Human Herpesvirus 7 in Allogeneic Hemopoietic Stem Cell Transplant Recipients in the Central Clinical Hospital in Warsaw: A Three-Year Survey

Objectives: Human herpesvirus 7 (HHV-7) is spread worldwide and has been described as a potential pathogen in immunosuppressed patients. Different clinical manifestations have been described including fever and skin rash; HHV-7 may also be a possible cofactor for cytomegalovirus disease in transplant recipients. Materials and Methods: A retrospective review of a group of 58 adult recipients of allogeneic hemopoietic stem cell transplantation was made. Serum samples taken in the range of 0–180 days after transplant were examined for presence of specific HHV-7 sequences using the quantitative real-time PCR method. Results: HHV-7 DNA was detected in plasma samples in 26 (45%) of the 58 recipients between day 20 and day 65 of transplantation. All of them developed fever of unknown origin; also HHV-5 DNA was detected in plasma samples collected from 11 HHV-7-positive patients. None of the described individuals died during detectable HHV-7 or HHV-5 viremia periods. Conclusions: There is a high frequency of Received: October 20, 2009 Accepted after revision: February 21, 2010 Published online: August 6, 2010 Tomasz Dzieciatkowski, MSc, PhD Department of Medical Microbiology Medical University of Warsaw 5 Chalubinski Str, PL–02-004 Warsaw (Poland) Tel./Fax +48 22 599 1778, E-Mail dzieciatkowski @ wp.pl © 2010 S. Karger AG, Basel 0300–5526/11/0541–0025$38.00/0 Accessible online at: www.karger.com/int D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 10 :4 3 P M Dzieciatkowski /Przybylski /Basak / Torosian /Jedrzejczak /Mlynarczyk Intervirology 2011;54:25–29 26 reports and small patient series have associated primary HHV-7 infection with hepatitis, upper respiratory tract infections and pancytopenia [2] . HHV-7 involvement has been also implicated in pityriasis rosea, based on the virus isolation from peripheral blood mononuclear cells (PBMC) of patients and molecular detection of HHV-7 DNA in plasma, PBMC and skin lesions [5] . All human  -herpesviruses – especially HHV-5 – have been recognized as important causes of morbidity and mortality in transplant recipients, but the number of studies of HHV-7 infection in immunosuppressed patients is very limited. One of the major problems is crossreaction between HHV-6 and HHV-7 antibodies, which might confuse the results of serological analyses [6] . Some investigators have suggested that HHV-7 may be a possible cofactor for cytomegalovirus disease in transplant recipients. Chan et al. [7] monitored 61 bone marrow transplant recipients by PCR analysis, and found that patients with cytomegalovirus disease were more likely to have concurrent HHV-7 DNA in PBMC prior to onset of disease than were patients with asymptomatic HHV-5 infection. Similar findings have also been demonstrated in renal and liver transplant recipients [8, 9] . In a recent study, we tried to summarize retrospective results of the determination of HHV-7 infection status in allogeneic stem cell transplantation recipients. As the infections with HHV-7 are rarely accompanied by clinical symptoms, our first aim was to compare infection status, measured as viral DNA presence in the blood, in the posttransplantation period. Materials and Methods This retrospective study involved patients who received an allogeneic hemopoietic stem cell transplantation (HSCT) and were hospitalized in the Hematology, Oncology and Internal Medicine Clinics, Medical University of Warsaw. In the considered period (from January 2006 to December 2008), there were 58 patients receiving allogeneic stem cell transplantations. Additional criteria for the study included appearance of at least one of the syndromes listed below in the period of 100 days after HSCT: appearance or intensification of graft versus host disease (GvHD), skin rush or neutropenic fever. Monitoring of clinical status of the patients and viral load in blood samples comprised the period of 180 days after HSCT. Collection of plasma samples from all patients for PCR investigations began at a median of 3 days after transplantation (range 1–7 days) and lasted until a median of 107 days (range 28–180 days). Presence of viral DNA was tested by real-time PCR in sera samples obtained once a week until the 100th day after allogeneic HSCT, and thereafter once every 2 weeks. The median number of blood samples per patient was 13 (range 4–24 samples). A total of 574 samples obtained from 58 patients were examined using realtime PCR. Viral DNA was extracted from 200  l of plasma, using a High Pure Viral Nucleic Acid Kit  (Roche Diagnostics) in accordance with the manufacturer’s instructions. For the detection of HHV-7, a real-time PCR (qPCR) assay with fluorescent TaqMan probes, complementary for the sequence lying within the amplified product, was used. Tests were run on the LightCycler 2.0 instrument (Roche Diagnostics) with a modified in-house method described below [10] . A highly conservative region of HHV-7 genome encoding major capside protein (U57) was chosen (GenBank, U43400), and a set of primers was developed, as well as the probe, labeled with the fluorophore reporter JOE on the 5 end and with TAMRA quencher on its 3 end (Oligo ; table 1 ). Investigations were performed using the reaction mixture TaqMan Master Kit  (Roche Diagnostics). Besides chemicals supplied by the kit producer, the final reaction mixture contained 5 l of isolated viral DNA, 2.75 M of HHV7-p1 primer, 3.25  M HHV7-p2 primer and 1.50 M HHV7-JOE probe, in a total volume of 20  l. Amplification was performed with activation of the thermostable hot-start DNA polymerase for 10 min at 95 ° , followed by 45 cycles comprising denaturation (15 s at 95 ° ), primer annealing (15 s at 60 ° ) and strand elongation (15 s at 72 ° ). After the end of cycling, the material was cooled down to 40 ° for 60 s. Fluorescence levels were read at 560-nm wavelength, specific for JOE fluorophore dye [10] . Every tested sample was amplified along with an internal control (positive control of DNA amplification process). For the assessment of reaction specificity, DNA isolated from the following viruses was used: HHV-1, HHV-5 and HHV-6. Each amplification reaction included, except tested samples, also positive HHV-7 controls (RK strain) in a range of 100– 100,000 copies/ml and a negative control of DNA extraction and the amplification process [10] . HHV-5 DNA was detected using the commercially available quantitative CMV Quant Kit (Roche Diagnostics) developed especially for monitoring cytomegalovirus disease with the LightCycler 2.0 instrument. The test uses SCORPIONS TM fluorescent probes. Analogically for HHV-7 detection, an internal control was added for every sample and amplification was performed in the presence of amplification-specific controls (positive, negative and extraction process control). Name Sequence (5 –3 ) HHV7-p1 CCC AAC TAT TTA CAG TAC GGT TGG T HHV7-p2 TTT AGT TCC AGC ACT GCA ATC HHV7-JOE JOE–CTA TTT TCG GTC TTT CCA ATG CAC GCA–TAMRA Table 1. Sequences of primers and the probe used in this work D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 10 :4 3 P M HHV-7 in Allogeneic HSCT Recipients: A 3-Year Survey Intervirology 2011;54:25–29 27 Statistics was done with the Kruskal-Wallis rank test. We compared viral load in samples taken from patients with a single virus with that from patients who had  -herpesvirus coinfection.