity. On multivariate analysis only rural residence and elevated ALT levels were independently associated with HEV seropositivity.

Table 1
Demographic data associated with HEV seropositivity in thalassemic children.

Demographic data			Anti-HEV IgG				Anti-HEV IgM
			Positive (n = 34)	Negative (n = 106)	p-Value		Positive (n = 4)	Negative (n = 136)	p-Value
Age range (years)	2–6		5 (14.7%)	33 (31.1%)	0.004a		0 (0.0%)	38 (27.9%)	0.044a
	6–12		14 (41.2%)	55 (51.9%)			1 (25.0%)		68 (50.0%)
	12–18		15 (44.1%)	18 (17.0%)			3 (75.0%)		30 (22.1%)
Sex	Female		16 (47.1%)	49 (46.2%)	0.933		1 (25.0%)	64 (47.1%)	0.383
	Male		18 (52.9%)	57 (53.8%)			3 (75.0%)		72 (52.9%)
Residence	Rural		28 (82.4%)	51 (48.1%)	<0.001b		3 (75.0%)	76 (55.9%)	0.447
	Urban		6 (17.6%)	55 (51.9%)			1 (25.0%)		60 (44.1%)

• n, number.
• ^a
  Significance <0.05.
• ^b
  High significance.

Table 2
Clinical parameters associated with HEV seropositivity in thalessemic children.

Clinical data			Anti-HEV IgG				Anti-HEV IgM
			Positive (n = 34)	Negative (n = 106)	p-Value		Positive (n = 4)	Negative (n = 136)	p-Value
Splen-ectomy	Yes		16 (47.1%)	42 (39.6%)	0.444		2 (50.0%)	56 (41.2%)	0.724
	No		18 (52.9%)	64 (60.4%)			2 (50.0%)	80 (58.8%)
HCV	Positive		11 (32.4%)	25 (23.6%)	0.309		1 (25.0%)	35 (25.7%)	0.974
	Negative		23 (67.6%)	81 (76.4%)			3 (75.0%)	101 (74.3%)
HBV	Positive		0 (0.0%)	0 (0.0%)	–		0 (0.0%)	0 (0.0%)	–
	Negative		34 (100.0%)	106 (100.0%)			4 (100.0%)	136 (100.0%)
ALT	Elevated		28 (82.4%)	47 (44.3%)	<0.001b		4 (100.0%)	71 (52.2%)	0.059
	Normal		6 (17.6%)	59 (55.7%)			0 (0.0%)	65 (47.8%)
AST	Elevated		23 (67.6%)	39 (36.8%)	0.002a		3 (75.0%)	59 (43.4%)	0.210
	Normal		11 (32.4%)	67 (63.2%)			1 (25.0%)	77 (56.6%)

• ALT, alanine transaminase; AST, aspartate transaminase; n, number.
• ^a
  Significance <0.05.
• ^b
  High significance.

Table 3
Univariate and multivariate analyses of factors associated with HEV seropositivity in thalassemic children.

	Univariate analysis				Multivariate analysis
	p Value	OR	95% C.I.		p Value	OR	95% C.I.
Age (years)	0.001a	1.18	1.07–1.29
Male sex	0.807	1.098	0.52–2.32
Rural Residence	0.001a	4.98	2.01–12.35		0.003a	4.277	1.65–11.09
Splenectomy	0.385	1.39	0.66–2.96
Abnormal AST	0.001a	3.97	1.79–8.80
Abnormal ALT	<0.001a	7.32	2.81–19.04		<0.001b	6.486	2.43–17.32
Amount of blood transfused (mL/year)	0.008a	1.00	1.00–1.00
Serum ferritin (µg/L)	0.313	1.00	1.00–1.00

• ALT, Alanine transaminase; AST, Aspartate transaminase; p, probability; OR, odd's ratio; CI, confidence interval.
• ^a
  Significance <0.05.
• ^b
  High significance.

