Transfusion and Apheresis Science xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey _ Berrin Uzun ⇑, Aslı Gamze Sß ener, Serdar Güngör, Ilhan Afsßar, Mustafa Demirci Izmir Katip Celebi University Ataturk Training and Research Hospital, Department of Medical Microbiology, Turkey
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 7 March 2014 Accepted 13 March 2014 Available online xxxx Keywords: Blood donors HBsAg reactivity Hepatitis delta virus Seroprevalence Transfusion-transmitted infections
a b s t r a c t Background: Hepatitis B virus (HBV) and hepatitis delta virus (HDV) infections are potentially dangerous complications of transfusion therapy. Objective: The aim of this study was to determine the prevalence of HDV markers examined by serological and molecular methods in hepatitis B surface antigen (HBsAg)-reactive sera among blood donors. Materials and methods: Samples from 88 HBsAg-reactive blood donors were investigated for total anti-delta antibody (anti-HDV) and HDV-RNA between April 2010 and February 2011. HBsAg screening tests were performed by ‘‘microparticle enzyme immunoassay’’ (MEIA) method using the AxSYM system (Abbott Laboratories, USA), and total anti-delta antibody tests were performed by MEIA method using the Alisei system (Radim, Italy). HDV-RNA was quantified using the polymerase chain reaction (PCR) method. Viral nucleic acid isolation system (Anatolia Geneworks) was used with Bosphore HDV quantification kit. Results: HBsAg reactivity was determined as 1% (124/12.423) among blood donors as a whole. Eighty-eight of these 124 samples were investigated further for HDV. Three (3.4%) of the 88 HBsAg-reactive serum samples were total anti-delta antibody-reactive. Of the 3 anti-HDV-reactive sera, 2 were reactive for HDV-RNA. Therefore, HDV-RNA reactivity was determined as 2.3% (2/88) in HBsAg-reactive donors as a whole. The 2 HDV-RNA-reactive donors were brothers. Conclusions: Investigation of HDV is important because HBV infection is endemic in Turkey. Intrafamilial transmission is important in HDV transmission. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction A number of viruses, bacteria and parasites can be transmitted through blood or blood products. Among these, hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) are mandatorily tested in blood donors worldwide due to the potential serious chronic clinical sequelae associated with these readily transmitted agents [1]. Infection with HBV, HCV or hepatitis D virus (HDV) is a potentially dangerous complication of ⇑ Corresponding author. Tel.: +90 533 7694571; fax: +90 232 2434848. E-mail address: [email protected] (B. Uzun).
transfusion therapy. Although effective serologic and molecular screening tools are employed to detect transfusion-transmitted agents among healthy blood donors, the transmission risk remains [2]. Hepatitis delta is caused by infection with HDV, which requires presence of HBV surface antigen (HBsAg) for transmission. The incidence of HDV infection is very high in the Mediterranean basin, Middle East, Central Asia, West Africa, and part of South America [3]. It was revealed that HDV infection was highly prevalent among HBV-infected blood donors [4]. The traditional methods for the diagnosis of HDV infection, such as detection of serum anti-HDV antibodies, are sufficient for the diagnosis of delta infection. Recent
http://dx.doi.org/10.1016/j.transci.2014.03.005 1473-0502/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
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B. Uzun et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
improvements in molecular techniques, such as HDV-RNA hybridization and reverse transcriptase polymerase chain reaction (RT-PCR), have provided increased diagnostic precision and a more thorough understanding of the natural course of HDV infection [5]. Several guidelines suggest that all HBsAg-reactive patients should be tested for anti-HDV antibodies [6,7]. However, anti-HDV antibodies may not be identified after recovery from infection. Hepatitis D infection should be confirmed by the detection of HDV-RNA [3]. In the present study, the prevalence of HDV markers was examined by serological and molecular approaches in HBsAg-reactive sera among blood donors.
2. Materials and methods The present study was conducted in the Ataturk Training and Research Hospital Blood Center in Izmir, Turkey. This hospital is an 1100-bed tertiary-care teaching hospital and the largest state referral center for all patients. The study was performed in the largest Blood Center in a government hospital in the Aegean region, with an average of 40,000 units of blood consumption annually. Seventeen thousand blood donors are accepted annually. Three different blood products are obtained from each received donation. The Center has been categorized as the regional blood center. Accepted donors applying to our center are asked to complete donor questionnaire forms, and then physicians at the Center interview the donors and perform physical examinations. Donors whose medical histories revealed any of the following – hepatitis, surgery in the previous year, a tattoo, acupuncture, piercing, risky sexual intercourse, or oral and/or intravenous narcotic addiction – were not accepted. All serum samples of healthy volunteer blood donors were examined for transfusion-transmitted infections. Serum samples were collected from a total of 12,423 volunteer blood donors between April 2010 and February 2011 at the Blood Center of Izmir Ataturk Training and Research Hospital. To separate serum, the blood samples were collected into a sterile Vacutainer without any anticoagulant, and the blood container was centrifuged at 800–1.600g for 20 min. The separated serum was transferred to polypropylene tubes and stored at 20 °C or lower, until use.
