Official Journal of the British Blood Transfusion Society
Transfusion Medicine
| SHORT COMMUNICATION
Molecular epidemiology of human Herpesviruses types 1–6 and 8 among Greek blood donors E. Rouka & D. Kyriakou Transfusion Medicine Department, University Hospital of Larisa, Larisa, Greece Received 1 January 2015; accepted for publication 13 April 2015
SUMMARY Background: Human Herpesviruses (HHVs) maintain life-long latent persistence in the majority of the adult population including blood donors. The necessity for their study resides in the potential risk of transfusion-associated infection and the subsequent complications in the immunocompromised host. We aimed to assess the prevalence of HHVs types 1–6 and 8 among healthy blood donors of Thessaly prefecture in order to evaluate the frequency distribution of HHVs in Greek population and to ascertain possible correlations with demographic factors. Materials and Methods: Polymerase chain reaction (PCR) detection of HHVs DNA was determined in 401 randomly selected consecutive blood donors of Central Greece. Epidemiological data were recorded through a well structured questionnaire. Results: The overall PCR positivity for HHVs was 25·4%. HHVs types 1–3 were not detected in any donor sample. A specimen with high level of HHV-6 DNA (1 580 400 copies per mL) was recorded. HHV-4 DNA positivity was significantly associated with rural residency. Conclusion: HHV-4 DNA is commonly detected in whole blood specimens of healthy individuals. HHVs types 5, 6 and 8 are rarely detected. However, the existence of a donor sample with high HHV-6 viral load raises questions regarding the potential risk of HHV-6 blood-borne infection and the safety of blood products. Key words: blood-borne infections, human herpesviruses, molecular testing.
INTRODUCTION Human herpesviruses (HHVs) are double-stranded DNA enveloped viruses which belong to the Herpesviridae family
Correspondence: Erasmia Rouka, Transfusion Medicine Department, University Hospital of Larisa, Larisa, Greece. Tel.: +30 2413502255; fax: +30 2413502260; e-mail: [email protected]
First published online 4 May 2015 doi: 10.1111/tme.12202
and can be transmitted by the transfusion of blood products. Seropositivity for HHVs in adults ranges significantly depending on geographical and socioeconomic conditions (Pamphilon et al., 1999; Qu et al., 2010). Infected individuals maintain a life-long carriage of latent viruses in different cells and organs. Although leukodepletion has diminished the risk of transmission of leukotropic viruses, transfusion-associated morbidity and mortality remains a clinical concern particularly for patients with immature (e.g. preterm infants and neonates) or compromised [organ transplant recipients and acquired immune deficiency syndrome (AIDS) patients] immune systems (O’Riordan et al., 2006; Hassan et al., 2007; Siakallis et al., 2009). Despite the systemic administration of prophylactic antiviral treatment in immunosuppressed patients, the appearance of HHVs-drug resistant mutants has been constantly reported and has emerged as a significant problem especially in the transplantation setting (Piret & Boivin, 2014). As regards cytomegalovirus (CMV) infection in particular, it is documented that the incidence of severe CMV-related disease in immunocompetent adults is greater than previously thought, which may be partly due to immune dysfunction related to comorbidities such as kidney disease or diabetes mellitus (Lancini et al., 2014). In addition, although conflicting data exist with respect to the incidence of transfusion-transmitted CMV remaining after the introduction of leukodepletion, it has been clearly shown that both prevalence and concentration of CMV DNA in peripheral blood are highest in newly seropositive donors. Therefore, it is suggested that the avoidance of blood products from these donors is the most important goal of any transfusion strategy (as reviewed by Ziemann & Hennig, 2014). The aim of this study was to determine the prevalence of HHVs types 1–6 and 8, namely Herpes Simplex Virus (HSV types 1 and 2), Varicella Zoster Virus (VZV), Epstein–Barr Virus (EBV), Human Cytomegalovirus (HCMV), Human Herpes Virus 6 (HHV-6) and Human Herpes Virus 8 (HHV-8) among healthy blood donors of Thessaly prefecture in order to estimate the frequency distribution of HHVs in the Greek population and to reveal probable correlations with epidemiological markers.
© 2015 British Blood Transfusion Society
Molecular epidemiology of human herpesviruses types 1–6 and 8 277
MATERIALS AND METHODS Subjects and samples Four hundred one (401) healthy and asymptomatic blood donors, residents of Central Greece were included in the study after they had given their informed consent. At enrollment trained medical doctors obtained the questionnaire data which included demographic variables, medical and sexual history, occupational and marital status. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes and stored at 4 ∘ C until processing. The study was approved by the local hospital ethics scientific committee.
