Human Herpesvirus 6: Infection and Disease Following Autologous

Human Herpesvirus 6: Infection and Disease Following Autologous and Allogeneic Bone Marrow Transplantation

By Madhavi P. Kadakia, Witold B. Rybka, John A. Stewart, Joanne L. Patton, Felicia R. Starney, Magdy Elsawy, Philip E. Pellett, and John A. Armstrong

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P = .005). The proportion of cultures positive increased after

UMAN HERPESVIRUS 6 (HHV-6) was first isolated from lymphocytes of patients with acquired immuno-

deficiency syndrome (AIDS) and lymphoproliferative disorders.’ It was later isolated from other patients with AIDSz4 and has been established as the principal causative agent of exanthem subitum (roseola infan t~m) .~ .~

Variation in restriction fragment profiles, growth properties, and reactivity to monoclonal antibodies (MoAbs) led to the recognition of two distinct yet closely related HHV-6 variants, A (eg, strains U1102 and GS) and B (eg, strain Z29).6*9“1

HHV-6 shares several molecular and biologic properties with human cytomegalovirus (HCMV), which is known to cause morbidity and mortality after transplantation, particularly bone marrow transplantation (BMT). HHV-6 has been associated with febrile episodes in renal transplantation patients.12 In BMT recipients, HHV-6 has been reported to be associated with the development of skin rash, fever, interstitial pneumonitis, and either the delay or suppression of marrow engraftment.I3-”

We describe here a prospective study of the frequency of active HHV-6 infection in BMT recipients and possible associations with disease.

HHV-6 infection was studied using four different methods: detection of HHV-6 DNA by polymerase chain reaction (PCR) from uncultured peripheral blood mononuclear cells (PBMC), virus isolation from PBMC by coculture with cord blood lymphocytes (confirmation by PCR and immunofluorescence [IFA]), serologic increases to HHV-6, and salivary shedding of HHV-6 DNA. The clinical events studied included fever or rash both separately and graft- versus-host disease (GVHD), pneumonia, adult respiratory distress syndrome (ARDS), sinusitis:’ central nervous system (CNS) involvement:’ marrow suppression, and delay in engraftment. l6

H

MATERIALS AND METHODS

Patients ’ Characteristics

All subjects scheduled to undergo BMT at the Pittsburgh Cancer Institute during the study period and their donors were informed of the study. The study protocol and the consent forms were approved

Blood, Vol 87, No 12 (June 15). 1996: pp 5341-5354

transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .W). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation ( P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event. This is 8 US government work. There 8re no restrictions On its use.

by the Biomedical Institutional Review Board of the University of Pittsburgh. All potential candidates consented to be in the study.

Patient characteristics as regards to age, sex, diagnosis, conditioning regimen, type of transplant, duration of follow-up, and the final outcome of the patient are summarized in Table 1. Fifteen of the 26 patients underwent allogeneic BMT and the remaining 11 underwent autologous BMT. Eight of the 15 allogeneic transplant patients received marrow from matched related donors (MRD) and the remaining 7 received marrow from matched unrelated donors (MUD). Six of the 11 autologous transplant patients received BM alone and the remaining 5 received peripheral blood stem cells (PBSC) alone.

Patients were enrolled sequentially in the study as they were ad- mitted to hospital for transplantation to minimize selection bias. The enrollment of patients occurred in two phases. The pilot phase included the first 7 patients enrolled over 3 months. The second phase consisted of 19 additional patients enrolled over 4 months.

Treatment Regimens The pretransplant conditioning regimen (Table 1) consisted of one

of the following regimens: ( l ) cyclophosphamide (Cy; 120 mgkg) and total body irradiation (TBI; 1,320 cGy); (2) etoposide (VP16; 60 mgkg) and TB1 (1,320 cGy); (3) Cy (120 mgkg) and busulfan (Bu; 16 mgkg); (4) VP16 (1,600 mg/m2), Cy (100 mgkg), and carboplatin (Cb; 1,500 mg/mz); and (5) Cy (6,000 mg/m’), thiopeta (500 mg/m’), and Cb (800 mg/m’). Allogeneic and autologous BMT recipients received 3 X lo8 nucleated marrow cells per kilogram of recipient body weight. PBSC transplant recipients received 7 X lo8 mononuclear cells per kilogram of body GVHD prophy-

From the Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pitts- burgh, PA; the Adult Bone Marrow Transplant Program, Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pitts- burgh, PA; and the Centers for Disease Control and Prevention, Atlanta, GA.

Submitted May 8, 1995; accepted January 9, 1996. Address reprint requests to Madhavi P, Kadakia, PhD, Depart-

ment of Surgery, E1512 Biomedical Science Tower, Desoto and Lothrop Street, University of Pittsburgh, Pittsburgh, PA 15261.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “adveaisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

This is a US govemment work. There are no restrictiom on its use. 0006-4971/96/8712-0005$0.00/0

5341

For personal use only.on April 13, 2019. by guest www.bloodjournal.orgFrom

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5342 KADAKIA ET AL

Table 1. Patient Characteristics

Patient Type of Pretransplantation Duration of Outcome No. Age (yr)/Sex Diagnosis Transplantation Regimen Follow-Up (day from transplant) Cause of Death

1 52/F AML Allo, MRD CY, Bu 90 Dead (174) Invasive pulmonary aspergillosis 2 52lM AML Auto, BM Cy, Bu 324 Alive - 3 32/F N H L Auto, BM Cy, TB1 68 Dead (82) CMV pneumonitis 4 34/F N H L Auto, PBSC Cy, TB1 175 Dead (199) Relapse 5 22/M ALL Allo, MUD Cy, TB1 56 Dead (60) Septic shock and ARDS

7 35/F AML Allo, MUD CY, Bu 91 Dead (102) a 39/F CML Allo, MRD CY, Bu 224 Dead (397) Relapse 9 60/F oc Auto, BM VP16, Cy, Cb 324 Alive

10 55/F CML Allo, MRD CY. Bu 151 Dead (157) Invasive aspergillosis 11 36lF ALL Allo, MRD CY. Bu 327 Alive - 12 37lF BC Auto, PBSC VP16, Cy, Cb 328 Alive

6 34lM AML Allo, MRD CY, Bu 153 Alive - Disseminated toxoplasmosis

-

13 51/M CML Allo, M U D CY. Bu 50 Dead (50) Sepsis and ARDS 14 4OlF BC Auto, PBSC VP16, Cy, Cb 58 Dead (259) Relapse 15 48/M CML Allo, M U D CY, Bu 202 Alive 16 34/F BC Auto, BM VP16, Cy, Cb 91 Dead (201) Relapse

-

-

17 27lF CML Allo, M U D CY. Bu 68 Dead (88) Sepsis 18 33lF BC Auto, BM VP16, Cy, Cb 30 1 Alive 19 41/M AML Allo, MRD VP16, TB1 16 Dead (16) ARDS 20 39/F CML Allo, MRD CY. Bu 62 Dead (87) Polymicrobial septic shock 21 41lM CML Allo, M U D Cy, Bu 54 Dead (78) Multiple organ failure 22 48lF N H L Auto, PBSC Cy, TB1 275 Dead (330) Relapse

-

23 50lM AML Allo, M U D CY. Bu 29 Dead (71) Septicemia 24 37lF N H L Auto, BM Cy, Bu 144 Alive 25 24lM ALL Allo, MRD CY, Bu 90 Dead (228) Relapse 26 36/F BC Auto, PBSC Cy, T, Cb 260 Alive -