2.1. Serological assay for HBV and HDV HBsAg screening tests were performed by ‘’microparticle enzyme immunoassay’’ (MEIA) method using the AxSYM system (Abbott Laboratories, USA). One hundred and twenty-four initially HBsAg-reactive samples were re-tested by the same method, same equipment and same serum sample to determine repeated reactivity. Samples that were repeatedly HBsAg-reactive were included. Eighty-eight serum samples were investigated for total anti-delta antibody using microplate analyzer and the Alisei system (Radim, Italy) according to the manufacturer’s protocol, with >98% sensitivity and specificity.
2.2. HDV-RNA detection HDV-RNA was quantified using the RT-PCR method. The viral nucleic acid isolation system (Anatolia Geneworks, Turkey) was used with Bosphore HDV quantification kit. RNA isolation and quantification were performed according to the manufacturer’s instructions. All analyses were done automatically. PCR was performed once. The parameters of the PCR experiment (standards, internal control, threshold, and Ct, etc.) were controlled individually. RNA isolation from the patient’s serum was done using the Magnesia™ viral nucleic acid extraction kit (Anatolia Geneworks, Turkey) isolation system with Bosphore™ HDV quantification kit. The PCR mix, containing HotStarTaq DNA polymerase, Probe RT-PCR buffer, and ROX passive reference dye, was prepared. The enzyme was activated after the reverse-transcription step by a 15-min, 95 °C incubation step. The hot start also inactivates the reverse-transcription enzymes, ensuring temporal separation of reverse transcription and PCR, and allowing both steps to be performed sequentially in a single tube. The RT mix, containing a unique Omniscript and Sensiscript blend, was prepared. Both enzymes exhibit a high affinity for RNA, facilitating transcription through secondary structures that may inhibit other reverse transcriptases. Detection mix 1 contains HDV-specific forward and reverse primers (25 lM each) and a dual-labeled probe (3.3 lM), while detection mix 2 contains internal controlspecific forward and reverse primers (3.3 lM each) and a dual-labeled probe (3.3 lM). An internal control, a synthetic DNA molecule derived from human genome, was included in the kit to control PCR inhibition. It was added directly into the RT-PCR master mix to control the PCR inhibition exclusively. For this purpose, 0.1 ll of internal control should be added into the master mix for each reaction. Analysis of the results was performed automatically. The analytical detection limit for BosphoreÒ HDV was found to be 120 copies/ml. The linear range of BosphoreÒ HDV Quantification-Detection Kit was determined to be 150 copies/ml to at least 1 108 copies/ml.
3. Results Of all donors, 11,305 (91%) were males and 1118 (9%) were females. The median age of the donors was 41 years (range, 18–64 years). HBsAg reactivity was 1% (124/12,423) among all blood donors. Totally, anti-HDV and HDV-RNA were studied in the serum of 88 donors. Other reactive serum samples were excluded due to insufficient serum. A total of 85 males (96%) and 3 females (4%) were admitted to the study group (n: 88). Total anti-HDV antibodies were detected in 3 (3.4%) of these 88 HBsAgreactive serum samples. HDV-RNA was detected in 2 of the 3 anti-HDV-reactive sera. Test results were 5.37 106 copies/ml and 5.10 106 copies/ml. It was probably a coincidence that the results were high and almost equal. Therefore, HDV-RNA reactivity among all HBsAg-reactive donors was determined to be 2.3% (2/88). The 2 donors with HDV-RNA-reactive serum samples were brothers, aged 30 and 31 years, from the Western region of Turkey.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
B. Uzun et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
All HBsAg-reactive/anti-HDV-nonreactive sera tested negative for HDV-RNA.
