Long-Lasting HHV-6 Reactivation in Long-Term Adult Survivors After Double Umbilical Cord Blood Allogeneic Stem Cell Transplantation

Long-Lasting HHV-6 Reactivation in Long-Term Adult Survivors After Double Umbilical Cord Blood Allogeneic Stem Cell Transplantation

JID Long-Lasting HHV-6 Reactivation in Long-Term Adult Survivors After Double Umbilical Cord Blood Allogeneic Stem Cell Transplantation Marina Illiaquer 2 Florent Malard 1 Thierry Guillaume 1 Berthe-Marie Imbert-Marcille 2 Jacques Delaunay 1 Amandine Le Bourgeois 1 Marie Rimbert 0 Celine Bressollette-Bodin 2 Cristina Precupanu 1 Sameh Ayari 1 Pierre Peterlin 1 Philippe Moreau 1 Mohamad Mohty 3 Patrice Chevallier () 1 0 Centre Hospitalier et Universitaire (CHU) de Nantes, Département d’Immunologie , Nantes 1 Centre Hospitalier et Universitaire (CHU) de Nantes, Hématologie Clinique, Centre d’Investigation Clinique en Cancérologie (CI2C) , Nantes 2 Centre Hospitalier et Universitaire (CHU) de Nantes, Département de Virologie, Nantes France and EA4271, Immunovirology and Genetic Polymorphism, University of Nantes 3 Hôpital Saint Antoine, Hématologie Clinique , Paris , France Higher incidence of human herpesvirus 6 (HHV-6) infection has been documented after umbilical cord blood allotransplant in adults. The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Long-term immune reconstitution does not explain this event, which remains to be explained. Serum samples taken up to 180 days after transplantation were examined with quantitative real-time PCR for measurement of viral load (CMV, HHV-6, and HHV-7). Of the remaining publications, only studies with (i) a study cohort of at least 15 HSCT patients with at least 1 being CB, (ii) a minimum of monthly surveillance for HHV-6 reactivation as an initial study parameter and (iii) independent data available for patients who received CB as the stem cell source vs non-CBT recipients were included in this review. Approximately 4% (four cases) of 89 infants with primary HHV-6 infection had signs of hepatic injury during viral infection. The titre of antibody against HHV-6 in group A (1:99) was significantly higher than in group B (1:15) (P=0.007).

Nevertheless, the clinical impact of HHV-6 reactivation remains debated [7]. HHV-6 reactivations occur within the first month post-transplant and may persist a few months after dUCB allo-SCT with little or no consequences [2]. To our knowledge, no data are known about the frequencies of HHV-6 reactivation far from transplantation and its clinical features. The aim of our study was to evaluate the long-term persistence of HHV-6 reactivation and its consequences for the patients by comparing frequencies of HHV-6 positive viremia and pattern of immune reconstitution between 2 groups of long-surviving patients who received either double UCB units or PBSC as stem cells source for allo-SCT. This prospective study included 48 adults with a median age of 54 years (range: 20–69) who received a dUCB (n = 23, males n = 10, median age, 54 years) or a PBSC (n = 25, males n = 12, median age: 53 years) allo-SCT between February 2006 and January 2012 at the CHU of Nantes. As inclusion criteria, all patients had to be alive at least 1 year after transplant. Also, all cases were required to be fit with no symptoms and in persistent complete remission at time of analyses.

1990, Wyatt & Frenkel 1992). The use of antithymocyte globulins (ATG) was significantly higher in the PBSC group: 93% of cases vs 10%, P < .0001. Samples were collected prospectively once (predominantly) or twice in each patient between October 2012 and June 2013. However, the use of random effects models should account for some of the between-study variation related to age and other demographic differences between study populations. All patients provided informed consent, and the study was approved by the Nantes University Hospital's institutional review board. Association with different infectious agents has been suggested, including: Epstein-Barr virus (EBV) (1–3), enterovirus (4) and more recently, human herpesvirus 6 (HHV-6) (5,6). HHV-6 DNA quantification was performed after total nucleic acids extraction from whole blood in recipients with a QIAsymphony instrument and the QIAsymphony DNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions and stored in a final volume of 100 µL at −20°C until further analysis. HHV-6 (U65-U66 gene) DNA quantifications were performed using in-house real-time polymerase chain reaction (PCR) procedures adapted from previously described protocols [2]. Viral loads were expressed as the number of viral DNA copies/mL of blood. Total lymphocytes counts (normal range: 1.5–4.0 Giga/L) were assessed for each patient by a standard numeration. The CD3+ T cells (normal range: 0.9–1.8 Giga/L), the CD4+ (normal range: 0.5–1.2 Giga/L), and CD8+ T (normal range: 0.3–0.7 Giga/L) cell subsets, the CD19+ B cells (normal range: 0.1–0.4 Giga/L), and the CD56+ natural killer (NK) lymphocytes (normal range: 0.1–0.4 Giga/L) were quantified using standard flow cytometry analysis. Absolute numbers of cells were calculated by multiplying the peripheral white blood cell count by the percentage of positive cells. The CD3+ T cells (normal range: 0.9–1.8 Giga/L), the CD4+ (normal range: 0.5–1.2 Giga/L), and CD8+ T (normal range: 0.3–0.7 Giga/L) cell subsets, the CD19+ B cells (normal range: 0.1– 0.4 Giga/L), and the CD56+ natural killer (NK) lymphocytes (normal range: 0.1–0.4 Giga/L) were quantified using standard flow cytometry analysis. (2003). For continuous data, comparison between regimens was performed using the Mann-Whitney test for patient age, median year of transplant, median time between the graft and the time of analyses, median HHV6 viral load, and median lymphocytes counts. A P value of < .05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Half of the patients (52%, n = 12) showed a long-lasting HHV-6 positive viremia in the dUCB group (median viral load: 2.7 log10copies/mL (range: 2–3.4) compared to only 4% of the patients (n = 1, viral load: 2 log10copies/mL) in the PBSC group (P < .0001). The sera were inactivated at 56°C for 30 mins and serial twofold dilutions were made from an initial concentration of 1:10. However, among 19 cases with HHV-6 reactivation within the first year post-transplant in the UCB group, 12 still showed HHV-6 reactivation far from transplant, whereas 7 were documented with negative HHV-6 PCR. Three patients in this same group had persistent negative HHV-6 PCR. In the PBSC group, 14 patients remained with negative HHV-6 PCR, whereas 1 positive PCR case became negative at a distance. Except a significant higher long-lasting B lymphocytes recovery in the dUCB group (median: 0.74 vs 0.49 Giga/L, P = .03), there were no significant differences regarding long-term immune reconstitution between both groups, where total lymphocytes, CD3+, CD4+, and CD8+ T cells, NK cells counts, and gammaglobulin rates were in the normal ranges (Figure 1). In the dUCB group, patients with higher B lymphocytes count (>median: 0.74 Giga/L) showed no higher incidence of HHV6 reactivation (56% vs 67%, P = 1). Furthermore, no statistically significant correlation was observed between B lymphocyte counts and HHV6 reactivation. Double UCB allo-SCT has been extensively used in recent years in adults lacking suitable related or unrelated donors, showing significantly higher incidence of HHV-6 reactivation compared to transplants using other sources of graft, mainly PBSC [ 2 ]. Healthy adults can shed virus intermittently, behaving as the epidemiological source of infection among human populations. σ Data are missing for 5 patients in the UCB group.