-

Abbreviations: Auto, autologous transplantation; Allo, allogeneic transplantation; AML, acute myelogeneous leukemia; CML, chronic myelogeneous leukemia; NHL, non-Hodgkin‘s lymphoma; ALL, acute lymphocytic leukemia; OC, ovarian carcinoma; BC, breast carcinoma; TBI, total body irradiation; VP16, etoposide; Cy, cyclophosphamide; Bu, busulfan; T, thiopeta; Cb, carboplatin.

laxis for allogeneic patients consisted of cyclosporine (1 year) and corticosteroids (6 months).z5

Experimental Design

Pretransplantation heparinized blood was obtained from allogeneic and autologous donors. In addition, samples of harvested BM used for transplantation were obtained for patients receiving BM and samples of the harvested PBSC used for transplantation were obtained from patients receiving PBSC. Heparinized blood was obtained from all the recipients twice before transplantation as baseline, weekly for 1 month, and monthly for up to 3 months. In addition, a late follow-up sample was obtained between 6 months and 1 year after transplantation when possible. In the pilot study (patients no. 1 through 7). urine and saliva were studied before transplantation from both the recipients and their donors. In the study phase (patients no. 8 through 26), no urine specimens were studied, but saliva specimens were collected before transplantation from donors and from recipients at all the times when blood was collected from recipients. When possible, bronchoalveolar lavage (BAL) specimens were obtained from recipients on whom BAL was performed when they developed pneumonia. Skin biopsy specimens were obtained if a patient developed skin rash. Clinical data were collected prospectively on all 26 recipients, summarized at the end of the study, and assessed to determine associations with active HHV-6 infection.

Collection and Storage of the Specimens Heparinized blood was collected and stored on a rocker at room

temperature for less than 24 hours. Cells saved for PCR were frozen

as cell pellets at -70°C until lysed for analysis. Plasma specimens were aliquoted and then stored at -70°C until diluted for enzyme immunoassay. Saliva specimens were collected in cups after the patient chewed paraffin wax and were stored at 4°C for less than 24 hours before being aliquoted and then stored at -70°C until being lysed for PCR analysis. BAL and BM specimens were collected as soon as they were available and were processed similarly to blood specimens. Skin biopsies were stored at -70°C until lysed for analysis.

Cells and Viruses The human T-cell line JJhan and phytohaemagglutinin (PHA)-

stimulated cord blood lymphocytes (CBL) were used for the routine propagation of laboratory strains HHV-6A(U1102)’ and HHV- 6B(Z29)? respectively. JJhan cells and HHV-6A(UllO2) were a gift from J. Black (Centers for Disease Control and Prevention, Atlanta, GA). Cord blood used for routine propagation of laboratory strains and for virus isolation studies was obtained from healthy placentas after delivery at Magee Women’s Hospital (Pittsburgh, PA).

Virus Isolation Virus isolation was attempted only from mononuclear cells ob-

tained from marrow or blood. Procedures for the isolation and identification of HHV-6 were as described elsewhere?6 Cultures were monitored weekly for growth of HHV-6 by observing the cytopathic effect. The medium was changed and fresh PHA-stimulated cord blood mononuclear cells were added weekly for up to 3 weeks. After 4 weeks, the cultures were frozen in liquid nitrogen. Virus isolation




HHV-6 IN BONE MARROW TRANSPLANTATION 5343

was confirmed when cultured material from the recipient was positive by PCR for HHV-6 DNA or HHV-6-specific proteins by immunofluorescence.

Immunofluorescence Assay Cells from the cultures at 2 and 4 weeks were monitored for the

presence of HHV-6 by staining with p 4 1 (9A5D12) MoAb (B0145; Universal Biotechnology, Rockville, MD), which detects an HHV- 6 early protein using an indirect immunofluorescence method.” When cells that had been reacted with HHV-6 specific MoAb exhibited dense nuclear staining and the normal mouse serum controls showed no staining, the culture was considered positive for presence of HHV-6 antigen. Mock-infected and HHV-6B(Z29)-infected CBL were tested with each batch of slides.

PCR Ampl$cation Approximately IO6 mononuclear cells obtained from blood, BM,

or BAL were used for hot-start PCR amplification from primary (direct PCR) and cultured material (weeks 2 and 4 of culture; cultured PCR). Cells were digested in 100 pL lysis buffer (10 mmol/ L Tris, pH 8.5, 1% 10-lauryl ether with 200 pg/mL proteinase K) at 65°C for 2 hours, followed by incubation at 98°C for 10 minutes to destroy the proteinase. Twenty-microliter (2 X lo5 cells equivalent) and 2-pL (2 X lo4 cells equivalent) aliquots of the cell lysate were used for PCR coamplification of HHV-6 and human P-globin DNA using two different sets of primers in the same reaction tube. HHV-6 2A and 2B primers were specific for HHV-6 DNA and KM38 and PC03 primers were specific for P-globin (internal con- tr01).~~ Reactions consisted of 50 mmol/L KCI; 10 mmol/L Tris, pH 8.5; 1.5 mmol/L MgCI,; 0.01% gelatin (patients no. 1 through 7 only; gelatin was eliminated in the remaining patients because it was shown to interfere with PCR reactions); 200 pmoVL each of dATP, dTTP, dCTP, and dGTP (Pharmacia, Piscataway, NJ); 2.5 U of Taq DNA polymerase (Amplitaq; Perkin Elmer, Norwalk, CT); and 1.0 pmol/L of each primer. The reaction mix was incubated at 80°C for 10 minutes before the cell lysate was added to prevent nonspecific amplification. Thermocycling conditions consisted of incubation at 80°C for 10 minutes, followed by 35 amplification cycles, each consisting of denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 1 minute. This was then followed by an extension step at 72°C for 7 minutes. Specific amplification products were 187 bp and 167 bp for HHV-6 and P- globin, respectively. Amplified products were electrophoresed in composite agarose gels (3% Nusieve GTG agarose and 1% SeaKem GTG agarose; FMC, Rockland, MD) for 3 hours at 5 V/cm (electrode to electrode) in 1 X tris borate EDTA buffer (TBE). Sensitivity controls consisting of lo4, lo3, IO’, 10, and 1 infected cells diluted in a constant number (1 X lo6 cells) of uninfected human lung fibroblast cells were amplified with each set of PCR reactions. Thus, the DNA and P-globin amplified product would be constant in all the sensitivity controls.

Saliva specimens (150 pL neat saliva) and urine specimens (pellet from 20 mL of urine centrifuged at 75,OOOg for 2 hours) were resuspended in 150 pL and 300 pL of lysis buffer, respectively, and digested as described above. Skin biopsies were teased into very small pieces using a scalpel and digested in 300 pL of lysis buffer, as described above.

Southern Blot Hybridization Gels were blotted bidirectionally onto two Nytran membranes

(Schleicher and Schuell, Keene, NH) and probed with 5’ end-labeled (Kinace It; Stratagene, La Jolla, CA) oligonucleotide probes (19A for P-globin-specific product and HHV-62P and HHV-62P2 for

HHV-6-specific product). HHV-62P reacts with HHV-6 variant B, whereas HHV-62P2 reacts with both HHV-6 variants. After overnight hybridization at 55°C. the membranes were washed 3 times for 10 minutes each at room temperature with 2 X SSC, 0.1 % sodium dodecyl sulfate (SDS) and twice for 1 hour at 55°C. Intensifying screens were used during autoradiography.