DNA extraction Nucleic acid extraction was performed on the automated Magtration system 12GC plus (Precision System Science, Co, Ltd, 88 Kamihongou, Matsudo-city, Chiba, 271-0064 JAPAN) with an extraction protocol based on magnetic particles technology for the isolation of DNA from 200 μL of whole blood into a final elution volume of 100 μL.
HHVs genome detection Presence of HHVs genome copies was evaluated using validated, single-virus quantitative real-time polymerase chain reaction (PCR) assays by Nanogen Advanced Diagnostics (Corso Torino, 89/d, 10090 Buttigliera Alta, Italy). These assays incorporate the TaqMan Minor Groove Binder technology as previously described (Petrisli et al., 2010). HHV-7 was not included in the study as the manufacturer did not have an available product at that time. Five microliters of DNA were used as a template. The primers and probes were specific for the exon 4 region of the CMV MIEA gene, the gene region coding EBNA-1 protein of EBV, the ORF 13 R region of HHV-6, the US6 region of HSV types 1 and 2, the ORF 29 region of VZV and for the gene region coding the capside protein of HHV-8. The promoter and 5′ -untranslated region (5′ UTR) region of the human beta globin gene were co-amplified in each reaction in order to validate extraction and detection. Tests were run on 96-well plates using a program of 50 ∘ C for 2 min, 95 ∘ C for 10 min, followed by 45 cycles of 95 ∘ C for 15 s and 60 ∘ C for 1 min on ABI 7300 Real Time PCR system (850 Lincoln Centre Drive, Foster City, CA, USA). For each virus specific standard curves were generated with four stabilised solutions of plasmid at known titre (105 –102 copies) containing part of the genome region targeted by the single quantitative real-time PCR reaction. Assays sensitivity is reported to be 10 copies per PCR which is equivalent to 1000 copies per mL of whole blood as the 200 μL sample was concentrated into a 100 μL eluate from which 5 μL were used as a template. Acceptance range of correlation coefficient (R2 ) was 0·990–1·000. Distilled water was used as negative control in all procedures. A second, multiplex PCR assay by Sacace Biotechnologies S.r.I (Via Scalabrini 44, Como, Italy) was applied for the detection
© 2015 British Blood Transfusion Society
and differentiation of CMV, EBV and HHV-6 targeting the MIE, LMP and pol gene of each virus, respectively. Human beta globin gene was used as endogenous internal control. Ten microliters of extracted DNA sample were used as a template. The amplification program for ABI 7300 Real Time PCR system was as follows: 95 ∘ C/15 min (1 cycle), [95 ∘ C/5 s, 60 ∘ C/20 s, 72 ∘ C/15 s] (5 cycles), [95 ∘ C/5 s, 60 ∘ C/30 s, 72 ∘ C/15 s] (40 cycles). Positive and negative controls were included in each reaction. The kit CMV/EBV/HHV6 Quant Real-TM is reported to have a sensitivity of five DNA copies per 105 cells (approximately 500 copies per mL).
Statistical analysis Possible associations of HHVs DNA detection with demographic variables were assessed by using odds ratios (ORs) and their associated 95% confidence intervals (CIs). Pearson 𝜒 2 test was used to compare percentages. Two-sided P values < 0·05 were considered to be statistically significant. The analyses were performed using the spss version 19·00 package (IBM SPSS Statistics) for windows.
RESULTS The main demographic features of the 401 subjects enrolled in this study are presented in Table 1. Beta globin gene sequences were amplified in all PCR procedures verifying the successful extraction of genomic DNA. All standards and negative controls gave the expected results. The two alternative commercial assays gave reproducible results. Among 401 individuals tested, 102 were HHVs DNA positive (25·4%). HSV-1,2 DNA and VZV DNA were not detected in any donor sample and CMV DNA was detected in a single specimen (0·2%). HHV-8 DNA was present in 2 of 401 samples (0·5%). In contrast, EBV DNA was detected in 85 of 401 samples (21·2%) while 14 of 401 blood specimens had detectable HHV-6 DNA (3·49%). Viral loads ranged from 2000 copies per mL for CMV and HHV-8 to 1 580 400 copies per mL for HHV-6. HHVs DNA detection was slightly lower among females (OR = 0·891). On the opposite, risk for HHVs DNA positivity tended to be higher among married individuals (OR = 1·068) and among village residents (OR = 1·398) while EBV DNA detection was correlated significantly with rural residency (OR = 1·471, P = 0·035).
