Hiroaki Ishiko

Detection of Human Bocavirus in Japanese Children with Lower Respiratory Tract Infections

ABSTRACT Human bocavirus (HBoV), a newly cloned human virus of the genus Bocavirus, was detected by PCR from nasopharyngeal swab samples (8 of 318; 5.7%) collected from children with lower respiratory tract infections. HBoV may be one of the causative agents of lower respiratory tract infections in young children.

Human Metapneumovirus Infection in Japanese Children

ABSTRACT Human metapneumovirus (hMPV) has been recently discovered as an etiological agent of acute respiratory infections. Our purpose was to asses the virological and clinical features of children with respiratory infections caused by hMPV. We examined 658 nasopharyngeal swab samples obtained from 637 children with respiratory infections for hMPV by using reverse transcription-PCR (RT-PCR). A total of 268 samples from 637 children were inoculated onto tertiary monkey kidney cells. A total of 36 serum samples (26 in the acute phase and 10 in the convalescent phase) from the 26 hMPV-positive children were tested for immunoglobulin G (IgG) and IgM antibodies to hMPV by using an indirect immunofluorescence assay. We detected hMPV in 57 (8.9%) of the 637 samples by using RT-PCR and isolated 7 (2.6%) hMPV strains of the 268 samples in cell cultures. A total of 12 (46.2%) of 26 hMPV-positive children were suspected to have primary infection with hMPV as determined by an indirect immunofluorescence assay. The infected children were diagnosed as having wheezy bronchitis (36.8%), upper respiratory tract infection (26.3%), bronchitis (22.8%), and pneumonia (14.0%). We showed that two hMPV groups were circulating in different regions during the same period and that reinfection with hMPV frequently occurs in childhood. The RT-PCR test is the most sensitive test for detection of hMPV, and a serological test may be useful to differentiate between primary infection and reinfection with hMPV.

Molecular Diagnosis of Human Enteroviruses by Phylogeny-Based Classification by Use of the VP4 Sequence

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.

Genetic diversity of enterovirus 71 associated with hand, foot, and mouth disease epidemics in Japan from 1983 to 2003

Background: Enterovirus 71 (EV71) is one of the major etiologic agents of hand, foot and mouth disease (HFMD). The surveillance data indicate that EV71 infection follows an epidemic mode of transmission, causing large outbreaks and then becoming quiescent for a few years. Methods: We investigated the genetic diversity of a total of 121 EV71 strains isolated from patients with HFMD in Fukushima, Japan, from 1983 to 2003 and compared their genetic relation with the 164 EV71 strains isolated in the world using phylogenetic analysis based on the VP4 sequence. Results: We observed EV71-related HFMD outbreaks in Fukushima in 1984, 1987, 1990, 1993, 1997, 2000 and 2003. Phylogenetic reconstruction of EV71 strains isolated in Fukushima demonstrated 8 genetically distinct clusters, including 6 subgroups previously designated as B-1, B-2 and 3, B-4, C-1, C-2, and C-3 and 2 subgroups newly designated as B-5 and C-4. Additional 2 indistinct clusters belonged to genogroup C and were named C-U1 and C-U2. Of those subgroups, B-1, C-U1, C-U2, C-2, B4, and C-4 and B-5 dominantly related to epidemics that occurred in the years 1984, 1987 and 1990, 1993, 1997, 2000 and 2003, respectively. EV71 strains derived from each outbreak in Fukushima formed a single cluster with those isolated during almost the same time period in other area of Japan and in other countries. Conclusions: Our results suggested that the repeated EV71 outbreaks might be the result of the worldwide transmission of the newly introduced genetically divergent EV71 strains.

Outbreak of severe neurologic involvement associated with enterovirus 71 infection

Enterovirus 71 has been associated with several outbreaks, as well as sporadic cases, of central nervous system infection and has a worldwide distribution. Seven children with encephalitis and five with aseptic meningitis caused by Enterovirus 71 were seen at Otsu Municipal Hospital during the summer of 1997. The infections were confirmed serologically, although detection of the viral genome in cerebrospinal fluid was unsuccessful. Seven children were diagnosed as having hand-foot-and-mouth syndrome, two were diagnosed as having herpangina, and three patients younger than 12 months old developed no eruptions. The skin or mucosal manifestations of this outbreak demonstrated considerable variation. The Enterovirus 71 strain that caused the outbreak had a strong neurovirulent tendency. Among the patients with encephalitis, symptoms originating from the impairment of diencephalon were seen in four patients, and those originating from cerebellar impairment were seen in two patients. Brain magnetic resonance imaging in one patient revealed an abnormality in the pons. The neurologic manifestations associated with Enterovirus 71 infection may be characterized by involvement of the cerebellum, brainstem, and diencephalon. Enterovirus 71 is one of the pathogenic viruses that cause hand-foot-and-mouth syndrome, as well as a variety of other clinical manifestations. The most important of these is neurologic disease, especially in infants and young children.