Primers and Probes HHV-6 primers and probe sequences were derived from a segment

of DNA sequence that maps near the left end of the unique segment (gene V,) of HHV-6B(Z29) genome (Dambaugh et al, unpublished data). No cross-reactivity was seen with herpes simplex viruses 1 and 2, varicella zoster virus, Epstein-Barr virus, HCMV, and human herpesvirus 7. HHV-62A. 5’-GGGCAAATGCCGCCTTTCCA- TAG-3‘; HHV-62B. 5”GGATCTATCTGGGTAAGAAAGA-3’; HHV-62P, 5”CCGAATTCGATCCAGCTGATCTC-3’; and HHV- 62P2, 5‘-GCGTCGAGGTGCAGCTGGCCTA-3’. Sequences of the primers Aubin A and C were as described?

Variant Analysis PCR amplification and restriction fragment-length polymorphism

(RFLP) of amplt$ed product for variant analysis of the isolates. The cultures that were positive by PCR with primer set HHV-62A, 2B were subjected to PCR with Aubin primers A and C. The reaction mixture was similar to that used with HHV-62A and 2B primer set, but the thermocycling conditions were as described.’ The 830-bp amplified product was subjected to restriction digestion with HindIII and HinfI enzymes that distinguish variants A and B based on the resulting profiles?

MoAb-based analysis. Cultures that were positive by IFA with MoAb 9A5D12 were then tested with HHV-6B-specific MoAb C3108-103 (diluted 1:500)29~30 against the virion protein, 101K. Cul- tures were also tested with 1:200 diluted MoAb against gp102 (B0151; Universal Biotechnology) specific for variants A and B of HHV-6 and gp 82 (B0150; Universal Biotechnology) specific for variant A of HHV-6.”

Restriction Length Polymorphism Analysis of the HHV-6 Isolates

Whole cell DNA was prepared from approximately 10 to 15 X

lo6 cells.3’ Four restriction endonucleases (BamHI, Sal I, Nci I, and Pst I) were used for the analysis. Digests were electrophoresed in 0.8% gels (SeaKem GTG Agarose) overnight at 1 V/cm and trans- ferred to nitrocellulose membranes (Schleicher and Schuell). Blots were hybridized with nick-translated gel-purified HHV-6 whole viral DNA, as previously described.”

Serology by Enzyme Immunoassay (EIA) HHV-6B(Z29)-infected CBL at a concentration of 2 X IO6 cells/

mL were washed, freeze-thawed, and sonicated. Aliquots of 50 pL from this suspension were added to one-half of the wells of a 96- well flat-bottom microtiter plate and air-dried. Uninfected CBL were prepared in an identical manner and added as controls to the other half of the plate. The plates were fixed with 10% formalin for 10 minutes at room temperature, washed with phosphate-buffered saline (PBS), and then incubated 30 minutes at room temperature with PBS-bovine serum albumin (PBS-BSA) to block nonreacted sites. Plasma specimens were diluted 1:100 in serum diluent (PBS, pH 7.4, 1% BSA, 0.05% Tween-20, and 2% [vol/vol] of a normal cell culture antigen prepared from the HEL strain of human lung fibro- blasts), added to two infected and two uninfected wells, and incubated for 1 hour in a 37°C water bath followed by an incubation with affinity-purified antibody phosphatase-labeled goat antihuman




5344 KADAKIA ET AL

Table 2. Scoring tho Association of Active HHV-6 Infection With Clinical Events

Propinquity of Active HHV-6 Infection ~ ~ ~~ ~~~~ ~~

to Clinical Event Other Causes” Score

Within 2 wk

2-4 wk >4 wk

No Probable (3) Yes Possible (2) Yes or no Possible (2) Yes or no Improbable (1)

++ Other infections or disease conditions.

IgG (7 ) (Kirkegaard & Perry Laboratories, Inc, Gaithersburg, MD). After washing, bound antigen-antibody complex was detected with alkaline phosphatase substrate. Optical density (OD) was read at 405 nm with a microplate reader. The net absorbance value for each serum sample was determined by subtracting the OD of the control wells from the OD of the viral antigen wells. The final EIA value for each sample was determined by a calibration procedure using four standardized control serum samples on each plate and standard linear regression analysis. The EIA value is proportional to the amount of antibody in the test plasma. A cut-off value of 0.080 was used to determine positivity.’* Plasma specimens were collected at two time points before transplantation to perform serologic analyses (baseline titers). A change in titer was considered significant if there was a 1.6-fold increase or decrease in the titer as measured by EIA OD value, from baseline titer determined at day - 1 (or day 0, before the transplant).

Criteria Established to Determine Association of Active HHV-6 Infection With Disease Events and Activation of Other Herpesviruses

The following terms were used to define the relationship between the virus and the host. All patients were seropositive to HHV-6 before BMT and were considered infected with HHV-6. Isolation of HHV-6 from blood, detection of HHV-6 DNA in the saliva, or a significant increase in HHV-6-specific antibody titers were considered as evidence of “active” HHV-6 infection. HHV-6 disease was defined as the presence of signs or symptoms attributed to active HHV-6 infection.

The clinical events examined for their relationship to active HHV- 6 infection were fever, skin rash, GVHD, pneumonia, ARDS, engraftment, marrow suppression, CNS involvement, sinusitis, oral mucositis, gastroenteritis, bacterial infection, and reactivation of other herpesvirus infections: herpes simplex virus (HSV), varicella zoster virus (VZV), and HCMV.

Associations were established between clinical events and active HHV-6 infection occurring at any time point during the study period using Fisher’s exact test (two-tailed, for 2 X 2 contigency tables). In addition, a scoring system was devised to determine whether clinically significant disease was likely to be caused by active HHV- 6 infection. All clinical events were scored as 1 “improbable”, 2 “possible”, or 3 “probable” based on the temporal association of the clinical event with evidence of active HHV-6 infection and the presence of other identifiable causes. The scoring criteria are summarized in Table 2.

Engraftment was defined as recovery of hematopoiesis in each of the myeloid cell lines: neutrophils, platelets, and red blood cells (RBC). The criterion for neutrophil engraftment was an absolute neutrophil count (ANC) 2 500/pL for 2 consecutive days within the study period, counting the first day as the day of engraftment. The criterion for platelet engraftment was a platelet count 220,000/ pL for 2 consecutive days independent of transfusion within the study period, counting the first day as the day of engraftment. The

criterion of RBC engraftment was an RBC count 2 3 X 10”/pL for 2 consecutive weeks independent of transfusion within the study period, counting the first day as the day of engraftment.

Patients were evaluable for assessment of marrow suppression if they fulfilled the criteria for engraftment and in addition exceeded an ANC of I,OOO/pL for 2 consecutive measurements, a platelet count of 50,0OO/pL for 2 consecutive measurements, and an RBC count of 3 X 106/pL for 2 consecutive weeks (the latter two being achieved independent of transfusion). Marrow suppression was defined as achieving the following criteria in evaluable patients: a decrease in ANC to less than 1,OOO/pL, platelets to less than 50,000/ pL, or RBC to less than 3 X 106/pL for 2 consecutive days without evidence of bleeding or hemolysis.