4. Discussion The incidence and prevalence of transfusiontransmitted infections are high in blood donors in developing countries. For this reason, some agents of infection or cases of infection will escape laboratory detection through error or because the infected donor has not yet seroconverted. Therefore, sporadic donors present a high risk for recipients. Routine screening of HBsAg from all blood donors has been used by all blood organizations in Turkey since 1983 [8]. The present study demonstrated that the seroprevalence rate of HBsAg in blood donors in western Turkey was low. In this region, the seroprevalence rate of HDV in donors was found to be lower when compared to the Eastern regions with high endemicity for HBV. In addition, the present study emphasized that intrafamilial transmission is important in HDV transmission. Gurol et al. [8] analyzed seroprevalence of HBsAg among blood donors over 16 years in different regions of Turkey. HBsAg reactivity was detected in 3.9–12.9% (the mean seroprevalence of HBV was found as 4.2%), and the seroprevalence of HBV among the blood donors was determined to have declined since 1997. With the exclusion of high-risk blood donors, the declining trend in HBsAg may be related to population screening before vaccination and immunization with hepatitis B vaccine. Furthermore, the sensitivity of the assays as well as the confirmatory testing used in the different studies might be different. Our study, with its determined low seroprevalence rate, confirms the declining trend in HBsAg. There have been some studies in our region, and HBsAg seropositivity were found as 1.1%, 1,3%, and 2.0%, respectively, by Kaya et al. [9], Balcı et al. [10] and Uzun et al. [11]. Afsar et al. [12] reported HBsAg seropositivity as ranging from 1.1% to 1.5% among donors from our center in previous years. These rates were similar to the findings of the current study. Awareness of HDV as well as HBV infection in the diagnosis and treatment of liver diseases in Turkey is critical [13]. In the present study, anti-HDV reactivity and HDV-RNA reactivity were found as 3.4% and 2.3%, respectively, among blood donors with reactive HBsAg. Of the 3 anti-HDV-reactive sera, 2 were reactive for HDV-RNA. A study performed by Mesße et al. [14] among HBsAg-reactive blood donors in Turkey reported anti-HDV and HDV-RNA reactivity as 8.8% and 1.3%, respectively. In addition, HDV-RNA reactivity was determined as 14.3% (2/14) among blood donors who were HBsAg- and total antiHDV-reactive in that study [14]. The different rates found in these studies may be due to regional differences. In our study, although anti-HDV reactivity rate was found lower, HDV-RNA reactivity was found higher when compared to Mesße et al.’s results. A study carried out by Doosti et al. [15] in Iran among volunteer blood donors reported the seroprevalence of HBsAg as 1.8%; the seroprevalence of anti-HDV among HBsAg-reactive cases was 3%. In Saudi Arabia, the
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rate of hepatitis D infection was detected as 3.3% among HBsAg-reactive blood donors [16]. Researchers in Mauritania reported an HBsAg reactivity rate of 15.3%, and 19.8% of blood donors who were accepted to the study were HDV antibodies-reactive. Among the HDV antibodies-reactive blood donors, HDV-RNA reactivity was 62.2% [4]. Seropositivity of HBsAg and anti-HDV among all donors was found to be low. When compared to the present study, HDV-RNA reactivity among the anti-HDV-reactive donors was similar [4]. Although demographic characteristics are not homogeneous among the different blood collection agencies in Turkey, donor enquiry forms and rules for selection are standardized. This may be attributed to the use of donor questionnaires that eliminate individuals in high-risk groups with respect to transfusion-transmitted infections. In Southern Europe, prevailing intrafamilial transmission is important in HDV diffusion [17]. Intrafamilial transmission of HBV and HDV infections is higher because of the multi-membered family structure throughout Turkey. In addition, the improvement in living conditions is important in terms of intrafamilial transmissions. The intrafamilial transmission rate of both HBV and HDV infections has been reported as 25% in Eastern and Southeastern Anatolia [18]. In the present study, the 2 donors with reactive HDV-RNA were brothers. In a different study, which was performed on volunteer blood donors and their families in Spain, none of the HBsAg-reactive blood donors or their relatives had antibodies against delta agent [19]. Many studies have been performed worldwide for HDV among asymptomatic HbsAg carriers, and the anti-HDV seropositivity rate was found as 21.8% in Bangladesh [20], 16.9% in Mongolia [21], 6% in Diyarbakır [22], 5.8% in Iran [23], and 5.8% in Iran [24]. The decline in HDV in developing countries is the result of the control of HBV infection. A marked reduction was achieved in the last two decades through better public health standards and universal HBV vaccination. The reduction in domestic pools of HBsAg carriers is depriving HDV of the HBV network necessary for its survival, thus diminishing the circulation and medical impact of the defective virus [17]. The transmission risk of HDV infections, as in other transfusion-transmitted infections, is increased in patients who use a large amount of blood and blood products. Screening for HBsAg provides a high degree of safety in preventing HDV infections. However, due to particular HBV infections (so-called occult HBV infections, or OBI), some HDV infections can be missed with HBsAg tests. The HDV infection is also remarkably common among hepatitis patients in the Western as well as Eastern and Southeastern regions of Turkey [13]. However, the number of studies performed among blood donors was minimal. The present study showed that the seroprevalence rate of HDV in donors was lower when compared to the Eastern regions with a high endemicity for HBV. HDV is important in Turkey because HBV infection is endemic. The determination of anti-HDV is a safe and cheap diagnostic method. However, additional HBV tests for detecting occult HBV are a real alternative, e.g. HBV-DNA testing or anti-HBc testing, and are less costly and also effective. These are only indirect tests to facilitate a reduction in HDV transmission.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
