Shiho Akimoto

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.

Variation of Human Herpesvirus 7 Shedding in Saliva

Sequential analysis of human herpesvirus 7 (HHV-7) shedding in saliva obtained from healthy adults was performed for 6 months; virus was isolated in 92 (34.1%) of the 270 saliva samples obtained. Frequency of HHV-7 shedding in serially obtained saliva samples varied among subjects. Associations between frequency of HHV-7 shedding and age, sex, and virus antibody titer were analyzed, and, although sex was not associated with frequency of shedding, young age (P=.031) and low antibody titer (P=.006) were correlated with frequent viral shedding (5 or 6 times/6 months).