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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. · 3.97 Impact Factor
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Takuji Kumagai, Tetsushi Yoshikawa,
Mariko Yoshida,
Toyo Okui,
Masaru Ihira,
Nobuo Nagata,
Shoki Yano,
Kimiyasu Shiraki,
Masao Yamada,
Kiyoshi Ichihara,
Yoshizo Asano
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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. · 2.82 Impact Factor
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Nippon rinsho. Japanese journal of clinical medicine 04/2006; 64 Suppl 3:466-71.
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Nippon rinsho. Japanese journal of clinical medicine 04/2006; 64 Suppl 3:485-9.
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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.
The Pediatric Infectious Disease Journal 03/2006; 25(2):186-7. · 3.58 Impact Factor
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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. · 2.01 Impact Factor
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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.
Dermatology 02/2006; 213(4):341-4. · 2.05 Impact Factor
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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. · 2.82 Impact Factor
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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. · 2.82 Impact Factor
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ABSTRACT: Three strains of human herpesvirus-6 (HHV-6) were isolated from peripheral blood mononuclear cells of a leukemic child with the antibody of HHV-6 before and after bone marrow transplantation (BMT); two strains were obtained before BMT and one after BMT. The DNA extracted from the three isolates was analyzed by six different restriction endonucleases. The cleavage profiles of two strains obtained before BMT were different, but the third strain isolated after BMT was identical with one of the two, which suggest reactivation of HHV-6 from the recipient's own body after BMT and possible mutation or superinfection of the virus in an immunocompromized patient. © 1992 Wiley-Liss, Inc.
Journal of Medical Virology 12/2005; 37(3):228 - 231. · 2.82 Impact Factor
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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. · 6.41 Impact Factor
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ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.
Medical Microbiology and Immunology 09/2005; 194(4):181-5. · 3.83 Impact Factor
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ABSTRACT: The herpes simplex virus (HSV) US3 gene encodes a serine/threonine protein kinase (PK). Although US3 PK is not essential for virus replication in cell culture, it plays an important role in the regulation of apoptosis in infected cells. However, the role of US3 PK in virus replication and pathogenicity is not well understood. The UL46 gene encodes virion tegument phosphoproteins, the properties and functions of which are poorly understood. In this study, it is shown that the UL46 protein of HSV type 2 (HSV-2) is affected strikingly by the presence of US3 PK. In the absence of US3 PK, UL46 protein was quite unstable, being much more susceptible to degradation. UL46 protein was undetectable in the extracellular virions of US3-deficient virus. Moreover, in vitro kinase assays using recombinant US3 PK show that UL46 protein is phosphorylated by the US3 PK, suggesting that UL46 can be a direct substrate for US3 PK in infected cells. Together, these findings shed new light on the physiological functions of US3 PK.
Journal of General Virology 08/2005; 86(Pt 7):1979-85. · 3.36 Impact Factor
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ABSTRACT: A 4-year-old Japanese girl developed a sore throat and high fever. Her tonsils were enlarged, red and covered with a thick white membrane. There was marked leukocytosis (26,600 leukocytes per mm) and elevated C-reactive protein levels (23.3 mg/dL). Rothia dentocariosa was recovered from the throat swab; many Gram-positive cocci were observed in the smear from the pseudomembrane covering the tonsil.
The Pediatric Infectious Disease Journal 06/2005; 24(5):466-7. · 3.58 Impact Factor
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ABSTRACT: This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.
Journal of Medical Virology 05/2005; 75(4):583-7. · 2.82 Impact Factor
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Yoshihiko Enomoto, Tetsushi Yoshikawa,
Masaru Ihira,
Shiho Akimoto,
Fumi Miyake,
Chie Usui,
Sadao Suga,
Kayoko Suzuki,
Takashi Kawana,
Yukihiro Nishiyama,
Yoshizo Asano
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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.
Journal of Clinical Microbiology 03/2005; 43(2):951-5. · 4.15 Impact Factor
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ABSTRACT: The aim of this study is to determine whether human herpesvirus 6 (HHV-6) and HHV-7 might play an important role in causing interstitial pneumonia in patients who have not undergone transplantation. HHV-6 and HHV-7 DNAs were quantitated by real-time polymerase chain reaction (PCR) in paraffin embedded lung tissues collected from 24 patients having the disease. Control tissues (without fibrosis) were also collected from 19 of the 24 patients. Statistical analysis was carried out by the Wilcoxon signed rank test or the Mann-Whitney U-test. HHV-6 DNA was detected in 3 (12.5%) of the 24 target tissues and 3 (15.8%) of the 19 control tissues, respectively. In contrast, HHV-7 DNA was detected in 19 (79.2%) of the 24 target tissues and 11 (57.9%) of the 19 control tissues. Neither HHV-6 DNA load (P = 0.6395) nor HHV-7 DNA load (P = 0.5966) in target tissues differed between males and females. Neither HHV-6 DNA load (P = 0.9589) nor HHV-7 DNA load (P = 0.7419) in target tissues differed between cases with and without underlying collagen disease. While HHV-6 DNA load did not differ between the target and control tissues (P > 0.9999), the HHV-7 DNA load was significantly higher in the target tissue than in the control tissue (P = 0.0298). This study suggests that HHV-7 may play an important role in causing interstitial pneumonia in patients who are not transplant recipients.
Journal of Medical Virology 01/2005; 75(1):70-5. · 2.82 Impact Factor
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ABSTRACT: Perilymph and peripheral blood mononuclear cells (PBMCs) from patients with bilateral severe sensorineural hearing loss (SNHL) were evaluated for the presence of DNA from cytomegalovirus (CMV), herpes simplex virus (HSV), and human herpesvirus (HHV)6.
A prospective clinical study.
The subjects were 14 patients who underwent cochlear implantation and 1 patient who underwent gentamicin injection in the inner ear. We attempted to detect viral DNA from perilymph and PBMCs by real-time polymerase chain reaction (rtPCR).
CMV DNA was detected in two perilymph specimens obtained from patients who were diagnosed as congenitally symptomatic CMV infection, although no CMV DNA was detected in PBMCs. Neither HSV DNA nor HHV6 DNA was detected in any other perilymph specimens. CMV DNA was detected in three PBMC samples, HSV DNA was detected in two samples, and HHV6 DNA was detected in six samples.
CMV may persistently infect the inner ear of patients with congenital CMV infection, and rtPCR analysis may prove to be a valuable tool for investigating the etiology of SNHL.
The Laryngoscope 01/2005; 114(12):2235-8. · 1.75 Impact Factor
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Masahiro Ohashi, Tetsushi Yoshikawa,
Katsuhiro Asonuma,
Takuya Iwasaki,
Yukihiro Nishiyama,
Yoshizo Asano,
Yasuo Kimoto,
Takahito Yagi,
Naoto Urushihara,
Noriaki Tanaka,
Kiyoshi Baba
Pediatrics International 01/2005; 46(6):730-2. · 0.63 Impact Factor
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ABSTRACT: The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA; no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity; 87.0%, specificity; 100%, positive predictive value; 100%, negative predictive value; 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction.
Journal of Medical Virology 01/2005; 74(4):677-82. · 2.82 Impact Factor
