Takashi Kawana

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.

Are smoking and chlamydial infection risk factors for CIN? Different results after adjustment for HPV DNA and antibodies.

To identify the risk factors for cervical intraepithelial neoplasia (CIN), we reanalysed the data from our previous case–control study by adjusting for human papillomavirus (HPV) antibodies. Unlike our previous study based only on HPV DNA, smoking and Chlamydia trachomatis infection were revealed as significant risk factors for CIN after adjustment for HPV antibodies.

Prognostic factors associated with the clinical outcome of cervical intraepithelial neoplasia: a cohort study in Japan

One hundred and eighty-five Japanese women with cervical intraepithelial neoplasia (CIN) were enrolled in this follow-up study. On the basis of the prevalence of human papillomavirus (HPV) DNA in Japanese cervical cancer patients, HPV types were categorized into three groups as follows: (1) high risk (types 16, 18, 33, 52, and 58), (2) intermediate risk (types 31, 35, 39, 51, 56, 59, 68, and 70), (3) low risk (type 6, 30, 42, 53, 54, 55, 66 and unclassified types). High-risk HPV infection was a risk factor for progression of the disease. The regression rate in the HPV negative group was higher (83.3%) than those in the HPV positive groups, but the differences in regression were no longer significant after adjustment for age and CIN grade. It is also noted that a lower cytomegalovirus IgG level and a smaller number of past pregnancies might be associated with the regression of CIN lesions.

Discrimination of Herpes Simplex Virus Type 2 Strains by Nucleotide Sequence Variations

ABSTRACT We determined the polymorphous 400-bp regions in UL53, US1, and US4 for the discrimination of herpes simplex virus type 2 (HSV-2) strains. Thirty-six HSV-2 clinical strains could be differentiated into 35 groups using these three regions and into 36 groups by additional analysis of three noncoding regions previously reported as polymorphous.

Evaluation of mixed infection cases with both herpes simplex virus types 1 and 2

Herpes simplex virus type 1 (HSV‐1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV‐2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV‐1 and HSV‐2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV‐1 and HSV‐2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV‐1 and HSV‐2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV‐1 and HSV‐2 genome by a quantitative real time PCR demonstrated that HSV‐1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV‐2 genome was present at a 4–40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV‐1 or HSV‐2 infections. This study indicated that the frequency of mixed infection with both HSV‐1 and HSV‐2 is comparatively higher than those of previous reports. The genome ratio of HSV‐1 and HSV‐2 reflects the preference of each HSV type for the target organ. J. Med. Virol. 80:883–887, 2008.

Examination of the correlation between the manual and automated serological testing methods for syphilis

We evaluated the correlation between the conventional manual serological testing method for syphilis and a novel automated serological testing method and between six different reagents used in the automated method. Twenty‐six serum samples, which were positive on non‐treponemal manual serological testing, were obtained from 19 patients with early syphilis. The samples were manually analyzed using the non‐treponemal serological test for syphilis kit and automatically analyzed using six different reagents approved by the Ministry of Health, Labor and Welfare in Japan. Statistically significant correlations were observed between most of the reagents used in the automated testing (r = 0.652–0.996, P < 0.001), except for one combination of the reagents. In the simple regression analysis, the slope of the simple regression line (range, 0.014–3.040) and some of the regression coefficients were not equal to 1.0. Therefore, it is recommended that when the automated serological testing method is used to test for syphilis, the same reagent should be consistently selected to evaluate the changes in antibody titers. Statistically significant correlations were also observed between the manual method and all the reagents used in the automated method (r = 0.682–0.811, P < 0.001). In this case, the regression coefficients ranged 0.375–6.270, and the simple regression line intercept ranged −71.926 to 4.184. The regression coefficient and the intercept between the manual method and some of the reagents used in the automated method were not similar to the values described in the documentation attached to the reagents used in this study.

Novel deletion in glycoprotein G forms a cluster and causes epidemiologic spread of herpes simplex virus type 2 infection

The herpes simplex virus type 2 (HSV‐2) glycoprotein G (gG‐2) gene of 106 clinical isolates was analyzed and six isolates were identified with 63 nucleotides comprising 21 amino acids (aa) deleted in the immunodominant region. Compared with strain HG52, variations in the gG‐2 gene were found at 276 and 27 sites in nucleotide and aa sequences, respectively, in the 106 strains. Significant variations in both nucleotides and aa were accumulated in the immunodominant region rather than in the other regions (P < 0.001), indicating that the immunodominant region might be indispensable in vivo and a hot spot for variation. The frequency of 21 aa‐deleted strains (HSVΔ21/gG‐2) among clinical isolates was 5%, indicating the advantage of this deletion of gG‐2 for epidemiological expansion. Phylogenetic analysis of the 106 strains indicated that the HSVΔ21/gG‐2 strains formed a cluster among the various variations but that their genomes showed different endonuclease digestion patterns. The antibody titers to total HSV antigens of patients infected with wild HSV‐2 and HSVΔ21/gG‐2 were similar, but patients with HSVΔ21/gG‐2 had a lower antibody titer to gG‐2 than those with wild HSV‐2 (P < 0.001). HSVΔ21/gG‐2 might be less immnunogenic and reduce antibody production to gG‐2, while its pathogenicity in humans was not distinguished in its clinical manifestations. Thus, infection with HSVΔ21/gG‐2 caused genital lesions similar to wild HSV‐2 infection, but evaded the immune response to gG‐2 to allow epidemiological spread, indicating the importance of this deletion in the immunodominant region of gG‐2 in the pathogenesis and transmission of genital herpes. J Med. Virol. 85:1818–1828, 2013.

Serologic and genotypic analysis of a series of herpes simplex virus type 1 isolates from two patients with genital herpes

Herpes simplex virus type 1 (HSV‐1) has been reported increasingly as a cause of genital herpes, although HSV‐1 is usually associated with oro‐labial herpes. In the present study, serum specimens and materials for viral isolation were obtained serially from two patients with recrudescent HSV‐1 genital infections to study serology and molecular epidemiology. Recurrent episodes, during which HSV‐1 was isolated, were followed by an increase in the level of anti‐HSV‐1 antibody, suggesting a booster effect from re‐exposure to viral antigens and the possible usefulness of the variation in the level of anti‐HSV‐1 antibody to diagnose recurrence. While genotypes of HSV‐1 isolates obtained from one patient were different from those from the other patient, genotypes of sequential HSV‐1 isolates obtained from the same patient were the same, implying that the recrudescent genital lesions of the two patients could be attributed to endogenous recurrence of a latent virus. Sera from one patient neutralized HSV‐1 isolates obtained from the other patient as well as HSV‐1 isolates obtained from the same patient. An HSV‐1 isolate obtained during a later episode in one patient was neutralized by sera taken before/during the later episode of the same patient, as effectively as an HSV‐1 isolate obtained during an earlier episode in the same patient; thus, in these two cases, HSV‐1 was assumed to have multiplied during recurrence despite the presence of an anti‐HSV‐1 antibody that could neutralize experimentally HSV‐1. J. Med. Virol. 81:1605–1612, 2009.