DISCUSSION In this study, real-time PCR technology was used in order to detect HHVs DNA in whole blood of healthy Greek qualified donors. So far, little attention has been paid to the alpha-subfamily members of Herpesviridae due to their neuronal tropism. More than 20 years have passed since the study that reported the detection of HSV DNA sequences in human blood and bone marrow cells suggesting an additional site of latency (Cantin et al., 1994). More recently, it has been
Transfusion Medicine, 2015, 25, 276–279
278 E. Rouka and D. Kyriakou Table 1. The main demographic features of the donor population of this study Characteristics
Frequency
Sex Male Female Marital status Married Unmarried Residence Urban Rural Age (years) 18–28 29–39 40–50 51–61
401 325 76 396 227 169 397 291 106 391 76 157 120 38
Percent (%)
81 19 57 · 3 42 · 7 73 · 3 26 · 7 19 · 4 40 · 2 30 · 7 9·7
documented that HSV DNAemia is possible but seems to be limited to primary but not recurrent infection (Juhl et al., 2010). This study demonstrates that latent-alpha herpesvirus DNA is undetectable in peripheral blood of healthy and asyptomatic blood donors thus, the potential of blood-borne viral infection is uncommon for HHVs types 1–3. This conclusion is in accordance with previous findings (Hudnall et al., 2008) regarding the population of Texas southeast region. The same investigators identified CMV DNA in a single specimen and that seems to be the case in this study. Another study (Roback et al., 2003), using validated PCR assays also concluded that CMV DNA is rarely detected in healthy seropositive blood donors although stating that the minimal CMV load required for transfusion transmitted CMV has not been determined. Recently, CMV DNA was detected in the cellular fraction of 4·3% of samples from donors in their 60s and in 1·0% of samples from donors younger than 60 years (Furui et al., 2013). In this study, HHV-8 DNA was identified in only 2 of 401 blood specimens. Three previous investigations reported a zero HHV-8 DNA positivity among healthy blood donors (Hudnall et al., 2003; Hudnall et al., 2008; Qu et al., 2010). The current results suggest that the prevalence of HHV-8 DNA sequences in Greek blood donors is very low although previous data (Zavos et al., 2005) indicated an overall HHV-8 DNA positivity rate of 9·6% in human immunodeficiency virus (HIV) negative Greeks without Kaposi’s sarcoma. HHV-6 DNA was detected in 3·49% of the blood donor samples. Previous studies on HHV-6 DNA positivity reported significantly variable rates (Cone et al., 1993; Wilborn et al., 1994; Luppi et al., 1995; Kozireva et al., 2001; Hudnall et al., 2008). It appears that more studies with larger sample size are
Transfusion Medicine, 2015, 25, 276–279
needed in order to clarify whether the higher probabilities of HHV-6 DNA positivity are due to a higher prevalence of infection in some regions compared with others or due to technical inter laboratory factors. Regarding the proportion of samples with high HHV-6 viral load, previous studies have reported ratios of 4 : 500 (Leong et al., 2007) and 1 : 100 (Hudnall et al., 2008). This study demonstrates a proportion of 1 : 401. Undoubtedly, the identification of healthy and asyptomatic carriers with high HHV-6 DNA copies number, that could not be recognised and therefore could not be excluded during donor selection raises serious questions regarding the transfusion safety. EBV DNA was detected in 85 of 401 blood donor specimens. Previous studies have reported rates ranging from 8 to 76% (Correa et al., 2004; Nishiwaki et al., 2006; Compston et al., 2008; Hudnall et al., 2008). As in the case of HHV-6 this variability probably reflects differences in regional epidemiology. The association of EBV DNA detection with rural residency should be studied further in combination with other social and demographic factors. Despite advances in the screening of donated blood for infectious agents, the risk of transfusion-transmitted viral, bacterial and protozoal infections, as well as newly emerging diseases, still persists. It has been recognised that no assay has a sensitivity of 100% or can absolutely eliminate the risk of infection with the screened agents (Cervia et al., 2007). Although several reports have outlined the benefit of leukoreduction in reducing the risk of CMV, EBV and HHV-8 transmission (as reviewed by Cervia et al., 2007), studies with respect to the incidence of blood-borne HHV-6 infection after leukoreduction are still lacking. This study has two significant limitations. The first resides in the fact that the PCR assays used are not sensitive enough to detect very low viral copy numbers, thus the prevalence of HHVs DNA may have been underestimated. In addition, due to financial constraints it was not possible to implement a second molecular assay for HHVs types 1–3 and 8. It is in our intentions to carry out the necessary additional experiments as soon as possible.
