Diagnosis of group A coxsackieviral infection using polymerase chain reaction.

AIMS: To examine the relation between enteroviral infection, especially group A coxsackieviral infection, and acute febrile illness over two summers using tissue culture and polymerase chain reaction (PCR). METHODS: Throat swabs were collected from 246 children from June to August 1997 and 1998. RESULTS: Enteroviruses were isolated from 33/246 samples and 35 other viruses were isolated. Enteroviral genomes were detected in 54/178 samples from which no virus was isolated. Of 41 enteroviral genotypes identified by sequence analysis of PCR products, 38 were group A coxsackieviruses, which are usually difficult to isolate using tissue culture. CONCLUSION: Results indicate that viral detection and identification based on PCR is useful in the diagnosis of group A coxsackieviral infection.

Polio vaccine virus-associated meningoencephalitis in an infant with transient hypogammaglobulinemia.

The case of an infant with transient hypogammaglobulinemia who developed meningoencephalitis, retinitis and sensorineural hearing loss is presented. The neurovirulent variant of the Sabin type 2 oral poliovirus vaccine was detected in cerebrospinal fluid and stool.

Association of Mycoplasma genitalium persistence in the urethra with recurrence of nongonococcal urethritis.

BACKGROUND: Most patients with recurrent symptomatic nongonococcal urethritis receive negative test results for Chlamydia trachomatis and Ureaplasma urealyticum, and the cause of such recurrence usually is unknown. GOAL: To assess the association of Mycoplasma genitalium with recurrent nongonococcal urethritis. STUDY DESIGN: In this study, 72 men with nongonococcal urethritis were treated with levofloxacin. Before and after treatment, symptoms and signs were assessed and first-pass urine was examined for C trachomatis, M genitalium, U urealyticum, and Mycoplasma hominis by polymerase chain reaction-based assays. RESULTS: In 6 of 45 men who had no symptoms and no evidence of inflammation after treatment, nongonococcal urethritis recurred. Of these 6 men, 5 had positive test results for M genitalium before levofloxacin treatment, which remained positive afterward. After the second treatment for recurrent nongonococcal urethritis, one man was still had a positive test result for the mycoplasma and experienced a subsequent recurrence. CONCLUSIONS: This study suggests that the persistence of M genitalium in the urethra may be associated with recurrence of nongonococcal urethritis.

Genetic analysis of recent Taiwanese isolates of a variant of coxsackievirus A24.

Epidemics of acute hemorrhagic conjunctivitis (AHC) caused by a variant of coxsackievirus A24 (CA24v) reappeared in Taiwan in 1990 and 1994, following the first two epidemics of 1985--86 and 1988--89. To analyze the genetic diversity of recent CA24v in Taiwan, 7 Taiwanese strains isolated during the 1990--94 period were studied together with one Japanese and two Thai strains isolated in 1993. A fragment of 674 nucleotides between the carboxy terminal 3A and the amino terminal 3D polymerase, including the entire 3C protease (3C(pro)), was amplified by a reverse transcription-polymerase chain reaction (RT-PCR) and the nucleotide sequences were determined. In the 549 nucleotides (183 amino acids) of the entire 3C(pro), we found nucleotide differences at 80 positions between 10 strains and the prototype strain, EH24/70, one of the earliest strains of CA24v. Most of the nucleotide changes were synonymous substitutions and only nine amino acid changes were found. The nucleotide sequence homologies among 71 strains worldwide were 88-100%. These 71 nucleotide sequences were then analyzed by Neighbor-joining method and phylogenetically separated into three distinct genotypes. Genotype I consisted of early strains isolated in 1970--71 from Singapore and Hong Kong. Genotype II included isolates from Singapore and Thailand obtained in 1975. Genotype III comprised strains from the eastern hemisphere isolated in 1985--94 from Japan, Taiwan, China, Hong Kong, Thailand, Singapore, Pakistan and Ghana. They were further divided chronologically into six clusters. The recent isolates from Taiwan obtained in 1985/1986, 1988/1989 and 1990--94 were classified into genotype III Clusters 1, 5, and 6 respectively. The evolutionary rate was re-estimated to be 3 x 10(- 3) 30 years after the emergence of the virus.