A significant increase in the HCMV titer (defined by a 1.6-fold increase in EIA optical density from baseline), seroconversion, or culture of HCMV from urine, throat, blood, BAL, lung biopsy, or biopsies of other diseased organs after transplantation were considered as evidence of active HCMV infection. Cultures for HCMV were performed only if HCMV disease was suspected in a patient.

Statistical Analysis Statistical analyses, including Fisher’s exact test (two-tailed) and

the Pearson x* test for contigency tables, linear regression analysis, Kaplan-Meier survival analysis, and the Log Rank test, were performed using the STATA program (STATA COT, College Station, TX).

RESULTS

Patient Population

Twenty-six patients were observed for a median of 118 days (range, 16 to 328 days). The median follow-up periods in allogeneic and autologous transplantation patients were 90 days (range, 19 to 327 days) and 260 days (range, 58 to 328 days), respectively. The mortality rate was higher in the allogeneic as compared with the autologous transplant patients (P = .03; Fig 1). The median survival time was 102

............................ .............................................

L 0.25

0 30 60 90 120 150 180

DAYS FROM TRANSPLANT

Fig 1. Kaplan-Maier survival curves in patients who had under- gona allogenaic and autologous transplantation. P h repre-nt alto- geneic (-) and autologous (. * .) transplantation m-- tively. Significance was determined by l o g rank taat (P = .W).





Table 3. A Summary of Active HHV-6 Infections in the Study Population

Posttransplantation Patient Type of HHV-6 Isolation HHV-6 Serologic Active No. Transplantation From Blood HHV-6 DNA in Saliva Increase Infection

1 Allo, MRD + (90) - 6 Allo, MRD + (48, 153) - 8 Allo, MRD - + (0, 7, 122, 171)

10 Allo, MRD + (132) + (6, 14, 20, 27, 62, 83, - +

11 Allo, MRD - + (64) 19 Allo, MRD - + (-7, -1) + (6) + 20 Allo, MRD -

- + + +

-

-

109, 132, 140) - +

- - - 25 Allo, MRD - + (-1, 13, 20, 24, 62, 90) + (13) +

+ 5 Allo, MUD - + (-6) 7 Allo, MUD + (24, 27) - + (27) +

13 Allo, MUD + (50) + (7, 14, 21, 29, 50) - + 15 Allo, MUD - + (0) + (20) + 17 Allo, MUD + (68) + (-8, -2, 12, 20, 27, 68) - + 21 Allo, MUD - + (-8, 21, 28, 54) - + 23 Allo, MUD - - - -

3 Auto, BM + (21) + (-7) + (21) + 9 Auto, BM + (15, 324) + (-7, -2, 15, 26) - +

16 Auto, BM - + (50, 91) - + 18 Auto, BM - + (-7, 13, 41) - +

-

24 Auto, BM + (28) + (28, 92) - + 2 Auto, PBSC + (198) - - + 4 Auto, PBSC - - -

12 Auto, PBSC + (13) + (38) + 14 Auto, PBSC - - + (30) + 22 Auto, PBSC + (20, 65) + (13) - + 26 Auto, PBSC - 0 - + All patients were seropositive to HHV-6 before transplantation. Numbers in parentheses are the days from transplantation. Abbreviations: Auto, autologous transplantation; Allo, allogeneic transplantation.

- -

days in allogeneic and greater than 182 days in autologous transplantation patients.

Direct Detection by PCR

Detection of HHV-6 DNA was attempted by direct PCR as well as by PCR on material cultured from transplant recipients (patients) and donors. Mononuclear cells were isolated from heparinized blood, marrow obtained from both donors and patients, and BAL from patients. One million cells of primary material were saved for direct PCR when possible. Primary material (mononuclear cells) was always negative when tested for HHV-6 DNA. In some patients, particularly the allogeneic transplantation patients who took longer to engraft, as few as 1 X lo5 cells were available. If more than 1 X lo6 cells were obtained, 1 X lo6 were saved for PCR and the remainder were cultured.

Virus Isolation

HHV-6 was identified by PCR in cultured PBMCs from 12 of 26 patients (Table 3). In 4 of these 12 patients, virus was isolated on two occasions (2 autologous and 2 allogeneic). In total, 16 isolates of HHV-6 were obtained. When cultures from patients no. 1 through 7 were analyzed, all the cultures positive at week 2 were also positive at week 4 of

the culture. PCR was therefore performed only on primary material and material cultured for 4 weeks in patients no. 8 through 26. For all of the patients from whom virus isolation was achieved, direct PCR was negative and only the cultured material was positive by PCR. A representative PCR analysis performed on patient no. 12 is shown in Fig 2. Of the 5 cultures established from patient no. 12 at days 6, 13, 20, 38, and 101 after transplantation, only the culture from day 13 was positive for HHV-6 DNA.

There were equal numbers of patients (6) with virus isolation in the autologous and allogeneic transplantation groups. Differences in the frequencies among the four subgroups of transplantation patients studied here were not statistically significant. In addition, we found no correlation between the frequency of HHV-6 isolation and the number of cells tested. The numbers of cells cultured were sorted into ascending order and divided into equal numbers of cultures in the lower cell range (2.5 X lo4 to 2.2 X lo6, n = 111) and higher cell range (2.3 X lo6 to 3.7 X lo’, n = 110). Cultures in the higher cell range were more frequently PCR positive (5/111 v 11/1 lo), but the difference was not statistically significant ( P = .128, Fisher’s exact test).

In addition to performing PCR to identify HHV-6, IFA was also performed on the cultured material at weeks 2 and 4 of culture. HHV-6 was identified by IFA in 8 of 12 PCR-




5346 KADAKIA ET AL

r 1 Y Y i

+

107 167 / I

l

2 3 4 6 8 7 8 0

l0 11 12 13

14 15 18 17 18 19 20 21 22 23 24 25 26 27 26

C t

2

3

1

5

8

7

8

0

l0

l1

l2

13

14

15

16

17

11

l0

B 21

P P H I I n I

107 I

Fig 2. Representative figure showing PCR analysis performed on the cultures obtained from the patients. Ethidium bromide-stained agarose gel of PCR-amplified products from primary (direct) and cultured (week 4 of culture) lymphocytes tested at two different cell concentrations of approximately 2 x lo5 (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20) and 2 x 10' (lanes 3, 5, 7, 9, 11, 15, 17, 19, and 21) cells (A). This is a representative gel of PCR performed with both HHV-6-specific primers 2A and 28 1187-bp amplimer) and /%globin (internal control, 167-bp amplimer) on cultures from patient no. 12 at different time points. The gel was Southern blotted and probed with internal probes specific for HHV-6 (B) and 0-globin IC) amplimers. The culture obtained from patient no. 12 at day 13 was positive for HHV-6-specific DNA.

positive cultured PBMCs. Cultures from patients no. 2, 6, 9, and 22 on days 198, 153, 324, and 65, respectively, were PCR positive but IFA negative. Thus, PCR was more sensitive (l6/22l cultures positive) than IFA (12/221 cultures positive) in detecting the presence of HHV-6 in cultured material ( P < .05, McNemar's test).

Salivary Shedding of HHV-6 DNA Eleven of the 26 patients (42%) showed evidence of sali-

vary shedding of HHV-6 before transplantation (Table 3). Posttransplantation analysis was limited to the 19 patients who were studied longitudinally. Twelve of 19 patients (63%) were positive for salivary shedding after transplantation. Posttransplantation, there was a statistically significant difference in the frequency of HHV-6 shedding between allogeneic (30/56 specimens [53%]) and autologous (9/31 specimens [29%]) patients ( P = .042). Pretransplantation, no such difference was observed, with the frequencies being 7 of 18 specimens (38%) in allogeneic and 4 of 13 specimens (30%) in autologous transplantation patients ( P > .05).

