[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
Rotavirus antigenemia is frequently observed in a patient's 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.
[show abstract][hide abstract] ABSTRACT: The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.
Journal of clinical microbiology 07/2008; 46(8):2665-70. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human herpesvirus 6 (HHV-6) is a ubiquitous virus with which infections have been associated with pathologies ranging from delayed bone marrow engraftment to a variety of neurological diseases. The lack of a standardized assay that can be used to detect and estimate HHV-6 DNA contents in various clinical specimens can lead and has led to discordant results among investigators and on the potential association of HHV-6 to diseases. To identify the most reliable and sensitive assays, an identical set of 11 coded serum samples spiked with various quantities of the HHV-6A variant (range, 4 to 400,000 genome copies/ml) was sent to eight independent laboratories around the world. Each laboratory was asked to estimate the HHV-6 DNA content by use of its own protocols and assays. Among the various assays, three TaqMan-based real-time PCR assays yielded quantities that were closest to the quantity of HHV-6 that had been spiked. To provide better homogeneity between the results from the different laboratories working on HHV-6, we propose that investigators interested in quantifying HHV-6 in clinical samples adopt one of these assays.
Journal of clinical microbiology 07/2008; 46(8):2700-6. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: The most extensive use of varicella vaccine has been in the United States and Canada, where it is universally recommended. However, a number of other countries now have recommendations for use of the vaccine, which has been expanding in Europe and Latin America. In this article, we review information concerning varicella vaccination in Japan, where the vaccine was first developed, and in South Korea and parts of Europe. Despite the worldwide availability of an efficient vaccine, varicella vaccination policy is highly variable from country to country. The recent development of a tetravalent vaccine against measles, mumps, rubella, and varicella could modify this variability in the future. It is evident that efforts to control varicella will spread gradually to all continents.
The Journal of Infectious Diseases 04/2008; 197 Suppl 2:S185-90. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 alpha 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.
[show abstract][hide abstract] ABSTRACT: The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method.
Journal of Virological Methods 10/2007; 144(1-2):79-85. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.
Journal of Clinical Microbiology 06/2007; 45(5):1426-32. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: A more rapid and easier method is needed for monitoring human herpesvirus 6 (HHV-6) infections. The loop-mediated isothermal amplification method (LAMP) can detect viral DNA with high specificity, efficiency, and speed under isothermal conditions. LAMP requires only simple equipment that is available in hospital laboratories.
We evaluated LAMP as a means of detecting HHV-6 DNA directly from patients' sera.
The sensitivity of the HHV-6 LAMP protocol without heat denaturation was 1000 copies/tube; with heat denaturation 10 copies/tube were detected. Three hundred serum samples from children with fever were analyzed. Using HHV-6 isolation as a definition of HHV-6 infection, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHV-6 LAMP method without DNA extraction were 95.5%, 95.2%, 94.0%, and 96.4%, respectively.
Direct detection of HHV-6 DNA in serum with a modified HHV-6 LAMP could be used for rapid diagnosis of exanthem subitum (ES).
Journal of Clinical Virology 06/2007; 39(1):22-6. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human herpesvirus 6 (HHV-6) reactivation has been suggested to modify the clinical features of drug induced hypersensitivity syndrome (DIHS). However, mechanisms for viral reactivation and modification of the clinical features remain unclear.
To determine whether cytokines play an important role in viral reactivation and modification of the clinical features.
We examined the kinetics of serum cytokines and viral load in HHV-6 infections of six patients with DIHS.
HHV-6 infection occurred three to four weeks after the onset of disease. Elevated TNF-alpha and IL-6 levels were observed to precede HHV-6 infection in four of the six patients. Although high levels of IL-6 were observed in samples collected prior to HHV-6 infection, the amounts of this cytokine significantly decreased to undetectable levels during viral infection in five of the six patients. Subsequently, serum IL-6 levels were increased after viral infection in five patients. IL-1beta levels were also increased at the time of viral infection in three of the six patients. Neither IL-4 nor IFN-gamma could be detected in any of the samples.
These results demonstrate that cytokines play an important role in HHV-6 reactivation in patients with DIHS.
[show abstract][hide abstract] ABSTRACT: The loop-mediated isothermal amplification (LAMP) method is a novel technique for the amplification of specific DNA sequences.
To establish the LAMP method for amplifying Epstein-Barr virus (EBV) DNA and to examine its reliability for the detection of EBV DNA in clinical specimens.
Sera from 108 patients, who were initially suspected of primary EBV infection, were tested by the EBV LAMP method, and the results were compared with those of the real-time PCR assay. Serological examination was regarded as the standard diagnostic method.