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References [1] Bihl F, Castelli D, Marincola F, Dodd RY, Brander C. Transfusiontransmitted infections. J Trans Med 2007;6:5–25. [2] Liu CJ, Chen DS, Chen PJ. Epidemiology of HBV infection in Asian blood donors: emphasis on occult HBV infection and the role of NAT. J Clin Virol 2006;36(1):33–44. [3] Wedemeyer H. Hepatitis D revival. Liver Int 2011:140–4. http:// dx.doi.org/10.1111/j.1478-3231.2010.02408. [4] Mansour W, Bollahi MA, Hamed CT, Brichler S, Le Gal F, Ducancelle A, et al. Virological and epidemiological features of hepatitis delta infection among blood donors in Nouakchott Mauritania. J Clin Virol 2012;55(1):12–6. [5] Modahl LE, Lai MM. Hepatitis delta virus: the molecular basis of laboratory diagnosis. Crit Rev Clin Lab Sci 2000;37(1):45–92. [6] European Association For The Study Of The Liver. EASL clinical practice guidelines: management of chronic hepatitis B. J Hepatol 2009;50:227–42. [7] Cornberg M, Protzer U, Dollinger MM, et al. Prophylaxis, diagnosis and therapy of hepatitis B virus (HBV) infection: upgrade of the guideline, AWMF-Register 021/011. Z Gastroenterol 2007;45: 525–74. [8] Gurol E, Saban C, Oral O, Cigdem A, Armagan A. Trends in hepatitis B and hepatitis C virus among blood donors over 16 years in Turkey. Eur J Epidemiol 2006;21:299–305. [9] Kaya S, Alanoglu G, Polat M, Sipahi T. Evaluation of serologic scanning tests results in blood donors in Suleyman Demirel university, hospital of medical faculty, blood center between 2000 and 2007. Med J Suleyman Demirel Univ 2009;16(2):13–5. [10] Isık Balcı Y, Polat Y, Ovet G, Karabulut A, Goncu F, Yıldırım K. The evaluation of HBsAg, Anti-HCV, Anti-HIV VE VDRL test results in blood donors applying to the blood bank of Denizli State Hospital. Turkish J Infect 2009;23(3):117–9. [11] Uzun B, Güngör S, Demirci M. Seroprevalence of transfusion transmissible infections among blood donors in western part of Turkey: a six-year study. Transfus Apher Sci 2013;49(3):511–5. [12] Afsar I, Gungor S, Sener AG, Yurtsever SG. The prevalence of HBV, HCV and HIV infections among blood donors in Izmir Turkey. Indian J Med Microbiol 2008;26(3):288–9.
[13] Dagartekin H, Yalcın K, Yakut M. The prevalence of hepatitis delta virus infection in acute and chronic liver diseases in Turkey: an analysis of clinical studies. Turky J Gastroenterol 2006;17(1):25–34. [14] Mese S, Bilek H, Gulhan B, Yalcın K, Gul K. Investigation of HDV in positive HBsAg blood donors by serological and molecular methods. Turkiye Klinikleri J Cardiovasc Sci 2009;21(2):198–202. [15] Doosti A, Amini-Bavil-Olyaee S, Tajbakhsh E, Adeli A, Mahboudi F. Prevalence of viral hepatitis and molecular analysis of HBV among voluntary blood donors in west Iran. New Microbiol 2009;32:193–8. [16] Al Traif I, Ali A, Dafalla M, Al Tamaimi W, Qassem L. Prevalence of hepatitis delta antibody among HBsAg carriers in Suadi Arabia. Ann Saudi Med 2004;24:334–43. [17] Rizzetto M. Hepatitis D: the comeback? Liver Int 2009;29(1):140–2. [18] Turkdogan MK, Bozkurt H, Uygan I, Tuncer I, Irmak H, Buzgan T, et al. Chronic hepatitis delta virus infection in Van region of eastern Turkey. Turk J Gastroenterol 2005;16(1):17–20. [19] Alvarez-Muñoz MT, Bustamante-Calvillo ME, Guiscafré-Gallardo JP, Muñoz O. Hepatitis B and delta: the prevalence of seroepidemiological markers in volunteer blood donors and their families. Gac Med Mex 1991;127(5):399–404. [20] Zaki H, Darmstadt GL, Baten A, Ahsan CR, Saha SK. Seroepidemiology of hepatitis B and delta virus infections in Bangladesh. J Trop Pediatr 2003;49(6):371–4. [21] Inoue J, Takahashi M, Nishizawa T, Narantuya L, Sakuma M, Kagawa Y, et al. High prevalence of hepatitis delta virus infection detectable by enzyme immunoassay among apparently healthy individuals in Mongolia. J Med Virol 2005;76(3):333–40. [22] Celen MK, Ayaz C, Hosoglu S, Geyik MF, Ulug M. Anti-hepatitis delta virus seroprevalence and risk factors in patients with hepatitis B in Southeast Turkey. Saudi Med J 2006;27(5):617–20. [23] Gholamreza R, Shahryar S, Abbasali K, Hamidreza J, Abdolvahab M, Khodaberdi K, et al. Seroprevalence of hepatitis B virus and its coinfection with hepatitis D virus and hepatitis C virus in Iranian adult population. Indian J Med Sci 2007;61(5):263–8. [24] Roshandel G, Semnani S, Abdolahi N, Besharat S, Keshtkar AA, Joshaqani H, et al. Prevalence of hepatitis D virus infection in hepatitis B surface antigen-positive subjects in Golestan province, northeast Iran. J Microbiol Immunol Infect 2008;41(3):227–30.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
Contents lists available at ScienceDirect
Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey _ Berrin Uzun ⇑, Aslı Gamze Sß ener, Serdar Güngör, Ilhan Afsßar, Mustafa Demirci Izmir Katip Celebi University Ataturk Training and Research Hospital, Department of Medical Microbiology, Turkey
