Ken Sugata

Analysis of rotavirus antigenemia and extraintestinal manifestations in children with rotavirus gastroenteritis.

OBJECTIVE. This study was conducted to examine the association between rotavirus antigenemia and clinical features, particularly extraintestinal manifestations, and the association between serum cytokine levels and rotavirus antigen quantity. METHODS. Sixty hospitalized children who received a diagnosis of acute rotavirus gastroenteritis were enrolled in this study. Paired serum samples were collected from the 60 children when admitted to and discharged from the hospital. Associations among viral antigen levels and fever, elevated transaminase levels, and seizures were evaluated to determine whether antigenemia correlated with disease severity. Viral antigen was measured by using an in-house enzyme-linked immunosorbent assay that detected VP6 antigen. A flow-cytometric bead array was used to measure serum cytokine levels. RESULTS. Rotavirus antigen levels were significantly higher in serum collected at the time of hospital admission than at the time of discharge. Serum rotavirus antigen levels peaked on day 2 of the illness (2.02 ± 0.73), followed by a gradual decrease in antigen levels to nearly undetectable levels by day 6. The quantity of rotavirus antigen was significantly higher in serum collected from patients with fever than those without fever. The presence or absence of elevated transaminase levels and seizures was not associated with serum rotavirus antigen levels. A weak but significantly positive association was observed between interleukin 8 levels and antigenemia. A weak but significantly negative association was observed between interleukin 10 levels and antigenemia. CONCLUSIONS. Rotavirus antigenemia is frequently observed in a patients serum during the acute phase, and viral antigen levels change dramatically during the acute phase of the illness. Because patients with fever had higher rotavirus antigen levels, antigenemia severity might contribute to fever. The host immune response plays an important role in controlling antigenemia levels.

Exanthem subitum-associated encephalitis: nationwide survey in Japan.

We sought to clarify clinical features of exanthem subitum associated-encephalitis/encephalopathy, generally caused by primary human herpesvirus-6 infection in Japan. A two-part questionnaire was sent to hospitals between January 2003-December 2004. Of 3357 questionnaires, 2357 (70.2%) were returned, and 2293 (68.3%) were eligible for analysis. Eighty-six cases of exanthem subitum-associated encephalitis/encephalopathy were reported. Seventy-seven (89.5%) of 86 patients were diagnosed with human herpesvirus-6 infection by virologic examination. Although 41 (50.6%) of 81 patients had no sequelae, 38 (46.9%) had neurologic sequelae. Moreover, two fatal cases (2.5%) were reported. Pleocytosis was evident in only 4 (7.5%) of 53 patients, and cerebrospinal fluid protein levels were within normal range (23.4 +/- 14.6 mg/dL S.D.) in all patients. Human herpesvirus-6 DNA was detected in 21 (53.8%) of 39 patients. Abnormal computed tomography findings were a predictor of neurologic sequelae (P = 0.0097). As a consequence of this survey, we estimate that 61.9 cases of exanthem subitum-associated encephalitis occur every year. The disease prognosis was unexpectedly poor.

Different characteristics of human herpesvirus 6 encephalitis between primary infection and viral reactivation

BACKGROUND Pathogenesis of human herpesvirus 6 (HHV-6) encephalitis, in particular difference between HHV-6 encephalitis at the time of primary infection and reactivation remains unclear. OBJECTIVES To elucidate the mechanism of HHV-6 encephalitis at the time of primary infection and reactivation. STUDY DESIGN Twenty-two HHV-6 encephalitis patients at the time of primary infection, 6 febrile convulsion (FC) patients caused by HHV-6 infection, and 14 FC patients without HHV-6 infection (non HHV-6 FC) were enrolled. Additionally, 7 stem cell transplant recipients with HHV-6 encephalitis and eight adult controls were also enrolled in this study. Cerebrospinal fluid (CSF) HHV-6 DNA copy numbers and biomarkers levels were compared. RESULTS Low copy number of CSF HHV-6 DNA was detected in 7 of the 22 patients with HHV-6 encephalitis in primary infection, whereas all seven CSF samples collected from post-transplant HHV-6 encephalitis patients contained high viral DNA copy numbers (P<0.001). CSF concentrations of IL-6 (P=0.032), IL-8 (P=0.014), MMP-9 (P=0.004), and TIMP-1 (P=0.002) were significantly higher in patients with HHV-6 encephalitis in primary infection than non-HHV-6 FC. CSF IL-6 (P=0.008), IL-8 (P=0.015), and IL-10 (P=0.019) concentrations were significantly higher in patients with post-transplant HHV-6 encephalitis than adult controls. CONCLUSION The present study suggests that the characteristics of HHV-6 encephalitis are different between HHV-6 encephalitis at the time of primary infection and reactivation in transplant recipients.