CONCLUSION In this study, we assessed the prevalence of HHVs DNA in Greek blood donors. Distribution of HHVs in blood donors of various origins is important as it is suggested that HHVs might be included in blood testing before transfusion especially for immunodeficient or immunocompromised patients. Given that, (i) HHVs recurrent infections show no apparent symptoms in most cases, (ii) a copies number cut-off value capable of distinguishing between HHVs latency and low grade reactivation events has not yet been determined, a discussion among transfusion medicine specialists about risk analysis for HHVs molecular and serology screening must be undertaken.
© 2015 British Blood Transfusion Society
Molecular epidemiology of human herpesviruses types 1–6 and 8 279
ACKNOWLEDGMENTS
CONFLICT OF INTEREST
K. D. designed the research; R. E. performed the research and wrote the manuscript. Both authors revised the paper critically and approved the submitted version.
The authors have no competing interests.
REFERENCES Cantin, E., Chen, J., Gaidulis, L., Valo, Z. & McLaughlin-Taylor, E. (1994) Detection of herpes simplex virus DNA sequences in human blood and bone marrow cells. Journal of Medical Virology, 42, 279–286. Cervia, J.C., Wenz, B. & Ortolano, G.A. (2007) Leukocyte reduction’s role in the attenuation of infection risks among transfusion recipients. Clinical Infectious Diseases, 45, 1008–1013. Compston, L.I., Sarkobie, F., Li, C., Candotti, D., Opare-Sem, O. & Allain, J.P. (2008) Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions. Journal of Virological Methods, 151, 47–54. Cone, R.W., Huang, M.L., Ashley, R. & Corey, L. (1993) Human herpesvirus 6 DNA in peripheral blood cells and saliva from immunocompetent individuals. Journal of Clinical Microbiology, 31, 1262–1267. Correa, R.M., Fellner, M.D., Alonio, L.V., Durand, K., Teyssie, A.R. & Picconi, M.A. (2004) Epstein-barr virus (EBV) in healthy carriers: distribution of genotypes and 30 bp deletion in latent membrane protein-1 (LMP-1) oncogene. Journal of Medical Virology, 73, 583–588. Furui, Y., Satake, M., Hoshi, Y., Uchida, S., Suzuki, K. & Tadokoro, K. (2013) Cytomegalovirus (CMV) seroprevalence in Japanese blood donors and high detection frequency of CMV DNA in elderly donors. Transfusion, 53, 2190–2197. Hassan, J., Dooley, S. & Hall, W. (2007) Immunological response to cytomegalovirus in congenitally infected neonates. Clinical and Experimental Immunology, 147, 465–471. Hudnall, S.D., Chen, T., Rady, P., Tyring, S. & Allison, P. (2003) Human herpesvirus 8 seroprevalence and viral load in healthy adult blood donors. Transfusion, 43, 85–90.
© 2015 British Blood Transfusion Society
Hudnall, S.D., Chen, T., Allison, P., Tyring, S.K. & Heath, A. (2008) Herpes virus prevalence and viral load in healthy blood donors by quantitative real-time polymerase chain reaction. Transfusion, 48, 1180–1187. Juhl, D., Mosel, C., Nawroth, F., Funke, A.M., Dadgar, S.M., Hagenstrom, H., Kirchner, H. & Hennig, H. (2010) Detection of herpes simplex virus DNA in plasma of patients with primary but not with recurrent infection: implications for transfusion medicine? Transfusion Medicine, 20, 38–47. Kozireva, S., Nemceva, G., Danilane, I., Pavlova, O., Blomberg, J. & Murovska, M. (2001) Prevalence of blood-borne viral infections (cytomegalovirus, human herpesvirus-6, human herpesvirus-7, human herpesvirus-8, human T-cell lymphotropic virus-I/II, human retrovirus-5) among blood donors in Latvia. Annals of Hematology, 80, 669–673. Lancini, D., Faddy, H.M., Flower, R. & Hogan, C. (2014) Cytomegalovirus disease in immunocompetent adults. The Medical Journal of Australia, 201, 578–580. Leong, H.N., Tuke, P.W., Tedder, R.S. et al. (2007) The prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of UK blood donors. Journal of Medical Virology, 79, 45–51. Luppi, M., Barozzi, P., Marasca, R., Ceccherini-Nelli, L., Ceccherelli, G. & Torelli, G. (1995) Human herpesvirus-6 (HHV-6) in blood donors. British Journal of Haematology, 89, 943–945. Nishiwaki, M., Fujimuro, M., Teishikata, Y. et al. (2006) Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay. Journal of Medical Virology, 78, 1635–1642. O’Riordan, D.P., Golden, W.C. & Aucott, S.W. (2006) Herpes simplex virus infections in preterm infants. Pediatrics, 118, 1612–1620.