a r t i c l e
i n f o
Article history: Received 4 October 2013 Received in revised form 7 March 2014 Accepted 13 March 2014 Available online xxxx Keywords: Blood donors HBsAg reactivity Hepatitis delta virus Seroprevalence Transfusion-transmitted infections
a b s t r a c t Background: Hepatitis B virus (HBV) and hepatitis delta virus (HDV) infections are potentially dangerous complications of transfusion therapy. Objective: The aim of this study was to determine the prevalence of HDV markers examined by serological and molecular methods in hepatitis B surface antigen (HBsAg)-reactive sera among blood donors. Materials and methods: Samples from 88 HBsAg-reactive blood donors were investigated for total anti-delta antibody (anti-HDV) and HDV-RNA between April 2010 and February 2011. HBsAg screening tests were performed by ‘‘microparticle enzyme immunoassay’’ (MEIA) method using the AxSYM system (Abbott Laboratories, USA), and total anti-delta antibody tests were performed by MEIA method using the Alisei system (Radim, Italy). HDV-RNA was quantified using the polymerase chain reaction (PCR) method. Viral nucleic acid isolation system (Anatolia Geneworks) was used with Bosphore HDV quantification kit. Results: HBsAg reactivity was determined as 1% (124/12.423) among blood donors as a whole. Eighty-eight of these 124 samples were investigated further for HDV. Three (3.4%) of the 88 HBsAg-reactive serum samples were total anti-delta antibody-reactive. Of the 3 anti-HDV-reactive sera, 2 were reactive for HDV-RNA. Therefore, HDV-RNA reactivity was determined as 2.3% (2/88) in HBsAg-reactive donors as a whole. The 2 HDV-RNA-reactive donors were brothers. Conclusions: Investigation of HDV is important because HBV infection is endemic in Turkey. Intrafamilial transmission is important in HDV transmission. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction A number of viruses, bacteria and parasites can be transmitted through blood or blood products. Among these, hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) are mandatorily tested in blood donors worldwide due to the potential serious chronic clinical sequelae associated with these readily transmitted agents [1]. Infection with HBV, HCV or hepatitis D virus (HDV) is a potentially dangerous complication of ⇑ Corresponding author. Tel.: +90 533 7694571; fax: +90 232 2434848. E-mail address: [email protected] (B. Uzun).
transfusion therapy. Although effective serologic and molecular screening tools are employed to detect transfusion-transmitted agents among healthy blood donors, the transmission risk remains [2]. Hepatitis delta is caused by infection with HDV, which requires presence of HBV surface antigen (HBsAg) for transmission. The incidence of HDV infection is very high in the Mediterranean basin, Middle East, Central Asia, West Africa, and part of South America [3]. It was revealed that HDV infection was highly prevalent among HBV-infected blood donors [4]. The traditional methods for the diagnosis of HDV infection, such as detection of serum anti-HDV antibodies, are sufficient for the diagnosis of delta infection. Recent
http://dx.doi.org/10.1016/j.transci.2014.03.005 1473-0502/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
2
B. Uzun et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
improvements in molecular techniques, such as HDV-RNA hybridization and reverse transcriptase polymerase chain reaction (RT-PCR), have provided increased diagnostic precision and a more thorough understanding of the natural course of HDV infection [5]. Several guidelines suggest that all HBsAg-reactive patients should be tested for anti-HDV antibodies [6,7]. However, anti-HDV antibodies may not be identified after recovery from infection. Hepatitis D infection should be confirmed by the detection of HDV-RNA [3]. In the present study, the prevalence of HDV markers was examined by serological and molecular approaches in HBsAg-reactive sera among blood donors.
2. Materials and methods The present study was conducted in the Ataturk Training and Research Hospital Blood Center in Izmir, Turkey. This hospital is an 1100-bed tertiary-care teaching hospital and the largest state referral center for all patients. The study was performed in the largest Blood Center in a government hospital in the Aegean region, with an average of 40,000 units of blood consumption annually. Seventeen thousand blood donors are accepted annually. Three different blood products are obtained from each received donation. The Center has been categorized as the regional blood center. Accepted donors applying to our center are asked to complete donor questionnaire forms, and then physicians at the Center interview the donors and perform physical examinations. Donors whose medical histories revealed any of the following – hepatitis, surgery in the previous year, a tattoo, acupuncture, piercing, risky sexual intercourse, or oral and/or intravenous narcotic addiction – were not accepted. All serum samples of healthy volunteer blood donors were examined for transfusion-transmitted infections. Serum samples were collected from a total of 12,423 volunteer blood donors between April 2010 and February 2011 at the Blood Center of Izmir Ataturk Training and Research Hospital. To separate serum, the blood samples were collected into a sterile Vacutainer without any anticoagulant, and the blood container was centrifuged at 800–1.600g for 20 min. The separated serum was transferred to polypropylene tubes and stored at 20 °C or lower, until use.