A statistically significant difference was observed between the number of saliva specimens positive in the autologous

transplantation patients who received BM ( 1 1/23 [47%]) when compared with those who received PBSCs (2/21 [9%]; P = .008). No such difference was observed within the two subgroups of allogeneic transplantation patients, ie, those who received BM from matched related donors (22/41 [53%]) and those who received BM from matched unrelated donors (15/33 [45%]).

A significant negative association was observed between the fraction of saliva specimens with HHV-6 DNA and the mean HHV-6 antibody levels in the 19 patients in whom salivary shedding was observed longitudinally ( P = .005, linear regression analysis).

HHV-6 Serology

All of the recipients and donors were seropositive for HHV-6 at the time of transplantation. A significant increase in HHV-6 antibody titer was seen in 7 of 26 (26%) recipients (Table 3). Four of these were allogeneic and the remaining three were autologous transplantation patients. In subjects in whom a significant change in antibody levels was seen, the median time to the increase in titer was 21 days (range, 6 to 38 days). Significant decreases in titer were seen in 5





Table 4. Frequency of Active HHV-6 infections

Evidence of Infection

Period Relative to Transplantation Frequency PValue*

Culture positive Pretransplantation 0164 Posttransplantation 1611 65 .007 Posttransplantation (early)t 2/52 Posttransplantation (late)t 141113 >.05

With ganciclovir 1/12 Without ganciclovir 151214 >.05 With acyclovir 31105 Without acyclovir 131123 .035

Pretransplantation 11/31 Posttransplantation 39/87 >.05 Posttransplantation (ear1y)t 10129 Posttransplantation (late)t 29/58 >.05 With ganciclovir 411 1

Without ganciclovir 461110 >.05 With acyclovir 24/53 Without acyclovir 26/65 >.05

Saliva positive

c Fisher's exact test (two-tailed). t Early and late period included 0 to 16 and greater than 16 days

after transplantation, respectively.

of 26 patients at a median time of 31 days (range, 14 to 140 days). No significant difference in the proportion of patients with increases in HHV-6 antibody titers were observed between allogeneic and autologous transplantation patients. Moreover, no statistically significant association was observed between virus isolation and increases in HHV-6 antibodies ( P > .05).

Frequency of Active HHV-6 Infection

BMT increases the frequency of HHV-6 isolation as shown by a significant proportion of cultures being positive after transplantation when compared with the absence of positive cultures before transplantation (P = .007; Table 4). As described above, differences in the proportion of saliva specimens positive during pretransplantation and posttransplantation periods were not statistically significant. The posttransplantation period was further divided into early posttransplantation (days 0 through 16) and late posttransplantation (> 16 days) based on the median time to neutrophil engraftment (day 16). No significant difference was observed in the proportion of cultures and saliva positive between the early and late posttransplantation period (Table 4).

The effects of therapy with the antivirals acyclovir and gancyclovir on HHV-6 isolation from cultures and its detection in saliva were studied by determining the proportion of cultures and saliva specimens positive for HHV-6 during periods of antiviral and no antiviral treatment (Table 4). The number of cultures positive during acyclovir treatment was significantly lower than that during periods with no acyclovir treatment. However, no significant difference was observed during gancyclovir treatment.

Detection of HHV-6 DNA in Marrow, BAL, Urine, and Biopsy Specimens

BM specimens from donors of allogeneic transplants as well as from autologous transplantation patients before trans-

plantation were negative for virus isolation. BAL specimens were obtained from only 2 recipients and were also negative. No HHV-6 DNA was detected in urine specimens from the first seven patients before transplantation. Thirty skin biopsies tested from a total of 12 patients were negative for HHV-6 DNA, except for 1 time point in patient no. 1. In that patient, the skin rash was considered to be a manifestation of GVHD. It is possible that, because HHV-6 DNA was present in the biopsy material, the skin rash was related to HHV-6 infection. In addition, HHV-6 DNA was detected by PCR in lung tissue obtained from patient no. 19 at autopsy. No other identifiable cause was found for the pneumonia and subsequent development of A D S and death. Therefore, HHV-6 may have been involved in the disease in this patient.

Variant Analysis of HHV-6 Isolates

RFLP of PCR amplimers. In the present study, all the cultures that were positive with primer set HHV-6 2A and 2B were also positive with the Aubin primers, except for the culture from patient no. 22 at day 65. In addition, restriction profiles of the PCR amplimers from the HindIII and HinfI digestions were characteristic of variant B.9 Thus, by both HindIII and HinfI digestion, all of the isolates of HHV-6 were the B variant.

MoAb analysis As discussed earlier in the section on detection of HHV-6 by immunofluorescence, four PCR-positive cultures were negative by F A . The remaining cultures reacted with the panel of MoAbs in a manner consistent with variant B specificity.

Heterogeneity Among Viral Isolates Studied by RFLP Analysis of Viral DNA

To show that the HHV-6 isolates were different from the laboratory strain HHV-6B(Z29) and were not cross-contami- nants from isolates obtained from other patients, RFLP studies were performed on materials from the patients for whom sufficient infected cells were available (Fig 3). As can be seen in Fig 3, although there are numerous differences between HHV-6B(Z29) and the BMT isolates, as well as among the BMT isolates, they all exhibited variant B pat- terns." The two isolates from the same patient (patient no. 7, days 24 and 27) were identical to each other but differed from the other isolates, as shown by restriction profiles with enzymes BamHI and Pst I.

Associations With Disease

All patients were seropositive to HHV-6 before BMT and thus all patients were considered to be infected with HHV- 6. During prospective clinical observation, no episodes of identifiable HHV-6 disease were directly attributed to active HHV-6 infection. After the observation period, clinical data collected from all of the patients were assessed for associations with episodes of active HHV-6 infection.

Table 5 shows the Erequency of the various clinical events as well as documented infections observed in the patients under study. Data collected on bacterial infections were useful in inkqreting either febrile events or pneumonia. Diarrhea and oral mucositis occurring early in the posm-ansplantation period




5348 KADAKIA ET AL

NciI B

m I 7 l 9 1022 g 9

Q a L u M k i kkUt&UL 90 24 27 15 13220 5

- c -

I3 14 I S

16 17

18

PstI San BamHI

ti 6 7 l 9 1022s W 1 7 9Ib22,- ti 1 7 l 9 1022 m‘ Q > Q Q > >

90 2427 IS l3220 5 48 24 21 15 132 20 5 24 21 15 132 20 3 I” “IF - 0

e 12

I3

14

1s

0

TERM P

P Q

Fig 3. Southern blot analysis of HHV-6 isolates. Restriction fragment polymorphism studies were performed on HHV-6 isolates using restriction endonucleases Nci 1, Pst I, Sa/ I, and BamHl on whole cell DNA preparations. HHV-GB (229) was included as a positive control. The blots were probed with HHV-6 nucleocapsid DNA. The restriction profile of all of the isolates differed from each other and the laboratory strain HHV-GB (229).

in the absence of cultured herpes simplex virus were considered to be gastrointestinal toxicity due to chemotherapy.