Pamphilon, D.H., Rider, J.R., Barbara, J.A. & Williamson, L.M. (1999) Prevention of transfusion-transmitted cytomegalovirus infection. Transfusion Medicine, 9, 115–123. Petrisli, E., Chiereghin, A., Gabrielli, L. et al. (2010) Early and late virological monitoring of cytomegalovirus, Epstein-Barr virus and human herpes virus 6 infections in small bowel/multivisceral transplant recipients. Transplantation Proceedings, 42, 74–78. Piret, J. & Boivin, G. (2014) Antiviral drug resistance in herpesviruses other than cytomegalovirus. Reviews in Medical Virology, 24, 186–218. Qu, L., Jenkins, F. & Triulzi, D.J. (2010) Human herpesvirus 8 genomes and seroprevalence in United States blood donors. Transfusion, 50, 1050–1056. Roback, J.D., Drew, W.L., Laycock, M.E., Todd, D., Hillyer, C.D. & Busch, M.P. (2003) CMV DNA is rarely detected in healthy blood donors using validated PCR assays. Transfusion, 43, 314–321. Siakallis, G., Spandidos, D.A. & Sourvinos, G. (2009) Herpesviridae and novel inhibitors. Antiviral Therapy, 14, 1051–1064. Wilborn, F., Schmidt, C.A., Zimmermann, R., Brinkmann, V., Neipel, F. & Siegert, W. (1994) Detection of herpesvirus type 6 by polymerase chain reaction in blood donors: random tests and prospective longitudinal studies. British Journal of Haematology, 88, 187–192. Zavos, G., Gazouli, M., Papaconstantinou, I., Lucas, J.C., Zografidis, A., Kostakis, A. & Nasioulas, G. (2005) Prevalence of human herpesvirus 8 DNA sequences in human immunodeficiency virus-negative individuals without Kaposi’s sarcoma in Greece. In Vivo, 19, 729–732. Ziemann, M. & Hennig, H. (2014) Prevention of transfusion-transmitted cytomegalovirus infections: which is the optimal strategy? Transfusion Medicine and Hemotherapy, 41, 40–44.
Transfusion Medicine, 2015, 25, 276–279
Transfusion Medicine
| SHORT COMMUNICATION
Molecular epidemiology of human Herpesviruses types 1–6 and 8 among Greek blood donors E. Rouka & D. Kyriakou Transfusion Medicine Department, University Hospital of Larisa, Larisa, Greece Received 1 January 2015; accepted for publication 13 April 2015
SUMMARY Background: Human Herpesviruses (HHVs) maintain life-long latent persistence in the majority of the adult population including blood donors. The necessity for their study resides in the potential risk of transfusion-associated infection and the subsequent complications in the immunocompromised host. We aimed to assess the prevalence of HHVs types 1–6 and 8 among healthy blood donors of Thessaly prefecture in order to evaluate the frequency distribution of HHVs in Greek population and to ascertain possible correlations with demographic factors. Materials and Methods: Polymerase chain reaction (PCR) detection of HHVs DNA was determined in 401 randomly selected consecutive blood donors of Central Greece. Epidemiological data were recorded through a well structured questionnaire. Results: The overall PCR positivity for HHVs was 25·4%. HHVs types 1–3 were not detected in any donor sample. A specimen with high level of HHV-6 DNA (1 580 400 copies per mL) was recorded. HHV-4 DNA positivity was significantly associated with rural residency. Conclusion: HHV-4 DNA is commonly detected in whole blood specimens of healthy individuals. HHVs types 5, 6 and 8 are rarely detected. However, the existence of a donor sample with high HHV-6 viral load raises questions regarding the potential risk of HHV-6 blood-borne infection and the safety of blood products. Key words: blood-borne infections, human herpesviruses, molecular testing.