2.1. Serological assay for HBV and HDV HBsAg screening tests were performed by ‘’microparticle enzyme immunoassay’’ (MEIA) method using the AxSYM system (Abbott Laboratories, USA). One hundred and twenty-four initially HBsAg-reactive samples were re-tested by the same method, same equipment and same serum sample to determine repeated reactivity. Samples that were repeatedly HBsAg-reactive were included. Eighty-eight serum samples were investigated for total anti-delta antibody using microplate analyzer and the Alisei system (Radim, Italy) according to the manufacturer’s protocol, with >98% sensitivity and specificity.
2.2. HDV-RNA detection HDV-RNA was quantified using the RT-PCR method. The viral nucleic acid isolation system (Anatolia Geneworks, Turkey) was used with Bosphore HDV quantification kit. RNA isolation and quantification were performed according to the manufacturer’s instructions. All analyses were done automatically. PCR was performed once. The parameters of the PCR experiment (standards, internal control, threshold, and Ct, etc.) were controlled individually. RNA isolation from the patient’s serum was done using the Magnesia™ viral nucleic acid extraction kit (Anatolia Geneworks, Turkey) isolation system with Bosphore™ HDV quantification kit. The PCR mix, containing HotStarTaq DNA polymerase, Probe RT-PCR buffer, and ROX passive reference dye, was prepared. The enzyme was activated after the reverse-transcription step by a 15-min, 95 °C incubation step. The hot start also inactivates the reverse-transcription enzymes, ensuring temporal separation of reverse transcription and PCR, and allowing both steps to be performed sequentially in a single tube. The RT mix, containing a unique Omniscript and Sensiscript blend, was prepared. Both enzymes exhibit a high affinity for RNA, facilitating transcription through secondary structures that may inhibit other reverse transcriptases. Detection mix 1 contains HDV-specific forward and reverse primers (25 lM each) and a dual-labeled probe (3.3 lM), while detection mix 2 contains internal controlspecific forward and reverse primers (3.3 lM each) and a dual-labeled probe (3.3 lM). An internal control, a synthetic DNA molecule derived from human genome, was included in the kit to control PCR inhibition. It was added directly into the RT-PCR master mix to control the PCR inhibition exclusively. For this purpose, 0.1 ll of internal control should be added into the master mix for each reaction. Analysis of the results was performed automatically. The analytical detection limit for BosphoreÒ HDV was found to be 120 copies/ml. The linear range of BosphoreÒ HDV Quantification-Detection Kit was determined to be 150 copies/ml to at least 1 108 copies/ml.
3. Results Of all donors, 11,305 (91%) were males and 1118 (9%) were females. The median age of the donors was 41 years (range, 18–64 years). HBsAg reactivity was 1% (124/12,423) among all blood donors. Totally, anti-HDV and HDV-RNA were studied in the serum of 88 donors. Other reactive serum samples were excluded due to insufficient serum. A total of 85 males (96%) and 3 females (4%) were admitted to the study group (n: 88). Total anti-HDV antibodies were detected in 3 (3.4%) of these 88 HBsAgreactive serum samples. HDV-RNA was detected in 2 of the 3 anti-HDV-reactive sera. Test results were 5.37 106 copies/ml and 5.10 106 copies/ml. It was probably a coincidence that the results were high and almost equal. Therefore, HDV-RNA reactivity among all HBsAg-reactive donors was determined to be 2.3% (2/88). The 2 donors with HDV-RNA-reactive serum samples were brothers, aged 30 and 31 years, from the Western region of Turkey.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
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All HBsAg-reactive/anti-HDV-nonreactive sera tested negative for HDV-RNA.
4. Discussion The incidence and prevalence of transfusiontransmitted infections are high in blood donors in developing countries. For this reason, some agents of infection or cases of infection will escape laboratory detection through error or because the infected donor has not yet seroconverted. Therefore, sporadic donors present a high risk for recipients. Routine screening of HBsAg from all blood donors has been used by all blood organizations in Turkey since 1983 [8]. The present study demonstrated that the seroprevalence rate of HBsAg in blood donors in western Turkey was low. In this region, the seroprevalence rate of HDV in donors was found to be lower when compared to the Eastern regions with high endemicity for HBV. In addition, the present study emphasized that intrafamilial transmission is important in HDV transmission. Gurol et al. [8] analyzed seroprevalence of HBsAg among blood donors over 16 years in different regions of Turkey. HBsAg reactivity was detected in 3.9–12.9% (the mean seroprevalence of HBV was found as 4.2%), and the seroprevalence of HBV among the blood donors was determined to have declined since 1997. With the exclusion of high-risk blood donors, the declining trend in