Effectiveness of Ganciclovir Against Human Herpesvirus-6 Excreted in Saliva in Stem Cell Transplant Recipients

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ORIGINAL ARTICLE Effectiveness of ganciclovir against human herpesvirus-6 excreted in saliva in stem cell transplant recipients

P Ljungman1, H Dahl2, Y-H Xu2, K Larsson1, M Brytting2 and A Linde2

1Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden and 2Swedish Institute for Infectious Disease Control, Karolinska Institutet, Stockholm, Sweden

The aim of this studywas to evaluate the effect of frequently detectable in saliva. We therefore wanted to ganciclovir on human herpesvirus-6 (HHV)-6. Forty evaluate the antiviral effectiveness by measuring the HHV- allogeneic stem cell transplant recipients were prospec- 6 viral load in saliva of SCT recipients receiving preemptive tivelystudied byrepeated sampling of the saliva. The ganciclovir therapy for CMV infections. saliva samples were assayed for HHV-6 by quantitative polymerase chain reaction. HHV-6 was detected in 33 patients. Ganciclovir was given as preemptive therapyfor cytomegalovirus infection during 15 episodes that were Patients and methods compared to 18 episodes without anyconcomitant antiviral therapy. The mean HHV-6 load decreased 0.49 Patients

(s.e. 0.31) log10/week in patients receiving ganciclovir Forty allogeneic SCT recipients were included in the study. whereas it increased 0.15 (s.e. 0.17) log10/week in episodes The transplant procedure has been described in detail without antiviral therapy( P ¼ 0.04). We conclude that elsewhere.11 Saliva samples were collected through mouth ganciclovir can decrease the HHV-6 viral load in saliva. rinses with water before, at 2, 4 and 12 weeks after SCT, Bone Marrow Transplantation (2007) 39, 497–499. before the start of antiviral therapy and weekly during the doi:10.1038/sj.bmt.1705617; published online 5 March 2007 therapy. The ethical committee at the Karolinska Institute Keywords: HHV-6; ganciclovir; stem cell transplantation approved the study. All participants gave written consent to participation after having received oral and written information about the procedures.

Introduction HHV-6 PCR assay Human herpesvirus-6 (HHV-6) is a b-herpesvirus closely DNA was extracted by a BioRobot M48 (QIAGEN- related to cytomegalovirus (CMV) and HHV-7. HHV-6 instruments AG). The viral load was determined by a exists in two subtypes that differ in 4–8% of the DNA. real-time polymerase chain reaction (PCR). The selected 0 0 Subtype B is the cause of exanthem subitum in childhood. primers (forward: 5 gaa tta tat gtt gat tgc tgt gct ttg 3 , 0 0 It is unclear what if any disease that is caused by the reverse: 5 ccg ccg ggt agc aca a 3 ) and minor groove 0 primary infection of subtype A. HHV-6 has a propensity binding probe (5 6-carboxy-fluorescein-tgt atc tga aaa gtt 0 for the CNS and several studies have shown that HHV-6 is aga gtt ggat 3 ) were amplifying the 10R gene and the an important cause of encephalitis in transplant recipi- sequences were selected with the primer express software ents.1–5 HHV-6 has further been associated with skin (ABI). For each well, 5 ml of the extracted sample was rashes, interstitial pneumonia, encephalitis, hepatitis and mixed with 12.5 ml Mastermix (2 Â , ABI) probe to give a bone marrow suppression after stem cell transplant final concentration of 300 nm, forward and reverse primers (SCT).3,6–10 to give final concentrations of 300 and 900 nm, respectively. There has been no controlled study of antiviral therapy Distilled water was added to a final volume of 25 ml. The in patients with HHV-6 infections. Salivary gland epithe- samples were analyzed in triplicates, including one inhibi- lium supports HHV-6 replication and infectious HHV-6 is tion control to which 0.5 ml positive control containing 15 viral copies was added to control for inhibitory substances in the samples giving a false-negative result. The viral load was calculated in comparison to an external standard in an Correspondence: Dr P Ljungman, Hematology Center, Karolinska ABI PRISM 7900H.12 The mean value of the duplicates University Hospital, Stockholm SE-14186, Sweden. E-mail: [email protected] was used in the calculations. One positive sample from each Received 7 November 2006; revised 9 January 2007; accepted 11 January of 18 patients was subtyped by qualitative type-specific 2007; published online 5 March 2007 PCR.13 Effectiveness of ganciclovir against HHV-6 P Ljungman et al 498 Preemptive antiviral therapy 5 Antiviral therapy was given against CMV as indicated based on a positive PCR for CMV DNA.14 Ganciclovir 4 5 mg/kg i.v. twice daily was the primary therapeutic option.