INTRODUCTION Human herpesviruses (HHVs) are double-stranded DNA enveloped viruses which belong to the Herpesviridae family
Correspondence: Erasmia Rouka, Transfusion Medicine Department, University Hospital of Larisa, Larisa, Greece. Tel.: +30 2413502255; fax: +30 2413502260; e-mail: [email protected]
First published online 4 May 2015 doi: 10.1111/tme.12202
and can be transmitted by the transfusion of blood products. Seropositivity for HHVs in adults ranges significantly depending on geographical and socioeconomic conditions (Pamphilon et al., 1999; Qu et al., 2010). Infected individuals maintain a life-long carriage of latent viruses in different cells and organs. Although leukodepletion has diminished the risk of transmission of leukotropic viruses, transfusion-associated morbidity and mortality remains a clinical concern particularly for patients with immature (e.g. preterm infants and neonates) or compromised [organ transplant recipients and acquired immune deficiency syndrome (AIDS) patients] immune systems (O’Riordan et al., 2006; Hassan et al., 2007; Siakallis et al., 2009). Despite the systemic administration of prophylactic antiviral treatment in immunosuppressed patients, the appearance of HHVs-drug resistant mutants has been constantly reported and has emerged as a significant problem especially in the transplantation setting (Piret & Boivin, 2014). As regards cytomegalovirus (CMV) infection in particular, it is documented that the incidence of severe CMV-related disease in immunocompetent adults is greater than previously thought, which may be partly due to immune dysfunction related to comorbidities such as kidney disease or diabetes mellitus (Lancini et al., 2014). In addition, although conflicting data exist with respect to the incidence of transfusion-transmitted CMV remaining after the introduction of leukodepletion, it has been clearly shown that both prevalence and concentration of CMV DNA in peripheral blood are highest in newly seropositive donors. Therefore, it is suggested that the avoidance of blood products from these donors is the most important goal of any transfusion strategy (as reviewed by Ziemann & Hennig, 2014). The aim of this study was to determine the prevalence of HHVs types 1–6 and 8, namely Herpes Simplex Virus (HSV types 1 and 2), Varicella Zoster Virus (VZV), Epstein–Barr Virus (EBV), Human Cytomegalovirus (HCMV), Human Herpes Virus 6 (HHV-6) and Human Herpes Virus 8 (HHV-8) among healthy blood donors of Thessaly prefecture in order to estimate the frequency distribution of HHVs in the Greek population and to reveal probable correlations with epidemiological markers.
© 2015 British Blood Transfusion Society
Molecular epidemiology of human herpesviruses types 1–6 and 8 277
MATERIALS AND METHODS Subjects and samples Four hundred one (401) healthy and asymptomatic blood donors, residents of Central Greece were included in the study after they had given their informed consent. At enrollment trained medical doctors obtained the questionnaire data which included demographic variables, medical and sexual history, occupational and marital status. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes and stored at 4 ∘ C until processing. The study was approved by the local hospital ethics scientific committee.
DNA extraction Nucleic acid extraction was performed on the automated Magtration system 12GC plus (Precision System Science, Co, Ltd, 88 Kamihongou, Matsudo-city, Chiba, 271-0064 JAPAN) with an extraction protocol based on magnetic particles technology for the isolation of DNA from 200 μL of whole blood into a final elution volume of 100 μL.
