Yoshihiko Enomoto

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.

Rapid Diagnosis of Human Herpesvirus 6 Infection by a Novel DNA Amplification Method, Loop-Mediated Isothermal Amplification

ABSTRACT A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the methods sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.

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.

Monitoring herpesviruses DNA in three cases of acute retinal necrosis by real-time PCR

Abstract Background: It is not clear whether quantitative analysis of viral DNA in ocular specimens is correlated with disease activities of acute retinal necrosis (ARN). Objectives: To monitor viral load in ocular specimens collected from patients with ARN by real-time polymerase chain reaction (PCR). Study design: Ocular samples (aqueous humor and vitreous) were serially collected from three patients with ARN. Viral load in those samples was evaluated by real-time PCR. Result and conclusion: In case 1, large amounts of varicella zoster virus (VZV) DNA (4.8×106 to 5.5×106 copies/ml) were detected in aqueous humor during the first 2 weeks after admission. The viral load in vitreous was higher than that in aqueous humor at the time of vitrectomy. As ophthamoscopic findings and visual acuity improved through acyclovir (ACV) treatment, the viral load in aqueous humor decreased dramatically. In case 2, the patient was treated with intravenous ACV at first, but clinical features did not improve. The herpes simplex virus (HSV)-2 viral load in aqueous humor remained stable (2.3×103 to 2.8×103 copies/ml) during the first 3 weeks after admission. The amount of HSV-2 DNA in vitreous was again higher than that in aqueous humor. Although neither clinical features nor viral load had changed by ACV, intra-ocular ganciclovir (GCV) injection improved clinical features, and decreased viral load to undetectable levels. In case 3, the patient developed ARN within 1 month after the onset of varicella and demonstrated only mild clinical symptoms. She was treated with ACV administration alone and recovered quickly. In contrast to case 1, the copy number of VZV DNA at the time of admission was low (9×102 copies/ml), and decreased quickly in response to the treatment. Correlation between viral load in ocular specimens and clinical course of the disease was demonstrated in these patients.

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.

Heat denaturation increases the sensitivity of the cytomegalovirus loop-mediated isothermal amplification method

A new protocol for CMV LAMP with an additional heat denaturation step was developed. While the sensitivity of the original CMV LAMP method was 500 copies/tube, sensitivity was increased by up to 100 copies/tube by additional heat denaturation. CMV DNA was detected in 103 of 350 samples (29.4%) by the original CMV LAMP procedure and 148 of 350 samples (42.3%) by the new CMV LAMP protocol. When the pp65 antigenemia assay was used as the standard method, the sensitivity, specificity, PPV, and NPV of the new protocol were 92.9%, 77.7%, 62.2%, and 96.5%, respectively.

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.

Direct detection of human herpesvirus 6 DNA in serum by variant specific loop-mediated isothermal amplification in hematopoietic stem cell transplant recipients.

A variant specific direct loop-mediated isothermal amplification (LAMP) method was developed to detect human herpesvirus-6 (HHV-6) variants in serum samples. Specific primers were designed against HHV-6 U86 gene. Initial validation analysis confirmed high specificity and sensitivity of the method. This method was shown to be highly reliable for monitoring active HHV-6 infection in hematopoietic stem cell transplant recipients.

Host factors associated with the kinetics of Epstein-Barr virus DNA load in patients with primary Epstein-Barr virus infection

The aims of this study were to elucidate the kinetics of Epstein‐Barr virus (EBV) DNA load in serially collected peripheral blood mononuclear cells of patients with primary EBV infection, and to determine the correlated host factors. Blood samples were collected from 24 patients with primary EBV infection. EBV DNA copy numbers were measured using real‐time polymerase chain reaction. Based on the kinetics of EBV DNA load, the 24 patients were divided into two groups: rapid regression and slow regression. Eighteen of the 24 patients (75%) were included in the slow regression and 6 (25%) in the rapid regression group. No statistically significant differences were observed between the two groups in clinical features and laboratory findings. However, acute phase (3 to 10 days after the onset of the illness) serum samples from six children in the slow regression and four in the rapid regression group revealed significantly higher serum interleukin (IL)‐1β (P= 0.018), IL‐12 (P= 0.009), tumor necrosis factor‐α (P= 0.019), interferon‐inducible protein 10, and monokine induced by interferon γ concentrations in the rapid regression than the slow regression group. On the other hand, sera from six children in the slow regression and four in the rapid regression group in the convalescent phase (14 to 21 days after the onset of the illness) showed no statistically significant differences between the two groups in these biomarker concentrations. Based on this, it was concluded that the kinetics of EBV DNA load can be divided to two different patterns after primary EBV infection, and immune response might be associated with viral clearance.

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.