Fumi Miyake

Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method

ABSTRACT Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.

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.

Detection of Human Herpesvirus 7 DNA by Loop-Mediated Isothermal Amplification

ABSTRACT The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electrophoresis, no HHV-7 LAMP product could be detected in plasma samples after a 30-min LAMP reaction. After a 60-min LAMP reaction, HHV-7 LAMP product could be detected in acute-phase plasma samples.

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.

Transient pancytopenia associated with parvovirus infection in a healthy child

Human parvovirus (HPV) B19 infection has been reported to be a cause of transient erythroblastopenia in patients with hemolytic disorders (aplastic crisis). 1 Human parvovirus B19 is directly cytotoxic for erythroid progenitor cells and inhibits erythropoiesis. 2 Infrequently, HPV B19 inhibits hematopoiesis of three cell lineages and causes transient pancytopenia in patients with hemolytic disorders. 3,4 In experimental infections with normal volunteers, reticulocytes and platelet counts in the peripheral blood decreased 10–14 days after intranasal inoculation, and these hematological abnormalities recovered spontaneously within a few weeks. 5 We report a boy without any underlying disease who developed transient pancytopenia. A HPV B19 infection was confirmed by serological studies and DNA analysis as the causative infectious agent associated with severe pancytopenia.

Single episode of Behcet's disease‐like symptoms caused by herpes simplex virus reactivation

The etiology of Behcet’s disease (BD) has been linked to viral infection, autoimmune disease, streptococcal-related antigens, specific major histocompatibility complex alleles, and hazardous chemicals. Because Herpes simplex virus (HSV) can cause stomatitis, many investigators have searched for an association between HSV and BD. Eglin et al. demonstrated HSV-1 gene fragments in the peripheral blood mononuclear cells of BD patients using in situ DNA-RNA hybridization. Moreover, serum anti-HSV-1 antibodies were found in a higher percentage of BD patients than in controls, and other groups have reported circulating immune complexes to HSV-1 antigen. In addition to the human studies, 30% of ICR mice inoculated with HSV were reported to exhibit Behcet’s disease-like symptoms, including genital ulcers and skin and eye lesions. Viral infection alone, however, cannot sufficiently explain BD pathogenesis. BD has a strong genetic component with familiar aggregation. Among the various genetic markers, a correlation with human leukocyte antigen (HLA) class 1 antigens B5 and the subclass B51 is the most commonly reported, and linkage of the HLA-B locus during BD has also been shown. Here, we describe a patient with the HLA-B51 haplotype with Behcet’s disease-like symptoms concurrent with HSV-1 reactivation.

Guillain-Barré syndrome after exanthem subitum

A female infant developed Guillain-Barré syndrome 20 days after having exanthem subitum confirmed serologically as human herpesvirus 6 infection. DNA of human herpesvirus 6 was detected in peripheral blood mononuclear cells collected on admission.