Naoko Tanaka

Quantitative analysis of cytomegalovirus load using a real-time PCR assay.

A novel real‐time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real‐time PCR assay could detect from 6 to over 106 copies of CMV‐DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 103.3 copies/106 cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (102.0 copies/106 cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 104.0 copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti‐CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV‐related diseases and monitoring the virus load in patients with CMV infections. J. Med. Virol. 60:455–462, 2000.

Prospective monitoring of the Epstein–Barr virus DNA by a real‐time quantitative polymerase chain reaction after allogenic stem cell transplantation

Epstein‐Barr virus (EBV)‐related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)‐HSCT patients, focusing on the use of anti‐thymocyte globulin (ATG). We used a recently developed real‐time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47·4% (nine patients) had a significant increase in EBV genome load (102·5 copies/µg DNA). Of these nine patients, two developed LPD. Therefore, 10·5% of the patients receiving allo‐HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 104·0 copies/µg DNA. On the other hand, the viral loads of most patients with no symptoms were < 102·5 copies/µg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 102·5 copies/µg DNA is the reactivation of EBV, and that an EBV load > 104·0 copies/µg DNA is indicative of developing LPD.

Detection of Herpesvirus DNA in the Serum of Immunocompetent Children

The DNA of herpesviruses such as Epstein‐Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus‐6 (HHV6), and human herpesvirus‐7 (HHV7) has been detected in the serum of patients with primary infection or with immunosuppression. However, it is unknown how frequently herpesvirus DNA can be detected in the serum of immunocompetent children, or whether the detection of herpesvirus DNA indicates an active infection or virus‐related diseases. Using a real‐time polymerase chain reaction assay, attempts were made to detect herpesvirus DNA in the serum of 176 ambulatory children who visited a hospital for various reasons. EBV was detected in 4 (2.2%), HHV6 in 4 (2.2%), and HHV7 in 2 (1.1%) of 176 children, but CMV was not detected. Of the 10 positive patients, only 4 were considered, by virtue of clinical and serological characteristics, to have primary infections. The other 4 positive patients had other infections, such as mycoplasma and salmonella. Although herpesvirus DNA could be detected in the serum of immunocompetent children, there was not always a relationship between clinical manifestations and the detection of virus DNA. When herpesvirus DNA is detected in the serum, a careful interpretation is necessary to diagnose a primary infection or a virus‐associated disease.

Monitoring of Active HHV-6 Infection in Bone Marrow Transplant Recipients by Real Time PCR; Comparison to Detection of Viral DNA in Plasma by Qualitative PCR

Twelve (46%) of the 26 patients had human herpesvirus 6 (HHV‐6) viremia after bone marrow transplant (BMT). All isolates were recovered from the samples obtained at 2 weeks after BMT. The sensitivity and the specificity of detection of viral DNA in plasma by qualitative polymerase chain reaction (PCR) for monitoring active virus replication were 92% and 97% respectively. Moreover, the positive (85%) and negative (99%) predictive values were also high. The patients with HHV‐6 viremia showed a clear peak in HHV‐6 DNA in peripheral blood mononuclear cells (PBMCs) at 2 weeks after BMT, which was measured by real time PCR. The virus DNA level in PBMCs between the two groups (patients with viremia and patients without viremia) was statistically different at 2 weeks after BMT (P = 0.033). In patients with HHV‐6 viremia, mean HHV‐6 DNA copy number was higher in the samples collected at 2 weeks after BMT than the samples collected at any other time period.

Prevalence of Maternal Cytomegalovirus (CMV) Antibody and Detection of CMV DNA in Amniotic Fluid

The prevalence of cytomegalovirus (CMV) IgG antibody was determined in 573 pregnant women in the first trimester. The overall prevalence of CMV IgG antibody was 77.5%. The rate of seropositivity was 67.7% in women < 25 yr, and increased with age to 85.7% in women ≧40 yr. These results imply that young women in Japan are at increased risk for primary CMV infection during pregnancy and that congenital CMV infection rates might increase in the future. We conducted a prospective study of 75 pregnant women who underwent amniocentesis for various indications to determine if CMV DNA could be detected in the amniotic fluid. None had symptoms associated with CMV infection, CMV IgM antibody, or seroconversion to CMV IgG antibody during pregnancy. CMV DNA was not detected in the amniotic fluid using a polymerase chain reaction assay. The 65 fetuses, including 3 sets of twins, were followed through birth. CMV DNA was not detected in urine samples obtained within the first 2 weeks of life. In conclusion, CMV DNA was not detected in the amniotic fluid of women who did not have CMV infection. These results, however, suggest that the negative predictive value of prenatal amniotic fluid analysis is high and that the presence of CMV DNA in the amniotic fluid has clinical significance for the diagnosis of congenital CMV infection if detected in pregnant women.

Vidarabine therapy for Severe chronic active Epstein-Barr virus infection

Purpose Severe chronic active Epstein–Barr virus infection (SCAEBV) is an intractable disease with a poor prognosis, and a definitive treatment has not been established. We administered vidarabine to patients with natural killer (NK) cell-type SCAEBV and evaluated clinical and virologic effects. Patients and Methods Four patients with SCAEBV were enrolled in this study. These patients had various symptoms, including fever, chronic hepatitis, hepatosplenomegaly, and hypersensitivity to mosquito bites. All patients had increased numbers of NK cells in their peripheral blood, and most of these were infected with EBV. Viral load was measured by in situ hybridization and quantitative polymerase chain reaction (PCR). Results The patients all responded to the therapy, and their symptoms improved. After the therapy, the number of NK cells in their peripheral blood decreased. In two patients who were closely monitored, the viral load measured by in situ hybridization and quantitative PCR decreased in parallel with the symptomatic improvement. After discontinuing this drug, the patients symptoms returned and the Epstein–Barr virus load increased again. Conclusion These results indicate that vidarabine therapy is a therapeutic choice to control SCAEBV, although its effect may be transient.

Expression of tegument protein pp65 of human cytomegalovirus (CMV) and its application to the analysis of viral-specific cellular immunity in CMV-infected individuals

Summary. Investigations into human cytomegalovirus (CMV)-specific cellular immunity are important to better understand and manage CMV infections. CMV phosphoprotein pp65 is thought to be a major antigen for CMV-specific cellular immunity. We newly synthesized protein pp65 with a baculovirus expression system and purified it via metal affinity chromatography in a soluble form. The recombinant protein pp65 was antigenic in an enzyme immuno-linked assay for pp65-specific IgG in sera from 196 children. Traditional lymphoproliferation assays have shown that pp65 protein promotes specific lymphoproliferation in CMV-seropositive donors. Using an intracellular cytokine detection system, we showed that this recombinant protein stimulated CD4-positive T cells to express interferon-γ. The results of these assays using protein pp65 were comparable with the use of CMV whole antigen. pp65- and CMV-specific cellular immunity, and CMV DNA load were also compared in four recipients of unrelated cord blood transplantation. The delay in re-constitution in CMV-specific cellular immunity was associated with reactivation of CMV. These results indicated that the recombinant protein pp65 can be used to study specific immunity in CMV infections.