Most of the patients experienced febrile episodes after transplantation (73%; Table 5). A high frequency of skin rash was observed in allogeneic transplantation patients (80%) compared with autologous patients (36%). Pneumo- nia and ARDS episodes were generally limited to allogeneic patients (60%). except for one autologous transplantation patient. Graft failures were limited to allogeneic patients receiving marrow from matched unrelated donors. Sinusitis episodes were observed at a higher frequency in autologous (55%) than in allogeneic transplantation patients (21%). CNS involvement manifested as encephalopathy or neuropathy was again observed at a higher frequency in allogeneic (21%) than in autologous transplantation patients (9%).

Simple Associations of Active HHV-6 Infection to Disease Events

A high proportion of patients had one or more manifesta- tions of active HHV-6 infection. Tests were also performed separately to detect associations with virus isolation from blood, the presence of viral DNA in saliva, and significant

serologic increases to HHV-6. Associations with fever, skin rash, GVHD, pneumonia, ARDS, and CNS disease were not significant. Sinusitis was significantly associated with HHV- 6 isolation in autologous transplant patients ( P = .002). Ac- tive HCMV infection was significantly associated with HHV-6 isolation in allogeneic transplantation patients.

Temporal Association of HHV-6 Infection With Disease Events

A summary of the temporal association of active HHV-6 infection to clinical events with a scoring for the possible causal relationship is shown in Table 6. HHV-6 infection was a “probable” cause of fever in 4 patients, skin rash in 3 patients, fever associated with skin rash in 3 patients, sinusitis in 2 patients, pneumonia with ARDS in 1 patient, and platelet and RBC suppression in 1 patient. In all, 9 of the 26 patients had some clinical event probably due to HHV-6 infection, as defined by events occurring within 2 weeks of documented infection in the absence of other de- monstrable causes. When the data of Table 6 (excluding marrow suppression) were tested for independence of col- umns (association scores) and rows (clinical events), no association was observed ( P = .773, Pearson x2 test). The





Table 5. Frequency of Documented Infections and Other Clinical Events After BMT

Auto (N = 11) A110 MRD (N = 8) Allo MUD (N = 7) Total (N = 26)

Disease events Fever 9 (82%) 6 (75%) 4 (57%) 19 (73%) Skin rash

With GVHD 0 (0%) 6 (75%) 6 (86%) 12 (46%) Without GVHD 4 (36%) 0 (0%) 1 (14%) 5 (19%)

Pneumonia With ARDS 1 (9%) 4 (50%) 5 (71%) 10 (38%) Without ARDS 0 (0%) 1 (13%) 1(14%) 2 (8%)

Sinusitis , 6 (55%) 1 (13%) 2 (29%) 9 (35%) Graft failure 0 (0%) 1 (13%) 4 (57%) 5 (19%)

Encephalopathy/neuropathy 1 (9%) 1 (13%) 2 (29%) 4 (15%)

Documented infections Bacterial infection 3 (27%) 6 (75%) 6 (86%) 15 (58%) Fungal infection 3 (27%) 3 (38%) 4 (57%) 9 (35%) Viral infections

HCMV infections 1 (9%) 6 (75%) 4 (57%) 11 (42%) VZV disease 1 (9%) 0 (0%) 1 (14%) 2 (8%) HSV disease 2 (18%) 2 (25%) 1(14%) 5 (19%) Hepatitis C 0 (0%) 1 (13%) 1(14%) 2 (8%) Adenovirus infection 1 (9%) 0 (0%) 0 (0%) 1 (4%)

Abbreviations: Auto, autologous transplantation; Allo MRD, allogeneic transplantation with matched related donor; Allo MUD, allogeneic transplantation with matched unrelated donor.

marrow suppression data at the bottom of the Table 6 was analyzed separately, because the defining criteria were different from those for the clinical events in the upper part of the table; no association was observed ( P = .860).

HHV-6 DNA was detected in the skin biopsy specimen of 1 patient with GVHD histologically diagnosed in the same specimen. Thus, HHV-6 was considered to be only a “possible” cause of skin rash in this case. In another patient, HHV- 6 DNA was detected by PCR in lung tissue obtained at the time of autopsy. This patient died of idiopathic pneumonia and HHV-6 was therefore considered as the “probable” cause of the pneumonia.

Table 6. Association of Active HHV-6 Infection With Clinical Events After BM1

Scores’

Probable Possible Improbable Clinical Events N (3) (2) (1)

Fever 19 4 10 5 Skin rash 17 3 11 3 Fever with rash 11 3 7 1 GVHD 12 0 9 3 Sinusitis 9 2 5 2 CNS involvement 4 0 3 1 Pneumonia 12 1 6 5 ARDS 10 1 5 4 Marrow suppression

WBC 9 0 3 6 Platelet 9 1 2 6 RBC 8 1 2 5

Total 120 16 63 41

See Table 2.

Effect of HHV-6 Infection on Marrow Engraftment and Suppression

Figure 4 shows the time course of engraftment for patients with and without HHV-6 isolation from the blood. There was no statistically significant difference in the rate of engraftment or frequency of patients with engraftment in any of the 3 lymphoid lineages in patients with virus isolation when compared with those without virus isolation (Fig 4, P > .05 by log rank test). The median times to neutrophil, platelet, and RBC engraftment were 16, 30, and 51 days, respectively, in patients with no virus isolation. The median times to neutrophil, platelet, and RBC engraftment in patients with virus isolation was 17, 58, and greater than 26 days, respectively.

No statistically significant association was observed between active HHV-6 infection and neutrophil, platelet, or RBC suppression. Of the 22 patients that were evaluated for neutrophil suppression, 9 showed suppression. Active HHV- 6 infection manifested by virus isolation was identified by the propinquity analysis as a “possible” and “improbable” cause of neutrophil suppression in 3 and 6 patients, respectively. Of the 15 patients evaluated for platelet suppression, 9 showed suppression. Active HHV-6 infection manifested by virus isolation was identified as the “probable,” “possible,” and “improbable” cause of platelet suppression in 1, 2, and 6 patients, respectively. Of the 13 patients evaluated for RBC suppression, 8 showed suppression. Active HHV- 6 infection manifested by virus isolation was identified as a “probable,” “possible,” and “improbable” cause of RBC suppression in 1, 2, and 5 patients, respectively (Table 6).

Activation of Other Herpesviruses

HCMV. Five of the 26 recipients were seronegative to HCMV and 21 were seropositive to HCMV before trans-




5350 KADAKIA ET AL

plantation. None of the five seronegative patients showed any evidence of HCMV infection after transplantation. Four of the five (80%) showed active HHV-6 infection after transplantation (Table 7). There was no difference in the frequency of active HHV-6 infection among HCMV-seronegative patients (80%) and HCMV-seropositive patients with or without evidence of posttransplantation activity (88% and 91%, respectively). HHV-6 was isolated from the peripheral blood at a higher frequency in patients with active HCMV infection @/l2 [66%]) than those with no evidence of active HCMV infection (3/14 [21%]), the difference being statistically significant (P = .04) in allogeneic transplantation patients.

HSV and VZV. In 6 of 26 (23%) and 2 of 26 (7%) recipients, active HSV and VZV infection were documented, respectively (Table 7). In 5 of 6 cases, active HSV infection followed active HHV-6 infection; in 1 of 6 recipients, it preceded active HHV-6 infection. One of the two cases of active VZV ihfection followed HHV-6 infection, whereas the other preceded HHV-6 infection. No statistically significant association was observed between active HHV-6 infection and activation of HSV or VZV.