Discussion

The prevalence of HEV antibodies among thalassemic children in our study was 27.15%, 24.29% anti-HEV IgG positive and 2.86% anti-HEV IgM positive. Results of previous studies showed seroprevalence of HEV antibodies of 2.4% in thalassemic patients in Scandinavia ²¹ and 10.7% in Saudi Arabia. ²² In Iran, the seroprevalence of HEV IgG and HEV IgM were 10% and 1.8%, respectively. ¹⁵ Reasons such as differences in socioeconomic, cultural, hygienic, and climatic factors across geographical areas may explain the lower prevalence rates in developed countries compared to developing countries. ²⁴ In our study, age was significantly associated with HEV antibodies. The highest HEV prevalence rate found in the age group 12-18 years indicated that the risk of HEV infection increased with age. Previous studies carried out in Egypt ²⁵ and in other geographic regions reported the same finding. ^26-28 This could be explained by the fact that older children had likely received more transfused blood, in addition to have been more exposed to contaminated junk food than younger children. In this study, the prevalence of HEV antibodies in rural areas (81.6%) was higher than in urban areas (18.4%). In rural Egypt, more than 60% of children with aged over 10 years had positive anti-HEV IgG. ²⁹ On the contrary, another study conducted on HCV infected thalassemic patients in Iran reported higher HEV seropositivity in thalassemic patients from urban regions than those from rural areas. ⁶ Regarding liver enzymes, 84.2% of patients with HEV seropositivity showed elevated ALT levels, compared to 68.4% of elevated AST levels. Liver enzymes were higher among those with non-HEV antibodies compared to those with HEV antibodies. This may be due to presence of other causes of hepatitis either viral causes, mostly HCV, or non-viral causes. Although 100% and 75% of anti-HEV IgM positive patients had elevated ALT and AST levels, respectively, there was no significant association between liver enzymes and anti-HEV IgM seropositivity, probably due to small number of anti-HEV IgM positive patients. Previous studies reported that subclinical HEV infection might be the cause of elevated ALT. ³⁰ Therefore, in cases of unexplained ALT and AST elevation, HEV may be the reasonable cause. In two studies conducted in Japan on voluntary blood donors with elevated ALT levels, anti-HEV IgG was positive in 3.7% and 7.1% of participants. ^31,32 These findings suggest that patients with ongoing subclinical HEV infection had the potential to transmit HEV through blood transfusion. Our study revealed that there was significant association between the amount of blood transfused per year and HEV seropositivity (p = 0.008). On the other hand, a previous study showed no significant association between the amount of blood transfused and anti-HEV seropositivity in thalassemic patients. ²¹ This difference may be due to geographic difference and use of assays with low sensitivity that could underestimate HEV prevalence. ³³ Studies conducted in blood banks of several European countries reported that about 0.02-0.14% of blood donations were HEV RNA positive. ^16,34 This finding supports the possibility of HEV transmission through blood transfusion. Since recently in Ireland, blood donations are routinely screened for HEV RNA. Also in 2017, the UK, and Netherlands began HEV RNA screening in blood donations. In France and Germany, screening selectively occurs in high-risk patients, while blood authorities in Italy, Spain, Portugal and Greece are assessing whether HEV screening should be implemented or not. ¹⁷ A previous study conducted in Egypt showed that about 0.45% of blood donors have ongoing subclinical HEV infection. ³⁵ It was reported that the risk of transmission through blood transfusion is increased in high-risk patients in developing regions than in other locations.²³ HEV is endemic in Egypt and besides the possibility of HEV transmission through blood, our study showed significant association between amount of blood transfused and HEV seropositivity. Therefore, further studies should be carried out to detect HEV infection in risk group patients and blood donors.

Conclusion

In conclusion, the seroprevalence of HEV in β-thalassemic patients was relatively high. Therefore, close monitoring of these patients in addition to HEV screening of blood donations should be taken into consideration. Frequent assessment of liver enzymes in β-thalassemic patients to monitor subclinical HEV infections and other viral infections is recommended. Public awareness about HEV endemicity, modes of transmission, and risk hazards, especially in high-risk groups should be done to reduce the disease burden.