HHVs genome detection Presence of HHVs genome copies was evaluated using validated, single-virus quantitative real-time polymerase chain reaction (PCR) assays by Nanogen Advanced Diagnostics (Corso Torino, 89/d, 10090 Buttigliera Alta, Italy). These assays incorporate the TaqMan Minor Groove Binder technology as previously described (Petrisli et al., 2010). HHV-7 was not included in the study as the manufacturer did not have an available product at that time. Five microliters of DNA were used as a template. The primers and probes were specific for the exon 4 region of the CMV MIEA gene, the gene region coding EBNA-1 protein of EBV, the ORF 13 R region of HHV-6, the US6 region of HSV types 1 and 2, the ORF 29 region of VZV and for the gene region coding the capside protein of HHV-8. The promoter and 5′ -untranslated region (5′ UTR) region of the human beta globin gene were co-amplified in each reaction in order to validate extraction and detection. Tests were run on 96-well plates using a program of 50 ∘ C for 2 min, 95 ∘ C for 10 min, followed by 45 cycles of 95 ∘ C for 15 s and 60 ∘ C for 1 min on ABI 7300 Real Time PCR system (850 Lincoln Centre Drive, Foster City, CA, USA). For each virus specific standard curves were generated with four stabilised solutions of plasmid at known titre (105 –102 copies) containing part of the genome region targeted by the single quantitative real-time PCR reaction. Assays sensitivity is reported to be 10 copies per PCR which is equivalent to 1000 copies per mL of whole blood as the 200 μL sample was concentrated into a 100 μL eluate from which 5 μL were used as a template. Acceptance range of correlation coefficient (R2 ) was 0·990–1·000. Distilled water was used as negative control in all procedures. A second, multiplex PCR assay by Sacace Biotechnologies S.r.I (Via Scalabrini 44, Como, Italy) was applied for the detection
© 2015 British Blood Transfusion Society
and differentiation of CMV, EBV and HHV-6 targeting the MIE, LMP and pol gene of each virus, respectively. Human beta globin gene was used as endogenous internal control. Ten microliters of extracted DNA sample were used as a template. The amplification program for ABI 7300 Real Time PCR system was as follows: 95 ∘ C/15 min (1 cycle), [95 ∘ C/5 s, 60 ∘ C/20 s, 72 ∘ C/15 s] (5 cycles), [95 ∘ C/5 s, 60 ∘ C/30 s, 72 ∘ C/15 s] (40 cycles). Positive and negative controls were included in each reaction. The kit CMV/EBV/HHV6 Quant Real-TM is reported to have a sensitivity of five DNA copies per 105 cells (approximately 500 copies per mL).
Statistical analysis Possible associations of HHVs DNA detection with demographic variables were assessed by using odds ratios (ORs) and their associated 95% confidence intervals (CIs). Pearson 𝜒 2 test was used to compare percentages. Two-sided P values < 0·05 were considered to be statistically significant. The analyses were performed using the spss version 19·00 package (IBM SPSS Statistics) for windows.
RESULTS The main demographic features of the 401 subjects enrolled in this study are presented in Table 1. Beta globin gene sequences were amplified in all PCR procedures verifying the successful extraction of genomic DNA. All standards and negative controls gave the expected results. The two alternative commercial assays gave reproducible results. Among 401 individuals tested, 102 were HHVs DNA positive (25·4%). HSV-1,2 DNA and VZV DNA were not detected in any donor sample and CMV DNA was detected in a single specimen (0·2%). HHV-8 DNA was present in 2 of 401 samples (0·5%). In contrast, EBV DNA was detected in 85 of 401 samples (21·2%) while 14 of 401 blood specimens had detectable HHV-6 DNA (3·49%). Viral loads ranged from 2000 copies per mL for CMV and HHV-8 to 1 580 400 copies per mL for HHV-6. HHVs DNA detection was slightly lower among females (OR = 0·891). On the opposite, risk for HHVs DNA positivity tended to be higher among married individuals (OR = 1·068) and among village residents (OR = 1·398) while EBV DNA detection was correlated significantly with rural residency (OR = 1·471, P = 0·035).