To diagnose primary EBV infection, the sensitivity of LAMP was 86.4% and the specificity was 100%. The sensitivity of the real-time PCR assay was 84.1% and the specificity was 98.4%. Longitudinal analysis showed that the detection rate of EBV DNA in serum by the LAMP method decreased with time in accordance with the decrease of the EBV load. EBV DNA could not be detected in serum 40 days after onset of symptoms.
These results indicate that the sensitivity and specificity of the LAMP method are comparable to those of real-time PCR and that detecting EBV DNA in serum by this method is potentially useful for diagnosing primary EBV infection.
Journal of Clinical Virology 11/2006; 37(2):128-33. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: The time-course of cell-mediated immunity in exanthema subitum is not well documented. The lymphoproliferative response to purified human herpesvirus 6 (HHV-6) antigen and to phytohemagglutinin was measured and natural killer (NK) cell activities determined in three consecutive specimens obtained biweekly from 18 young children and infants with exanthema subitum. Virus isolation and PCR detection of virus DNA and determination of neutralization antibody to HHV-6 and -7 were also carried out. The magnitude of the HHV-6 specific lymphoproliferative response varied; however, in most cases the time course kinetics revealed a low response in the acute phase with a subsequent gradual increase. In contrast, NK cell activities were high in the acute phase and declined gradually during convalescence. The lymphoproliferative response to phytohemagglutinin did not show a consistent trend in kinetics of time; however, dynamic changes in activity were observed in patients during the acute and convalescent periods. The results suggest that NK cells play a major role in resolving acute phase infection while specific lymphocyte activity develops later. The cause of the delayed development of HHV-6 specific lymphoproliferative response is unknown. The lymphoproliferative response to phytohemagglutinin ratios implied that HHV-6 infection has some impact on host T-cell immunity during the course of exanthema subitum.
Journal of Medical Virology 07/2006; 78(6):792-9. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: We present a case of pneumonia with marked pleural effusion caused by Aspergillus infection in a 2-year-old Japanese girl with Down's syndrome. The patient was previously diagnosed with acute myeloid leukemia, developing the pneumonia during induction treatment. Although no pathogens could be isolated from any clinical specimens, 135,000 copies/mL of Aspergillus DNA were detected in the pleural fluid using real time polymerase chain reaction. The copy numbers of DNA decreased rapidly after appropriate antifungal treatment.
[show abstract][hide abstract] ABSTRACT: Cytomegalovirus (CMV) loop-mediated isothermal amplification (LAMP) was performed on DNA extracted from CMV (AD-169)-, herpes simplex virus (HSV) 1 (KOS)-, HSV-2 (186)-, varicella-zoster virus (Oka-vaccine)-, human herpesvirus (HHV)-6 A (U1102)-, HHV-6 B (Z29)-, and HHV-7 (RK)-infected cells. Although amplified CMV demonstrated typical ladder patterns, no LAMP product was detected in reactions performed with other viral DNAs. The sensitivity of the CMV LAMP was 500 copies/tube, as determined by either agarose gel electrophoresis or turbidity assay. To determine whether CMV LAMP could be used for quantitative analysis of viral DNA, threshold times, defined as the time (in seconds) to reach the threshold level (0.1), were measured by amplification of serial dilutions of the plasmid DNA. The standard curve exhibited a correlation coefficient of 0.944, a slope of -208.1, and a y-intercept of 3261.4. Following these initial validation experiments, we analyzed 180 samples collected serially from 20 pediatric hematopoietic stem cell transplant recipients. Detection of CMV DNA in whole blood (WB) was tested by CMV LAMP and real-time polymerase chain reaction (PCR). When >500 copies/tube (>5000 copies/200 microl of WB) was defined as positive for CMV infection, the sensitivity, specificity, positive predictive value, and negative predictive values of the CMV LAMP were 80.0, 98.9, 66.7, and 99.4%, respectively.
Journal of Virological Methods 03/2006; 132(1-2):216-21. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been suggested that reactivation of human herpesvirus 6 (HHV-6) infection may be involved in the pathogenesis of drug-induced hypersensitivity syndrome. We report a 45-year-old Japanese man who developed a generalized papuloerythematous rash, fever, hepatitis, lymphadenopathy and lymphocytosis with an increased number of atypical lymphocytes. He was diagnosed with DIHS due to mexiletine hydrochloride based on laboratory data, results of a patch test and the clinical course of his complaint, and was treated with systemic steroids. In order to determine whether HHV-6 or -7 was associated with the patient's disease, serological assays and PCR were carried out. Significant increases in antibody titers against HHV-6 and -7 were observed from day 12 to 24. From PCR analysis, none of the peripheral blood mononuclear cells or skin tissue samples contained HHV-6 DNA. All samples, however, were found to contain HHV-7 DNA. Reactivation of HHV-7 could be responsible for drug-induced hypersensitivity syndrome.