HBsAg may be related to population screening before vaccination and immunization with hepatitis B vaccine. Furthermore, the sensitivity of the assays as well as the confirmatory testing used in the different studies might be different. Our study, with its determined low seroprevalence rate, confirms the declining trend in HBsAg. There have been some studies in our region, and HBsAg seropositivity were found as 1.1%, 1,3%, and 2.0%, respectively, by Kaya et al. [9], Balcı et al. [10] and Uzun et al. [11]. Afsar et al. [12] reported HBsAg seropositivity as ranging from 1.1% to 1.5% among donors from our center in previous years. These rates were similar to the findings of the current study. Awareness of HDV as well as HBV infection in the diagnosis and treatment of liver diseases in Turkey is critical [13]. In the present study, anti-HDV reactivity and HDV-RNA reactivity were found as 3.4% and 2.3%, respectively, among blood donors with reactive HBsAg. Of the 3 anti-HDV-reactive sera, 2 were reactive for HDV-RNA. A study performed by Mesße et al. [14] among HBsAg-reactive blood donors in Turkey reported anti-HDV and HDV-RNA reactivity as 8.8% and 1.3%, respectively. In addition, HDV-RNA reactivity was determined as 14.3% (2/14) among blood donors who were HBsAg- and total antiHDV-reactive in that study [14]. The different rates found in these studies may be due to regional differences. In our study, although anti-HDV reactivity rate was found lower, HDV-RNA reactivity was found higher when compared to Mesße et al.’s results. A study carried out by Doosti et al. [15] in Iran among volunteer blood donors reported the seroprevalence of HBsAg as 1.8%; the seroprevalence of anti-HDV among HBsAg-reactive cases was 3%. In Saudi Arabia, the
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rate of hepatitis D infection was detected as 3.3% among HBsAg-reactive blood donors [16]. Researchers in Mauritania reported an HBsAg reactivity rate of 15.3%, and 19.8% of blood donors who were accepted to the study were HDV antibodies-reactive. Among the HDV antibodies-reactive blood donors, HDV-RNA reactivity was 62.2% [4]. Seropositivity of HBsAg and anti-HDV among all donors was found to be low. When compared to the present study, HDV-RNA reactivity among the anti-HDV-reactive donors was similar [4]. Although demographic characteristics are not homogeneous among the different blood collection agencies in Turkey, donor enquiry forms and rules for selection are standardized. This may be attributed to the use of donor questionnaires that eliminate individuals in high-risk groups with respect to transfusion-transmitted infections. In Southern Europe, prevailing intrafamilial transmission is important in HDV diffusion [17]. Intrafamilial transmission of HBV and HDV infections is higher because of the multi-membered family structure throughout Turkey. In addition, the improvement in living conditions is important in terms of intrafamilial transmissions. The intrafamilial transmission rate of both HBV and HDV infections has been reported as 25% in Eastern and Southeastern Anatolia [18]. In the present study, the 2 donors with reactive HDV-RNA were brothers. In a different study, which was performed on volunteer blood donors and their families in Spain, none of the HBsAg-reactive blood donors or their relatives had antibodies against delta agent [19]. Many studies have been performed worldwide for HDV among asymptomatic HbsAg carriers, and the anti-HDV seropositivity rate was found as 21.8% in Bangladesh [20], 16.9% in Mongolia [21], 6% in Diyarbakır [22], 5.8% in Iran [23], and 5.8% in Iran [24]. The decline in HDV in developing countries is the result of the control of HBV infection. A marked reduction was achieved in the last two decades through better public health standards and universal HBV vaccination. The reduction in domestic pools of HBsAg carriers is depriving HDV of the HBV network necessary for its survival, thus diminishing the circulation and medical impact of the defective virus [17]. The transmission risk of HDV infections, as in other transfusion-transmitted infections, is increased in patients who use a large amount of blood and blood products. Screening for HBsAg provides a high degree of safety in preventing HDV infections. However, due to particular HBV infections (so-called occult HBV infections, or OBI), some HDV infections can be missed with HBsAg tests. The HDV infection is also remarkably common among hepatitis patients in the Western as well as Eastern and Southeastern regions of Turkey [13]. However, the number of studies performed among blood donors was minimal. The present study showed that the seroprevalence rate of HDV in donors was lower when compared to the Eastern regions with a high endemicity for HBV. HDV is important in Turkey because HBV infection is endemic. The determination of anti-HDV is a safe and cheap diagnostic method. However, additional HBV tests for detecting occult HBV are a real alternative, e.g. HBV-DNA testing or anti-HBc testing, and are less costly and also effective. These are only indirect tests to facilitate a reduction in HDV transmission.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