• Funding
  The authors received no specific funding for this work.

References

• ¹
  Romano' L, Paladini S, Tagliacarne C, Canuti M, Bianchi S, Zanetti AR. Hepatitis E in Italy: a long-term prospective study. J Hepatol. 2011;54:34-40.
• ²
  Arends JE, Ghisetti V, Irving W, et al. Hepatitis E: an emerging infection in high income countries. J Clin Virol. 2014;59:81-8.
• ³
  Balayan MS. Epidemiology of hepatitis E virus infection. J Viral Hepatitis. 1997;4:155-65.
• ⁴
  Clemente Casares P, Ramos Romero C, Ramirez Gonzalez E, Mas A. Hepatitis E virus in industrialized countries: the silent threat. BioMed Res Int. 2016;:9838041.
• ⁵
  Amer AF, Zaki SA, Nagati AM, Darwish MA. Hepatitis E antibodies in Egyptian adolescent females: their prevalence and possible relevance. J Egypt Public Health Assoc. 1996;71:273-84.
• ⁶
  Elizee PK, Alavian SM, Miri SM, et al. The seroprevalence of entrically transmitted viral hepatitis in HCV infected thalassemia and hemophilia patients in Iran. Jundishapur J Microbiol. 2013;6:e9091.
• ⁷
  Mushahwar IK. Hepatitis E virus: molecular virology clinical features, diagnosis, transmission, epidemiology, and prevention. J Med Virol. 2008;80:646-58.
• ⁸
  Lewis HC, Wichmann O, Duizer E. Transmission routes and risk factors for autochthonous hepatitis E virus infection in Europe: a systematic review. Epidemiol Infect. 2010;138:145-66.
• ⁹
  Feray C, Pawlotsky JM, Roque-Afonso AM, Samuel D, Dhumeaux D. Should we screen blood products for hepatitis E virus RNA?. Lancet. 2014;383:218.
• ¹⁰
  Quek L, Thein SL. Molecular therapies in beta-thalassaemia. Br J Haematol. 2007;136:353-65.
• ¹¹
  Hamamy HA, Al-Allawi NA. Epidemiological profile of common haemoglobinopathies in Arab countries. J Commun Genet. 2013;4:147-67.
• ¹²
  El-Beshlawy A, Youssry I. Prevention of hemoglobinopathies in Egypt. Hemoglobin. 2009;33(Suppl 1):S14-20.
• ¹³
  Cho HC, Paik SW, Kim YJ, et al. Seroprevalence of anti-HAV among patients with chronic viral liver disease. World J Gastroenterol. 2011;17:236-41.
• ¹⁴
  Morteza P, AbdolReza S, Hamid N. Hepatitis E virus infection in hemodialysis patients: a seroepidemiological survey in Jahrom, Southern Iran. Hepatitis Mon. 2009;3(Summer):232-5.
• ¹⁵
  Jahromi AS, Ahmadi-Vasmehjani A, Zabetian H, et al. Sero-epidemiological study of hepatitis E virus among Thalassemia as high risk patients: a cross-sectional survey in Jahrom, Southern Iran. Global J Health Sci. 2016;8:245-50.
• ¹⁶
  Gallian P, Couchouron A, Dupont I, et al. Comparison of hepatitis E virus nucleic acid test screening platforms and RNA prevalence in French blood donors. Transfusion. 2017;57:223-4.
• ¹⁷
  Domanovic D, Tedder R, Blumel J, et al. Hepatitis E and blood donation safety in selected European countries: a shift to screening?. Euro Surveill. 2017;22:30514.
• ¹⁸
  Zakaria S, Fouad R, Shaker O, et al. Changing patterns of acute viral hepatitis at a major urban referral center in Egypt. Clin Infect Dis. 2007;44:e30-6.
• ¹⁹
  El-Tras WF, Tayel AA, El-Kady NN. Seroprevalence of hepatitis E virus in humans and geographically matched food animals in Egypt. Zoonoses Public Health. 2013;60:244-51.