Quantitative Detection of Mycoplasma genitalium from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR

ABSTRACT We developed a TaqMan-based real-time PCR assay for quantifying Mycoplasma genitalium. This assay is able to specifically quantify concentrations of the M. genitalium 16S rRNA gene ranging from 107 to 10 copies/reaction. Using the TaqMan assay, we quantified the M. genitalium 16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for M. genitalium by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for M. genitalium. The mycoplasma load for each specimen was <5 × 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for M. genitalium. One man had an M. genitalium load of <5 × 10 copies/ml, and six men had loads ranging from 1.1 × 107 to 2.7 × 102 copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for M. genitalium. The mycoplasma loads for these men ranged from 3.3 × 106 to 2.3 × 102 copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 × 102 and <5 × 10 copies/ml. The patients with NGU had significantly higher concentrations of M. genitalium in their first-pass urine than did men with GU (P < 0.01) or asymptomatic men (P < 0.05). In addition, M. genitalium loads were significantly higher in men with NCNGU than those in asymptomatic men (P < 0.05). The quantitative assessment of M. genitalium loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.

Seroepidemiology of Human Bocavirus in Hokkaido Prefecture, Japan

ABSTRACT A new human virus, provisionally named human bocavirus (HBoV), was discovered by Swedish researchers in 2005. A new immunofluorescence assay using Trichoplusia ni insect cells infected with a recombinant baculovirus expressing the VP1 protein of HBoV was developed, and the levels of immunoglobulin G antibody to the VP1 protein of HBoV in serum samples were measured. The overall seroprevalence rate of antibodies against the VP1 protein of HBoV in a Japanese population aged from 0 months to 41 years was 71.1% (145 of 204). The seropositive rate was lowest in the age group of 6 to 8 months and gradually increased with age. All of the children had been exposed to HBoV by the age of 6 years. A rise in titers of antibody against the VP1 protein of HBoV during the convalescent phase was observed for four patients with lower respiratory tract infections, and HBoV DNA was detected in nasopharyngeal swab and serum samples from all four patients. These results suggest that HBoV is a ubiquitous virus acquired early in life and that HBoV might play a role in the course of lower respiratory tract infections.

Association of Ureaplasma urealyticum (biovar 2) with nongonococcal urethritis.

Background The tiny (T)-strain mycoplasmas, designated in 1974 as Ureaplasma urealyticum, have been divided into 2 species, Ureaplasma parvum (biovar 1) and U. urealyticum (biovar 2), but association of each of these species with nongonococcal urethritis (NGU) remains unclear. Goal The goal of this study was to determine whether U. parvum (biovar 1) or U. urealyticum (biovar 2) is associated with NGU. Study Design The prevalences of U. parvum (biovar 1) and U. urealyticum (biovar 2) in 572 patients with urethritis were compared with those in 141 men without urethritis. Results The prevalence of U. urealyticum (biovar 2) in men with NGU (15.8%) or with nonchlamydial NGU (18.0%) was significantly higher than that in men without urethritis (7.8%). The prevalence of U. parvum (biovar 1) in men with NGU (8.5%) or with nonchlamydial NGU (11.1%) did not differ significantly from that in men without urethritis (13.5%). Conclusion Our results showed a significant association between U. urealyticum (biovar 2) and NGU. They also suggest that the presence of U. parvum (biovar 1) in the male urethra might be the result of colonization.

Rapid Detection of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum Organisms in Genitourinary Samples by PCR-Microtiter Plate Hybridization Assay

ABSTRACT We present a method for detecting the presence of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.

Quantitative Detection and Rapid Identification of Human Adenoviruses

ABSTRACT We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 × 102 to 1.6 × 106 copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.