Elevated serum cytokine levels are associated with human herpesvirus 6 reactivation in hematopoietic stem cell transplantation recipients

Although it has been demonstrated that human herpesvirus 6 (HHV-6) reactivation generally occurs approximately 2-3 weeks after transplantation in the hematopoietic stem cell transplantation (HSCT) recipients, the mechanism of viral reactivation remains unclear. To explore the relationship between HHV-6 reactivation and plasma cytokine levels, 24 HSCT recipients underwent measurements of plasma proinflammatory cytokine levels (IL-6, TNF-alpha, IL-1 beta, and IFN-gamma), viral isolation, and serological assays. Of these patients, 14 developed an HHV-6 reactivation, and 9 developed HHV-6 viremia approximately 2-3 weeks after transplantation. IL-6 levels were significantly higher in the recipients with an HHV-6 reactivation than in the subjects without an HHV-6 reactivation at 1 week, 2 weeks, and 4 weeks after transplantation. In addition, the level of TNF-alpha was significantly higher in recipients with an HHV-6 reactivation than in those without an HHV-6 reactivation at 2 weeks post-transplantation. Low levels of IL-1 beta and IFN-gamma were detected in a small number of the plasma samples, although there were no significant differences between the two groups in the levels of these cytokines. These results imply that proinflammatory cytokines, in particular IL-6 and TNF-alpha, play a role in the pathogenesis of HHV-6 reactivation after HSCT.

Human herpesvirus 6 infection in adult living related liver transplant recipients

To analyze human herpesvirus 6 (HHV‐6) infection in adult living related liver transplantation, we performed a virological analysis, including viral isolation, serological assay, and real‐time polymerase chain reaction, of serially collected blood samples from 67 recipients. In addition, cytokine levels were measured to determine their role in viral reactivation. HHV‐6 was isolated from only 4 recipients (6.0%), and viral DNA was detected in 15 (22.4%) of the 67 recipients. A significant increase in HHV‐6 immunoglobulin G antibody titers was observed in 19 (28.4%) of the 67 recipients. Finally, 26 recipients (38.8%) had HHV‐6 reactivation 2‐6 weeks after transplantation. HHV‐6 associated clinical features were analyzed in the 17 recipients presenting with either viremia or DNAemia. Two recipients with viremia and 3 recipients with DNAemia had unexplained fever at the time of viral infection. An increase in aminotransferase levels was observed in 2 recipients with viremia and 3 recipients with DNAemia. Recipients with liver cirrhosis caused by hepatitis B virus or hepatitis C virus infection as the underlying disease were more likely to have HHV‐6 infection (P = 0.025). Mortality at the last follow‐up in recipients with HHV‐6 reactivation was significantly higher than in those without viral reactivation (P = 0.0118). Plasma interleukin‐6 levels were significantly higher in the recipients with HHV‐6 viremia than in the recipients without viremia at 4 weeks post‐transplant (P = 0.0411). Moreover, tumor necrosis factor α levels were also higher in recipients with HHV‐6 viremia (P < 0.0001) or reactivation (P = 0.0011) than in recipients without viremia or reactivation 4 weeks post‐transplant. Liver Transpl, 2007.

Evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza A/H1N1 2009 virus†

Two genetic diagnosis systems using reverse transcription‐loop‐mediated isothermal amplification (RT‐LAMP) technology were evaluated: one for detecting the HA gene of the pandemic influenza A/H1N1 2009 virus (H1pdm RT‐LAMP) and the other for detecting the matrix gene of the influenza A virus (TypeA RT‐LAMP). The competence of these two RT‐LAMP assay kits for the diagnosis of the pandemic influenza A/H1N1 2009 virus was compared using real‐time RT‐PCR assays developed recently on viruses isolated and clinical specimens collected from patients with suspected infection. TypeA RT‐LAMP and H1pdm RT‐LAMP showed almost the same sensitivity as real‐time RT‐PCR for viruses isolated. The sensitivity and specificity of TypeA RT‐LAMP and H1pdm RT‐LAMP were 96.3% and 88.9%, respectively, for clinical specimens. Considering that the ability of the two RT‐LAMP assay kits for detection of the pandemic influenza A/H1N1 2009 virus was comparable to that of the real‐time RT‐PCR assays, and that the assays were completed within 1 hr and did not require any expensive equipment, these two RT‐LAMP assays are promising rapid diagnostic tests for the pandemic influenza A/H1N1 2009 virus at the hospital bedside. J. Med. Virol. 83:10–15, 2011.