• ²⁰
  Hasan G, Assiri A, Marzuuk N, et al. Incidence and characteristics of hepatitis E virus infection in children in Assiut Upper Egypt. J Int Med Res. 2016;44:1115-22.
• ²¹
  Psichogiou M, Tzala E, Boletis J, et al. Hepatitis E virus infection in individuals at high risk of transmission of non-A, non-B hepatitis and sexually transmitted diseases. Scand J Infect Dis. 1996;28:443-5.
• ²²
  Al-Fawaz I, Al-Rasheed S, Al-Mugeiren M, Al-Salloum A, Al-Sohaibani M, Ramia S. Hepatitis E virus infection in patients from Saudi Arabia with sickle cell anaemia and beta-thalassemia major: possible transmission by blood transfusion. J Viral Hepatitis. 1996;3:203-5.
• ²⁴
  Junaid SA, Agina SE, Abubakar KA. Epidemiology and associated risk factors of hepatitis e virus infection in plateau state Nigeria. Virology (Auckl). 2014;5:15-26.
• ²⁵
  Stoszek SK, Engle RE, Abdel-Hamid M, et al. Hepatitis E antibody seroconversion without disease in highly endemic rural Egyptian communities. Trans R Soc Trop Med Hyg. 2006;100:89-94.
• ²⁶
  Dong C, Dai X, Shao JS, Hu K, Meng JH. Identification of genetic diversity of hepatitis E virus (HEV) and determination of the seroprevalence of HEV in eastern China. Arch Virol. 2007;152:739-46.
• ²⁷
  Taremi M, Mohammad Alizadeh AH, Ardalan A, Ansari S, Zali MR. Seroprevalence of hepatitis E in Nahavand Islamic Republic of Iran: a population-based study. East Mediterr Health J. 2008;14:157-62.
• ²⁸
  Anderson DA 7th. Hepatitis E virus. In: Anderson DA 7th, editor. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Churchill Livingstone Elsevier; 2010. p. 2411–23.
• ²⁹
  Fix AD, Abdel-Hamid M, Purcell RH, et al. Prevalence of antibodies to hepatitis E in two rural Egyptian communities. Am J Trop Med Hyg. 2000;62:519-23.
• ³⁰
  Gao D, Peng G, Zhu J, Sun L, Zheng Y, Zhang J. Investigation of sub-clinical infection of hepatitis E virus in blood donors. Chin J Hepatol. 2004;12:11-2.
• ³¹
  Fukuda S, Sunaga J, Saito N, et al. Prevalence of antibodies to hepatitis E virus among Japanese blood donors: identification of three blood donors infected with a genotype 3 hepatitis E virus. J Med Virol. 2004;73:554-61.
• ³²
  Gotanda Y, Iwata A, Ohnuma H, et al. Ongoing subclinical infection of hepatitis E virus among blood donors with an elevated alanine aminotransferase level in Japan. J Med Virol. 2007;79:734-42.
• ³³
  Avellon A, Morago L, Garcia-Galera del Carmen M, Munoz M, Echevarria JM. Comparative sensitivity of commercial tests for hepatitis E genotype 3 virus antibody detection. J Med Virol. 2015;87:1934-9.
• ³⁴
  Hogema BM, Molier M, Sjerps M, et al. Incidence and duration of hepatitis E virus infection in Dutch blood donors. Transfusion. 2016;56:722-8.
• ³⁵
  Ibrahim EH, Abdelwahab SF, Nady S, et al. Prevalence of anti-HEV IgM among blood donors in Egypt. Egypt J Immunol. 2011;18:47-58.

Publication Dates

• Publication in this collection
  20 May 2019
• Date of issue
  Jan-Feb 2019

History

• Received
  22 Oct 2018
• Accepted
  3 Jan 2019
• Published
  28 Feb 2019