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B. Uzun et al. / Transfusion and Apheresis Science xxx (2014) xxx–xxx
References [1] Bihl F, Castelli D, Marincola F, Dodd RY, Brander C. Transfusiontransmitted infections. J Trans Med 2007;6:5–25. [2] Liu CJ, Chen DS, Chen PJ. Epidemiology of HBV infection in Asian blood donors: emphasis on occult HBV infection and the role of NAT. J Clin Virol 2006;36(1):33–44. [3] Wedemeyer H. Hepatitis D revival. Liver Int 2011:140–4. http:// dx.doi.org/10.1111/j.1478-3231.2010.02408. [4] Mansour W, Bollahi MA, Hamed CT, Brichler S, Le Gal F, Ducancelle A, et al. Virological and epidemiological features of hepatitis delta infection among blood donors in Nouakchott Mauritania. J Clin Virol 2012;55(1):12–6. [5] Modahl LE, Lai MM. Hepatitis delta virus: the molecular basis of laboratory diagnosis. Crit Rev Clin Lab Sci 2000;37(1):45–92. [6] European Association For The Study Of The Liver. EASL clinical practice guidelines: management of chronic hepatitis B. J Hepatol 2009;50:227–42. [7] Cornberg M, Protzer U, Dollinger MM, et al. Prophylaxis, diagnosis and therapy of hepatitis B virus (HBV) infection: upgrade of the guideline, AWMF-Register 021/011. Z Gastroenterol 2007;45: 525–74. [8] Gurol E, Saban C, Oral O, Cigdem A, Armagan A. Trends in hepatitis B and hepatitis C virus among blood donors over 16 years in Turkey. Eur J Epidemiol 2006;21:299–305. [9] Kaya S, Alanoglu G, Polat M, Sipahi T. Evaluation of serologic scanning tests results in blood donors in Suleyman Demirel university, hospital of medical faculty, blood center between 2000 and 2007. Med J Suleyman Demirel Univ 2009;16(2):13–5. [10] Isık Balcı Y, Polat Y, Ovet G, Karabulut A, Goncu F, Yıldırım K. The evaluation of HBsAg, Anti-HCV, Anti-HIV VE VDRL test results in blood donors applying to the blood bank of Denizli State Hospital. Turkish J Infect 2009;23(3):117–9. [11] Uzun B, Güngör S, Demirci M. Seroprevalence of transfusion transmissible infections among blood donors in western part of Turkey: a six-year study. Transfus Apher Sci 2013;49(3):511–5. [12] Afsar I, Gungor S, Sener AG, Yurtsever SG. The prevalence of HBV, HCV and HIV infections among blood donors in Izmir Turkey. Indian J Med Microbiol 2008;26(3):288–9.
[13] Dagartekin H, Yalcın K, Yakut M. The prevalence of hepatitis delta virus infection in acute and chronic liver diseases in Turkey: an analysis of clinical studies. Turky J Gastroenterol 2006;17(1):25–34. [14] Mese S, Bilek H, Gulhan B, Yalcın K, Gul K. Investigation of HDV in positive HBsAg blood donors by serological and molecular methods. Turkiye Klinikleri J Cardiovasc Sci 2009;21(2):198–202. [15] Doosti A, Amini-Bavil-Olyaee S, Tajbakhsh E, Adeli A, Mahboudi F. Prevalence of viral hepatitis and molecular analysis of HBV among voluntary blood donors in west Iran. New Microbiol 2009;32:193–8. [16] Al Traif I, Ali A, Dafalla M, Al Tamaimi W, Qassem L. Prevalence of hepatitis delta antibody among HBsAg carriers in Suadi Arabia. Ann Saudi Med 2004;24:334–43. [17] Rizzetto M. Hepatitis D: the comeback? Liver Int 2009;29(1):140–2. [18] Turkdogan MK, Bozkurt H, Uygan I, Tuncer I, Irmak H, Buzgan T, et al. Chronic hepatitis delta virus infection in Van region of eastern Turkey. Turk J Gastroenterol 2005;16(1):17–20. [19] Alvarez-Muñoz MT, Bustamante-Calvillo ME, Guiscafré-Gallardo JP, Muñoz O. Hepatitis B and delta: the prevalence of seroepidemiological markers in volunteer blood donors and their families. Gac Med Mex 1991;127(5):399–404. [20] Zaki H, Darmstadt GL, Baten A, Ahsan CR, Saha SK. Seroepidemiology of hepatitis B and delta virus infections in Bangladesh. J Trop Pediatr 2003;49(6):371–4. [21] Inoue J, Takahashi M, Nishizawa T, Narantuya L, Sakuma M, Kagawa Y, et al. High prevalence of hepatitis delta virus infection detectable by enzyme immunoassay among apparently healthy individuals in Mongolia. J Med Virol 2005;76(3):333–40. [22] Celen MK, Ayaz C, Hosoglu S, Geyik MF, Ulug M. Anti-hepatitis delta virus seroprevalence and risk factors in patients with hepatitis B in Southeast Turkey. Saudi Med J 2006;27(5):617–20. [23] Gholamreza R, Shahryar S, Abbasali K, Hamidreza J, Abdolvahab M, Khodaberdi K, et al. Seroprevalence of hepatitis B virus and its coinfection with hepatitis D virus and hepatitis C virus in Iranian adult population. Indian J Med Sci 2007;61(5):263–8. [24] Roshandel G, Semnani S, Abdolahi N, Besharat S, Keshtkar AA, Joshaqani H, et al. Prevalence of hepatitis D virus infection in hepatitis B surface antigen-positive subjects in Golestan province, northeast Iran. J Microbiol Immunol Infect 2008;41(3):227–30.
Please cite this article in press as: Uzun B et al. Evaluation of hepatitis delta virus (HDV) infection in blood donors in western Turkey. Transf Apheres Sci (2014), http://dx.doi.org/10.1016/j.transci.2014.03.005
![[Detection of hepatitis delta virus (HDV) markers in HBV carriers].](/assets/img/hold.png)