DISCUSSION

HHV-6 in BMT Recipients

The present study involved 26 adult BMT patients (1 1 autologous and 15 allogeneic) observed prospectively for up to 1 year after transplantation. Previous studies examining the frequency of HHV-6 infections were nearly exclusively limited to allogeneic transplantation patients because they are more immunosuppressed than the autologous transplantation patients and are considered to be at a greater risk of primary as well as reactivated herpesvirus In addition, in the studies by Yoshikawa et all' and Drobyski et a l , I 6 25 pediatric BMT patients (24 allogeneic and 1 autologous) and 16 adult BMT patients (all allogeneic) were observed for 60 and 100 days, respectively. Drobyski et all6 routinely attempted HHV-6 isolation only after the first 3 weeks after transplantation; thereafter, HHV-6 isolation was attempted only during febrile episodes with no identifiable cause. In our study, we examined all the patients routinely as per the experimental design unless the patient died or was lost to follow-up; both autologous and allogeneic transplantation patients were studied. Thus, this study differs from previous studies with respect to study design and duration of follow-up, as well as the composition of the transplantation population.

HHV-6 was isolated from the cultured blood mononuclear cells of 12 of 26 (46%) patients during the study period, which is comparable to the frequency reported by others, ie, 10 of 25 patients (40%)'' and 6 of 16 patients (37%).16 In the remaining two studies, HHV-6 was isolated from 3 of 3 patients (lOO%)I3 and 2 of 2 patients (lOO%).I4

In the present study, all patients and donors were HHV- 6 seropositive but culture negative before transplant. Three of the 12 patients (25%) from whom virus was isolated also showed significant increases in HHV-6-specific antibody titers after transplantation, as measured by enzyme immuno-

11 I

A = Neutrophils 0 ' 1 I I I I 1

0 30 60 90 120 150 180 '1 3 0.75

z

B = Platelets 30 60 90 120 150 180

0.75 l 1

I I

......................................................... 0.25 0.51 I-' C = Red Blood Cell

- 1 l I I I I I

0 30 60 90 120 150 180

DAYS TO ENGRAFTMENT Fig 4. Establishment of time to engraftment in 3 different lineagea

IANC, platelets, and RBC) in the presence or absence of HHV-6 bola- tion using Kaplan-Meier survival analysis. (-) Patients with engraftment (ANC, platelet, and RBC) in absence of virus. (. . . ) Patients with engraftment in presence of virus isolation. Signifkance was determined by log rank test, and in each of the 3 cases P > .05.

assay. This is lower, but not statistically different from that observed by Yoshikawa et a l l * and Asano et al," in which 5 of 10 patients (50%) and 2 of 3 patients (66%) with virus isolation, respectively, showed fourfold increases in the neutralizing antibody titers. In addition, in the present study, 4 of 14 patients (29%) from whom no virus was isolated also showed significant increases to HHV-6 after transplantation. This is a higher proportion than that observed by Yoshikawa et al," in which 2 of 15 patients (13%) with no virus isolation showed 16-fold increases in the neutralizing antibody titers. In total, 7 of 26 of the patients (27%) in the present study showed significant increases in HHV-6-specific antibody titers, which is similar to the result observed by Yoshikawa et al.'' Because allogeneic BMT patients are compromised in their humoral responses, the frequency of infection may be underestimated by analysis of serology. Therefore, serology





Table 7. Activation of Human Herpesviruses in the Study Population

Pre-Tx Active Patient Type of HCMV HCMV Active HSV Active V N Active HHV-6

No. Transplantation Serology Infection Infection Infection Infection

1 Allo, MRD + V V

2 Auto, BM + A V V V 3 Auto, BM + V 4 Auto, PBSC -

5 Allo, MUD + -

6 Allo, MRD + V 7 Allo, MUD + A. V V - V, A

S 9 Auto, BM + - v, S

10 Allo, MRD + V V v, S 11 Allo, MRD + V - S 12 Auto, PBSC - - V. A 13 Allo, MUD + V - v. S 14 Auto, PBSC - - - A

15 Allo, MUD + V - V S. A 16 Auto, BM - - S 17 Allo, MUD + V - - v. S 18 Auto, BM + - V - S 19 Allo, MRD + - - 20 Allo, MRD + V - -

21 Allo, MUD + - - - S 22 Auto, PBSC + - - - v. S 23 Allo, MUD + - -

24 Auto, BM + - V - v, S 25 Allo, MRD + V - - S, A 26 Auto, PBSC - -

Abbreviations: Auto, autologous transplantation; Allo, allogeneic transplantation: V, virus isolation; A, significant antibody increases after

- -

- - V, S. A

S V

- - - - - -

- -

a Allo, MRD + - V -

- -

-

-

- -

-

-

- -

- S, A -

- -

- - S

transplantation; S, salivary shedding of HHV-6 DNA.

cannot be used as a sole method in documenting active HHV- 6 infection. It is also possible to show false-positive serology in patients receiving large volumes of IVIG transfusion. We did not observe any effect of IVIG transfusion on serologic analysis (data not shown).

Direct PCR

We were unable to detect HHV-6 DNA by direct PCR of mononuclear cells with primer set HHV-62N2B. However, previous studies have shown the presence of HHV-6-specific DNA in the PBMCs from healthy The differences between the above two studies and this study could be the number of cells tested and the primer sets used for PCR analysis. Jarrett et a134 showed that DNA equivalent to 5 X lo4 to lo5 cells was required to detect HHV-6-specific DNA in the PBMCs. Cone et a133 tested DNA obtained from 7.5 mL of blood (approximately 7 X lo6 cells). In our study, we tested a median of 1.93 X lo5 by direct PCR from the 16 blood specimens that were PCR positive on culture. Fur- thermore, we used a different primer set for PCR analysis. It is possible that our primer set is not as sensitive in detecting HHV-6 DNA as those used by others. Support for the latter hypothesis comes from the observation that primary materials from patients no. 6 and 17 at days 48 and 68, respectively, were negative when tested by PCR with the primer set H62N2B but were positive when tested with primer set described by Aubin et a19 on ethidium bromide

staining. On the other hand, the culture from patient no. 22 on day 65 was positive with primer set H62N2B but negative with the primer set described by Aubin et al .9 Direct PCR of blood cells from seropositive subjects would presum- ably be positive in all cases if sufficient numbers of cells were tested and, unless quantitative, probably provides little information. Virus isolation, which we assume detects pro- ductively infected cells or, perhaps, free virus, may provide a significant measure of active infection, albeit at the expense of considerable effort.

Viral isolates obtained from patient no. 7 at two different time points (days 24 and 27) after transplantation were identical to each other. No virus was isolated during the pretransplantation period from the recipients or from the donors in this study, which prevented transmission studies.

Effect of Antibody on Salivary Shedding

In addition to virus isolations and significant increases in HHV-6 titers, we tested for HHV-6 DNA in saliva. This has not been previously studied in transplantation patients. The HHV-6 DNA detected in the saliva may be cell associated and may not represent active HHV-6 infection. Jarrett et a134

and Cone et a133 have reported that 18 of 20 (90%) and 16 of 17 (85%) healthy adults, respectively, had HHV-6-specific DNA in their saliva specimens and that 10 of 10 and 14 of 17 individuals, respectively, remained positive for HHV- 6 DNA in their saliva specimens upon subsequent testing.