3 Definitions An episode of ‘HHV-6 excretion with ganciclovir therapy’ was defined as 14 days of antiviral therapy in a patient with 2 HHV-6 shedding before the initiation of ganciclovir therapy and during which at least two saliva samples were 1 collected. An ‘untreated episode’ of HHV-6 excretion was Log salivaLog viral load defined as at least a 2-week period, during which at least two saliva samples, of which at least one must be HHV-6 0 positive, were collected and no antiviral therapy was given. At least 2 weeks had to pass between the end of ganciclovir –1 treatment and the beginning of a new episode either treated Start End Start End or untreated. The change in the viral load was calculated by GCV No antiviral therapy subtracting the viral load of the last sample from the viral Figure 1 The mean viral load at the start and end of episodes of viral load in the first sample of the episode divided by the DNA detection in saliva. The boxes represent s.e. and the bars confidence number of weeks of observation. A viral load decrease was intervals of the mean. defined as at least a 50% reduction in the viral load during the episode. during treatment. When analyzing individual episodes, the Statistics saliva viral load decreased in 12/15 (80%) patients during Mann–Whitney U-test was used for comparing the change ganciclovir therapy. in viral loads between treated and non-treated episodes. Fisher’s exact test was used to compare the number of HHV-6 saliva levels in absence ofantiviral therapy episodes with HHV-6 viral load decrease. Eighteen untreated episodes of HHV-6 secretion in saliva were documented. The mean viral load at was 2.6 and 3.05

log10 copies/ml in the beginning and the end of the episode, Results respectively (Figure 1). The mean viral load increased with

0.15 (s.e. 0.17) log10/week during these episodes (P ¼ 0.04 Detection ofHHV-6 in saliva compared to ganciclovir-treated episodes). When analyzing Two hundred and fifty-nine saliva samples were collected individual episodes, the saliva viral load decreased in 7/18 from the 40patients. The median number of samples per (39%) episodes (P ¼ 0.03 compared to ganciclovir-treated patient was seven. Thirty-three of the 40patients had episodes). HHV-6 detected at least once in the saliva. A total of 41 The untreated episodes occurred slightly earlier (mean HHV-6 shedding episodes from 30of the patients were 21 days s.d.724.6) compared to the ganciclovir-treated identified. In the other three patients, a follow-up sample episodes (mean 41 days; s.d.727.5; P ¼ 0.07). after the HHV-6-positive sample was either not taken or was taken too long after the positive sample. Overall, 114 of the 259 (44.0%) samples were positive for HHV-6 DNA Discussion and the median number of positive samples per patient was two. The strains from 18 samples were subtyped. Sixteen Only limited data exist regarding the efficacy of antiviral strains were subtype B whereas two strains could not be therapy on HHV-6. We chose to study saliva for a ‘proof of typed. No symptoms attributable to HHV-6 were seen. concept’ study as HHV-6 excretion in saliva is very common and in order to get a large enough sample to Antiviral therapy allow comparisons between ganciclovir treated and non- Ganciclovir was given during 15 of the HHV-6 shedding treated patients. It could be argued that the sampling episodes, 18 episodes were untreated and during eight procedure was not standardized and that variations in the episodes another antiviral drug was given (foscarnet in procedure might explain the results. This is of course three episodes; cidofovir in two and valacyclovir in three possible but we tried to as far as possible standardize the episodes). These latter eight episodes were not further procedure using similar volumes of mouth rinsing fluid. analyzed owing to the low number for each antiviral drug. Furthermore, in our opinion, the same risk for variation in Fifteen ganciclovir treatment episodes from 13 patients the sampling procedures existed in patients treated with were analyzed. The mean viral load at the start of antiviral ganciclovir as in non-treated patients. We choose saliva as

therapy was 2.95 log10 copies/ml saliva and had decreased our study material as HHV-6 is more frequently found in

after therapy to a mean viral load of 1.86 log10 copies/ml saliva than in plasma and it would therefore give us a better saliva (Figure 1); a 12-fold decrease. The saliva viral load chance of an effect of antiviral therapy than if we would

decreased a mean of 0.49 (s.e. 0.31) log10 copies per week have used plasma or whole blood. It has also been shown