Long-Lasting HHV-6 Reactivation in Long-Term Adult Survivors After Double Umbilical Cord Blood Allogeneic Stem Cell Transplantation
*P < .05. Abbreviations: PBSC, peripheral blood stem cells; UCB, umbilical cord blood; WB, whole blood. This is the first study to our knowledge evaluating the long persistence of HHV-6 reactivation after dUCB allo-SCT. All patients in groups A and B fulfilled the two major CDC criteria for CFS. Here we were able to demonstrate that HHV-6 positive viremia persists in half of the cases receiving a dUCB allo-SCT up to a median of 4 years post-transplant. As expected, this reactivation seems to have no clinical impact. Even if it was part of inclusion criteria, absence of symptoms in patients reactivating HHV-6 may be explained by the low level of HHV-6 detection here. More surprisingly, this phenomenon was not explained by the particular immune status of patients, namely, a higher B lymphocyte count far after transplant. The reason for such an event remains unexplained. Indeed, increased risk of infections is generally limited to the first 3 months after UCB allo-SCT [8], whereas B-cell deficiency has been described as the main factor for late infection occurrence [9]. Interestingly, we also were able to evaluate and compare for the first time the very long-term immune status of patients after dUCB allo-SCT vs PBSC allo-SCT. After stratifying by age (10-year interval), no significant differences were found in prevalence rates (p > 0.05). However, weaker immune status should also be associated with other detectable complications, but it was not the case here. It remains that cells originated from UCB graft; probably (especially) CD4+ T cells, the main targets of the virus, have particular long-term susceptibility to HHV-6.

Recently, CD134, a member of the tumor necrosis factor (TNF) receptor superfamily, has been identified as a specific receptor for HHV-6B [10]. CD134 expression in particular should be studied in patients not only early but also later after UCB allo-SCT in comparison to cases receiving PBSC transplants in order to demonstrate whether higher antigen detection exists for the former. Unfortunately, the minor criteria are numerous and nonspecific. Indeed, such results are generally provided within the first year post-transplant, and only a few series have explored the long-term immune status after UCB allo-SCT but only up to 2–3 years post-transplant [8, 11]. It is known that early faster B and NK cells reconstitutions and delayed T-cell reconstitution is the hallmark of patients within the first year after UCB allo-SCT [2, 8, 12]. These differences can persist thereafter, especially higher B-cell counts [8]. The reason for the persistence of a higher B-cell count far from UCB transplant also remains unexplained but could be the key to long-term survival as all of our patients were fit without any medication at time of analyses [9].

In conclusion, HHV-6 reactivation persists for a very long time in half of the patients after dUCB allo-SCT but is not clinically relevant. Long-term immune reconstitution does not explain this event. designed, performed, and coordinated the research, collected, contributed, analyzed and interpreted the data, and wrote the manuscript. 2002). M. I. and P.

C. designed, performed, and coordinated the research, collected, contributed, analyzed and interpreted the data, and wrote the manuscript. F. M. performed the statistical analyses and critically revised the manuscript. T. G., B.

M. B. In our study, the chosen clinical specimen was the whole saliva: a non-invasive sample that is easy to collect. D., A. L. B., M. R., C.

B. B., C. P., S. A., P. P., P. M., and M. M.

o x f o r d j o u r n a l s . Although diagnosis is best made by comparing antibody titers of paired sera and demonstrating seroconversion to low avidity antibodies, it has been proposed that the detection of HHV-6B DNA in saliva by PCR would be adequate to diagnose early primary infections as well as for epidemiological studies. Accepted February 5, 2014. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oup.com.

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