5352 KADAKIA ET AL

Infectious salivary HHV-6 has been detected only infre- q~ently.’”’~ Jarrett et a134 observed no correlation between antibody titer and the ability to isolate HHV-6 or detect

In 15 of 19 patients (78%) from this study, there was salivary shedding at least once and usually on multiple occasions, and it was shown to have a significant negative association with the mean antibody titer. Although other investiga- tors have not seen a correlation between HHV-6 shedding and HHV-6-specific antibody titer,’4 it is important to note that none of the previous studies observed the subjects as intensively as in this study. Also, it is important to study healthy adults longitudinally to show if the correlation with the antibody titer is seen in this group as well.

Association of Active HHV-6 Infection With Disease

HHV-6 DNA.

Active HHV-6 infection has been previously associated with several clinical events in the setting of solid organ transplantation as well as BMT, including fever, skin rash, pneumonitis, and BM suppression.’”’‘,l8 We examined associations between active HHV-6 infection between these and other clinical events. Of the associations tested, HHV-6 isolation was found to be significantly associated only with sinusitis ( P = .002) and active HCMV infection ( P = .04) in autologous and allogeneic transplantation patients, respectively.

The association of sinusitis with active HHV-6 infection is surprising. However, the virus has been associated with sinusitis as well as otitis media.*’ Three allogeneic transplantation patients also had episodes of sinusitis, but the association with active HHV-6 infection was not statistically significant. Possibly, a higher frequency of sinusitis may have been missed as these patients experience a greater number of concurrent complications. In addition, the lack of other identifiable causes of sinusitis might be due to difficulty in obtaining appropriate cultures from the sinuses in neutro- penic and thrombocytopenic patients. Nevertheless, the temporal association of HHV-6 infection and the onset of sinusitis is intriguing and bears further investigation. In allogeneic transplantation patients, active HCMV infection was also shown to be significantly associated with HHV-6 isolation, suggesting coactivation of these two viruses under immunosuppressed conditions.

Based on temporal association of active HHV-6 infection with clinical events, HHV-6 infection was identified as the probable cause of fever, skin rash, sinusitis, pneumonia, ARDS, platelet suppression, and RBC suppression in 9 of 26 patients. We resorted to the term probable because an unequivocal association could not be made.

We found no simple association of active HHV-6 infection with GVHD. However, the high frequency of active HHV- 6 infection in our patient group would hide any direct association with GVHD. Wilborn et all7 detected HHV-6 DNA at a higher frequency (urine, buffy coat, or oral lavage fluid) in BMT patients with moderate or severe GVHD than in those with mild or no GVHD. This could be explained by increased viral activity during a more immunosuppressed state. We found no statistically significant association between GVHD and active HHV-6 infection. Nonetheless,

from one patient with histologically diagnosed GVHD, HHV-6 DNA was detected in a skin biopsy, suggesting a possible etiologic association between HHV-6 and skin rash or GVHD. Previously, HHV-6 DNA has been detected in the skin biopsy of BMT patients with exanthematous rash.”.” In both of the above studies, patients had exanthematous rash at the time of skin biopsy. These studies and our study suggests that a virologic analysis should be performed on skin biopsy in addition to the pathologic analysis performed for GVHD. Although a high frequency of pneumonia episodes was observed in allogeneic (12 of 15) compared with autologous (1 of 1 1) transplantation patients, no simple association was observed between active HHV-6 infection and development of pneumonitis. However, there was one patient in whom HHV-6 DNA was found in the lung tissue obtained at autopsy and who died of idiopathic pneumonia.

It has been suggested that HHV-6 might be responsible for idiopathic pneumonia after BMTi4.I5 and delays in engraftment and marrow suppression.I6 However, in our study population, there was no statistically significant association between active HHV-6 infection and either delayed engraftment or marrow suppression in any of the 3 lineages studied (neutrophil, platelet, and RBC). However, in two patients, HHV-6 was considered as a probable cause of platelet and RBC suppression.

The Role of Immunosuppression

The frequency of HHV-6 shedding in saliva may also be dependent on the degree of immunosuppression. Thus, allogeneic transplantation patients, who are more immunosuppressed, showed significantly higher levels of salivary shedding compared with autologous transplantation patients ( P = .035). The correlation noted above between the frequency of salivary shedding and antibody levels was due to the contribution of autologous transplantation patients ( P < .05, linear regression analysis) but not allogeneic transplantation patients (P > .05). The lack of correlation observed in the case of allogeneic transplantation patients might be due to passive antibodies received by those patients in the form of IVIG transfusions for prophylaxis of HCMV disease. No IVIG transfusions were administered to autologous transplantation patients after transplantation unless the patient developed HCMV disease.

There was no statistically significant difference in salivary shedding between allogeneic transplantation patients receiving marrow from living related donors and matched unrelated donors, but a significant difference was observed between autologous transplantation patients who received marrow versus those who received PBSCs. The difference in the latter two groups is that, in patients receiving marrow, immune reconstitution takes place from the progenitor cells alone, whereas in the patients receiving PBSCs, immune reconstitution is due to stem cells with the addition of mature leukocytes of a variety of lineages, which accelerates engraftment.

The Role of Antivirals In our study, the number of PCR-positive cultures was

significantly lower during periods of acyclovir treatment





when compared with periods with no acyclovir treatment. This suggests that acyclovir has some antiviral activity against HHV-6 in BMT recipients. These results are counter to expectation, in as much as ganciclovir has a marked effect on HHV-6 in vitro, whereas acyclovir has much less effect.39 Similar observations have been made with respect to effect of acyclovir on HCMV infection and disease. Acyclovir has no significant effect on HCMV in cell cultures and patients4 However, a study by Meyers et a14' in BMT patients showed that patients receiving acyclovir treatment were shown to have significantly decreased levels of HCMV disease when compared with patients with no acyclovir treatment. This suggests that acyclovir may have an antiviral effect on both HHVd and HCMV, the mechanism of which is not yet understood.

HHV-6 Variants

Similar to the observations of Drobyski et al,'6,42 all the HHV-6 isolates from this study were variant B. HHVdB consists of two subgroups of viruses on the basis of restriction profile of PCR amplimers.' In this study, we identified representatives of both classes.

In summary, active HHV-6 infections occurred frequently after transplantation. Virus isolation with PCR confirmation is more sensitive than monitoring cultures by IFA. There was no statistically significant difference between the overall frequency of active HHV-6 infection, manifested as virus isolation, increases in HHV-6-specific antibody titer, and salivary shedding of HHV-6 DNA, between allogeneic and autologous transplantation patients. Because of the relatively high frequency of active HHV-6 infection in this group of patients, simple temporal correlations with clinical events are not sufficient to establish causality until the spectrum of HHV-6 disease has been more clearly defined. Nevertheless, active HHV-6 infection was implicated as a potential cause of disease in a substantial proportion of our patients. A de- finitive correlation would require a much larger sample size than our study allowed. Despite these limitations, our find- ings suggest that HHV-6 infection and disease should be important considerations in assessing patients after BMT and that further studies are warranted for the pathogenic role of HHV-6 in BMT patients.

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1996 87: 5341-5354

ArmstrongMP Kadakia, WB Rybka, JA Stewart, JL Patton, FR Stamey, M Elsawy, PE Pellett and JA and allogeneic bone marrow transplantationHuman herpesvirus 6: infection and disease following autologous

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