DISCUSSION In this study, real-time PCR technology was used in order to detect HHVs DNA in whole blood of healthy Greek qualified donors. So far, little attention has been paid to the alpha-subfamily members of Herpesviridae due to their neuronal tropism. More than 20 years have passed since the study that reported the detection of HSV DNA sequences in human blood and bone marrow cells suggesting an additional site of latency (Cantin et al., 1994). More recently, it has been
Transfusion Medicine, 2015, 25, 276–279
278 E. Rouka and D. Kyriakou Table 1. The main demographic features of the donor population of this study Characteristics
Frequency
Sex Male Female Marital status Married Unmarried Residence Urban Rural Age (years) 18–28 29–39 40–50 51–61
401 325 76 396 227 169 397 291 106 391 76 157 120 38
Percent (%)
81 19 57 · 3 42 · 7 73 · 3 26 · 7 19 · 4 40 · 2 30 · 7 9·7
documented that HSV DNAemia is possible but seems to be limited to primary but not recurrent infection (Juhl et al., 2010). This study demonstrates that latent-alpha herpesvirus DNA is undetectable in peripheral blood of healthy and asyptomatic blood donors thus, the potential of blood-borne viral infection is uncommon for HHVs types 1–3. This conclusion is in accordance with previous findings (Hudnall et al., 2008) regarding the population of Texas southeast region. The same investigators identified CMV DNA in a single specimen and that seems to be the case in this study. Another study (Roback et al., 2003), using validated PCR assays also concluded that CMV DNA is rarely detected in healthy seropositive blood donors although stating that the minimal CMV load required for transfusion transmitted CMV has not been determined. Recently, CMV DNA was detected in the cellular fraction of 4·3% of samples from donors in their 60s and in 1·0% of samples from donors younger than 60 years (Furui et al., 2013). In this study, HHV-8 DNA was identified in only 2 of 401 blood specimens. Three previous investigations reported a zero HHV-8 DNA positivity among healthy blood donors (Hudnall et al., 2003; Hudnall et al., 2008; Qu et al., 2010). The current results suggest that the prevalence of HHV-8 DNA sequences in Greek blood donors is very low although previous data (Zavos et al., 2005) indicated an overall HHV-8 DNA positivity rate of 9·6% in human immunodeficiency virus (HIV) negative Greeks without Kaposi’s sarcoma. HHV-6 DNA was detected in 3·49% of the blood donor samples. Previous studies on HHV-6 DNA positivity reported significantly variable rates (Cone et al., 1993; Wilborn et al., 1994; Luppi et al., 1995; Kozireva et al., 2001; Hudnall et al., 2008). It appears that more studies with larger sample size are
Transfusion Medicine, 2015, 25, 276–279
needed in order to clarify whether the higher probabilities of HHV-6 DNA positivity are due to a higher prevalence of infection in some regions compared with others or due to technical inter laboratory factors. Regarding the proportion of samples with high HHV-6 viral load, previous studies have reported ratios of 4 : 500 (Leong et al., 2007) and 1 : 100 (Hudnall et al., 2008). This study demonstrates a proportion of 1 : 401. Undoubtedly, the identification of healthy and asyptomatic carriers with high HHV-6 DNA copies number, that could not be recognised and therefore could not be excluded during donor selection raises serious questions regarding the transfusion safety. EBV DNA was detected in 85 of 401 blood donor specimens. Previous studies have reported rates ranging from 8 to 76% (Correa et al., 2004; Nishiwaki et al., 2006; Compston et al., 2008; Hudnall et al., 2008). As in the case of HHV-6 this variability probably reflects differences in regional epidemiology. The association of EBV DNA detection with rural residency should be studied further in combination with other social and demographic factors. Despite advances in the screening of donated blood for infectious agents, the risk of transfusion-transmitted viral, bacterial and protozoal infections, as well as newly emerging diseases, still persists. It has been recognised that no assay has a sensitivity of 100% or can absolutely eliminate the risk of infection with the screened agents (Cervia et al., 2007). Although several reports have outlined the benefit of leukoreduction in reducing the risk of CMV, EBV and HHV-8 transmission (as reviewed by Cervia et al., 2007), studies with respect to the incidence of blood-borne HHV-6 infection after leukoreduction are still lacking. This study has two significant limitations. The first resides in the fact that the PCR assays used are not sensitive enough to detect very low viral copy numbers, thus the prevalence of HHVs DNA may have been underestimated. In addition, due to financial constraints it was not possible to implement a second molecular assay for HHVs types 1–3 and 8. It is in our intentions to carry out the necessary additional experiments as soon as possible.
CONCLUSION In this study, we assessed the prevalence of HHVs DNA in Greek blood donors. Distribution of HHVs in blood donors of various origins is important as it is suggested that HHVs might be included in blood testing before transfusion especially for immunodeficient or immunocompromised patients. Given that, (i) HHVs recurrent infections show no apparent symptoms in most cases, (ii) a copies number cut-off value capable of distinguishing between HHVs latency and low grade reactivation events has not yet been determined, a discussion among transfusion medicine specialists about risk analysis for HHVs molecular and serology screening must be undertaken.
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ACKNOWLEDGMENTS
CONFLICT OF INTEREST
K. D. designed the research; R. E. performed the research and wrote the manuscript. Both authors revised the paper critically and approved the submitted version.
The authors have no competing interests.
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