[show abstract][hide abstract] ABSTRACT: To determine the cell populations in peripheral blood that are infected latently with human herpesvirus 7 (HHV-7), the real-time polymerase chain reaction (PCR) was used to determine the quantities of viral DNA in adherent and non-adherent cells from 71 healthy volunteers. Real-time PCR, which detected the U31 gene of HHV-7, was developed to measure viral load. The majority of non-adherent cells (14/16; 87.5%) contained HHV-7 DNA, while most of the adherent cells did not (1/16; 6.3%). HHV-7 viral load in non-adherent cells was significantly higher than that in adherent cells (P < 0.0001). Then, HHV-7 DNA load was compared between the CD4-positive and -negative cell fractions derived from the non-adherent cells of 26 healthy adults. As in the previous experiment, only 2 (7.7%) of the 26 adherent cell specimens contained small amounts of HHV-7 DNA (27.7 copies/1 x 10(6) cells and 208.7 copies/1 x 10(6) cells). In contrast, 88.5% of CD4(+) T cell samples (23/26 specimens) were positive for HHV-7 DNA, ranging from 0.4 to 3,542.8 copies/1 x 10(6) cells. Viral DNA was detected in only 3 (11.5%) of the 26 CD4(-) T cell specimens, with 8.4, 63.5, and 74.1 copies/1 x 10(6) cells. HHV-7-positive DNA loads were significantly higher in the CD4(+) T cells than those observed in the CD4(-) T cells (P = 0.0005). The relationship between HHV-7 viral loads in non-adherent cells and those in saliva was investigated. Comparison of HHV-7 DNA load between blood CD4(+) T cells and saliva revealed that the HHV-7 DNA load in saliva correlated with that present in CD4(+) T cells (r = 0.415; P = 0.0174).
Journal of Medical Virology 01/2006; 78(1):112-6. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: The isolation of human herpesvirus 7 (HHV-7) from saliva and blood, and the prevalence of antibodies to the virus in healthy individuals were investigated in Japan. By cocultivating samples with phytohemagglutinin-P-stimulated cord blood mononuclear cells, HHV-7 was isolated from the saliva of 1 of 20 children and from 4 of 38 adults but not from their blood. The isolates were confirmed as closely related to RK strain of HHV-7, but not to U1102 (human herpesvirus 6, HHV-6 type A) or Z29 (HHV-6 type B) strains by restriction cleavage patterns of the DNA. The virus antibody of 330 healthy children and adults was measured with an indirect immunofluorescence assay, using one of our isolates (FG7-6). The positivity rate of antibody was 40% in the first 2 months of life, declined during the first 6 months, then gradually increased and was 45% at 1-4 years of age. It reached the highest level (60%) at 11-13 years of age and was maintained until the end of the third decade, then decreased thereafter. Additionally, no simultaneous rise in the antibody titers was observed in 7 virologically confirmed exanthem subitum patients.
Journal of Medical Virology 12/2005; 41(4):319 - 323. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Whether an association exists between infection with beta -herpesviruses and connective tissue diseases remains unclear, as are the mechanisms for the regulation of these infections in the salivary glands.
Human herpesvirus (HHV)-7 was isolated and viral DNA was quantified by real-time polymerase chain reaction (PCR) in serially collected saliva samples, to determine whether viral load correlated with infectivity. Then, to examine the role played by beta -herpesviruses in connective tissue diseases, cytomegalovirus, HHV-6, and HHV-7 DNA loads were examined by real-time PCR in serially collected saliva samples from 21 patients with connective tissue diseases.
Although subjects with frequent HHV-7 shedding were more likely to have a high viral load than were other subjects, high viral loads were detected in saliva samples from a portion of the subjects with low viral shedding rates. No significant difference between the quantity of HHV-7 DNA in saliva samples from which active virus was isolated and that amplified from samples without detectable virus was observed. Patients with adult-onset Still disease consistently had high HHV-7 DNA loads, in contrast to patients with other connective tissue diseases (P=.0003) and healthy adults (P=.0224). The mean HHV-6 (P=.012) and HHV-7 (P<.0001) DNA loads in patients with connective tissue diseases were lower than those in healthy adults.
These data suggest that a number of host factors in patients with adult-onset Still disease may function to accelerate HHV-7 replication in the salivary glands.
The Journal of Infectious Diseases 11/2005; 192(9):1530-6. · 5.85 Impact Factor