Development of a Human Herpesvirus 6 Species-Specific Immunoblotting Assay

ABSTRACT In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts†‡

The monitoring of active human herpesvirus 6 (HHV‐6) B infection is important for distinguishing between the reactivation and latent state of the virus. The aim of this present study is to develop a quantitative reverse transcription polymerase chain reaction (RT‐PCR) assay for diagnosis of active viral infection. Primers and probes for in house quantitative RT‐PCR methods were designed to detect the three kinetic classes of HHV‐6B mRNAs (U90, U12, U100). Stored PBMCs samples collected from 10 patients with exanthem subitum (primary HHV‐6B infection) and 15 hematopoietic stem cell transplant recipients with HHV‐6B reactivation were used to evaluate reliability for testing clinical samples. Excellent linearity was obtained with high correlation efficiency between the diluted RNA (1–100 ng/reaction) and Ct value of each gene transcript. The U90 and U12 gene transcripts were detected in all of the peripheral blood mononuclear cells (PBMCs) samples collected in acute period of primary HHV‐6B infection. Only one convalescent PBMCs sample was positive for the U90 gene transcript. Additionally, the reliability of HHV‐6B quantitative RT‐PCRs for diagnosis of viral reactivation in hematopoietic transplant recipients was evaluated. Relative to virus culture, U90 quantitative RT‐PCR demonstrated the highest assay sensitivity, specificity, positive predictive value, and negative predictive value. Thus, this method could be a rapid and lower cost alternative to virus culture, which is difficult to perform generally, for identifying active HHV‐6B infection. J. Med. Virol. 84:1388–1395, 2012.

Serum biomarker kinetics with three different courses of HHV-6B encephalitis

Human herpesvirus-6B (HHV-6B) encephalitis can clinically manifest as hemorrhagic shock and encephalopathy syndrome (HSES), acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), and acute necrotizing encephalopathy (ANE). To compare the underlying pathophysiology, we measured several biomarkers of interest in patients with these three different courses. Based on their clinical course and neuroimaging analysis, Cases 1, 2 and 3 were diagnosed as HSES, AESD, and ANE, respectively. HHV-6B was isolated from peripheral blood obtained during the acute phase in all three patients, and was detected in the cerebrospinal fluid of Cases 2 and 3. In Case 1, a marked increase in levels of several serum cytokines (IL-1β, IL-6, and IL-10) and chemokines (IL-8, MIG, MCP-1, and IP-10) was observed at disease onset. Subsequently, serum cytokine levels gradually became undetectable and chemokine levels stabilized by day 11 of illness. In Case 2, only two cytokines (IL-6 and IL-10) were slightly elevated at disease onset. In Case 3, the kinetics appeared to follow an up-and-down pattern. Additionally, in all three patients, TIMP-1 concentrations remained high during the observation period, and MMP-9 decreased quickly a few days after disease onset, and then returned to normal level.

Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B

Genotyping of human herpesvirus 6 (HHV-6) is important clinically, particularly for the diagnosis of neurological diseases. The objective of this study was to establish a rapid HHV-6 genotyping method using the loop-mediated isothermal amplification (LAMP) method. An AccI site is located in the target sequence of HHV-6 B, but not in HHV-6 A. LAMP products were digested with the AccI enzyme and then separated by agarose gel electrophoresis to differentiate the digest pattern of the two variants. The fragment patterns were clearly different between HHV-6 A and B. In order to evaluate the reliability of this HHV-6 genotyping method for use in the clinical laboratory, serum samples from 20 patients with either primary HHV-6 infection or viral reactivation were collected and analyzed. HHV-6 DNA was amplified directly from the serum samples and all 20 LAMP products were positive for HHV-6 B.