Bone Marrow Transplantation Effectiveness of ganciclovir against HHV-6 P Ljungman et al 499 previously that HHV-6 is detected in plasma early after with ganciclovir. Bone Marrow Transplantation 1997; 20: transplant at a time when CMV is more uncommonly 905–906. detected. We did in fact also analyze plasma and could only 5 Drobyski WR, Knox KK, Majewski D, Carrigan DR. Brief document two episodes timed so that the effect of GCV on report: fatal encephalitis due to variant B human herpesvirus-6 HHV-6 could have been studied. infection in a bone marrow-transplant recipient. N Engl J Med Our results show that preemptively given ganciclovir 1994; 330: 1356–1360. 6 Carrigan DR, Drobyski WR, Russler SK, Tapper MA, were able to reduce the saliva viral load in 80% of the Knox KK, Ash RC. Interstitial pneumonitis associated with episodes. Furthermore, the mean HHV-6 viral load human herpesvirus-6 infection after marrow transplantation. decreased in the ganciclovir-treated episodes whereas it Lancet 1991; 338: 147–149. increased in the non-treated episodes strongly supporting 7 Drobyski WR, Dunne WM, Burd EM, Knox KK, Ash RC, an antiviral effect of ganciclovir on HHV-6. Most of our Horowitz MM et al. Human herpesvirus-6 (HHV-6) infection HHV-6 strains were subtype B. In vitro studies have given in allogeneic bone marrow transplant recipients: evidence of a partly diverging results for the two subtypes. Long et al.15 marrow-suppressive role for HHV-6 in vivo. J Infect Dis 1993; showed a similar effectiveness of ganciclovir, cidofovir and 167: 735–739. foscarnet against the HHV-6 subtype A but poor efficacy of 8 Ljungman P, Wang FZ, Clark DA, Emery VC, Remberger M, Ringden O et al. High levels of human herpesvirus 6 DNA in foscarnet against subtype B. De Clercq et al.16 showed in peripheral blood leucocytes are correlated to platelet engraft- contrast similar efficacy of ganciclovir, foscarnet and ment and disease in allogeneic stem cell transplant patients. cidofovir on both HHV-6 subtypes. In vivo studies have Br J Haematol 2000; 111: 774–781. suggested efficacy of both ganciclovir and foscarnet in 9 Wang FZ, Linde A, Hagglund H, Testa M, Locasciulli A, treatment of HHV-6 encephalitis although these data come Ljungman P. Human herpesvirus 6 DNA in cerebrospinal from anecdotal reports and small patient series.9,17 fluid specimens from allogeneic bone marrow transplant Tokimasa et al.18 reported a lower risk for HHV-6 patients: does it have clinical significance? Clin Infect Dis reactivation among patients receiving ganciclovir prophy- 1999; 28: 562–568. laxis against CMV. Would other antiviral agents have been 10Carrigan DR, Knox KK. Bone marrow suppression by human similarly or more effective? We have data on a few episodes herpesvirus-6: comparison of the A and B variants of the virus Blood when foscarnet, cidofovir or valacyclovir was given but the (letter; comment). 1995; 86: 835–836. 11 Le Blanc K, Remberger M, Uzunel M, Mattsson J, Barkholt L, number of episodes is too low to allow any conclusions. Ringden O. A comparison of nonmyeloablative and reduced- Thus, ganciclovir may inhibit HHV-6 replication in the intensity conditioning for allogeneic stem-cell transplantation. salivary glands. The methodology described might be used Transplantation 2004; 78: 1014–1020. for further assessments of the efficacy of ganciclovir and 12 Enbom M, Strand A, Falk KI, Linde A. Detection of Epstein– that of other antiviral agents against HHV-6. Barr virus, but not human herpesvirus 8, DNA in cervical secretions from Swedish women by real-time polymerase chain reaction. Sex Transm Dis 2001; 28: 300–306. 13 Wang FZ, Dahl H, Linde A, Brytting M, Ehrnst A, Ljungman P. 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