Molecular epidemiology of enterovirus 71 in Taiwan.

Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5-24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 x 10(-3) vs. 1.4 x 10(-3)) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance.

Association of nonpolio enteroviral infection in the central nervous system of children with febrile seizures.

OBJECTIVE: To clarify the relationship between enteroviral infection and febrile seizures. STUDY DESIGN: Cerebrospinal fluid (CSF), serum, throat swab, and rectal swab samples were collected for virologic examination from 67 children with febrile seizures from April 1997 to March 1999. Those samples were examined for the presence of enterovirus using cell culture and 2 polymerase chain reaction (PCR) methods. RESULTS: No enterovirus was isolated from cell culture of CSF, throat swab, or rectal swab samples. All samples were screened for the presence of enteroviral sequences using a sensitive PCR method (PCR-Fukushima). We obtained positive results from 14 of 67 CSF samples, 10 of 62 serum samples, 12 of 64 throat swab samples, and 13 of 64 rectal swab samples. Of 21 patients in whom febrile seizures had developed during the summer months (June through August), 13 (61.9%) had positive PCR results in the CSF. Forty-seven of the 49 samples with a positive result using PCR-Fukushima were reexamined independently for the presence of the enteroviral genome using another PCR method (PCR-Mitsubishi). PCR-Mitsubishi had slightly lower sensitivity than PCR-Fukushima but identified genotypes of enterovirus by subsequent sequence analysis of the PCR products. The presence of the enteroviral genome was confirmed in 39 of the samples (83.0%). In 8 of the 9 enteroviruses detected in the CSF and/or serum samples using PCR-Mitsubishi, the genotypes were identified as coxsackieviruses group A, which are usually difficult to isolate using cell culture methods. CONCLUSIONS: These findings proved that the causative agents of febrile illness associated with seizures in summer were primarily enteroviruses, especially coxsackieviruses group A, and that febrile seizures might be caused by enteroviral infection in the central nervous system.

Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

Squamous cell carcinoma of the vulva and adjacent lesions treated at Nagoya University Hospital from 1965 to 1997.

Japan has a lower incidence of vulvar squamous cell carcinoma (VSCC) than Western nations. To pin-point the reasons for this, we reviewed biopsy samples from all cases treated at Nagoya University Hospital over the past 33 years in order to investigate the background lesions for VSCC. Two of 36 VSCC patients had adjacent or coexisting lichen sclerosus (LS), 5 had squamous cell hyperplasia (SCH), and 16 had vulvar intraepithelial neoplasia (VIN). There were 8 cases in which these lesions were thought to be the origin of the VSCC, 1 in which keratinizing squamous cell carcinoma (KSC) was seen in LS, 1 in which verrucous SCH was the origin, and 6 in which 4 basaloid carcinoma and 2 warty carcinoma developed from basaloid VIN and warty VIN, respectively. Although 8 other cases of keratinizing or non-keratinizing squamous cell carcinomas (NSC) coexisted with VIN NOS (not otherwise specified), differentiated VIN or basaloid VIN, we could not be histologically certain of the origin. Among 22 VSCC patients tested for HPV DNA, only an 84-year-old woman presenting a histological feature of KSC tested positive by in situ hybridization (ISH). It was considered that LS and SCH had little and VIN considerable capacity to cause the malignancy of VSCC. We surmise that in Japan the majority of squamous cell carcinoma is unrelated to HPV. One reason for the low incidence of VSCC is largely due to race; the homogeneous, monoethnic Japanese population, as well as the few cases of HPV-related VSCC.

Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen.

The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65-85%), but a low aa identity with genogroup I NV (44-46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self-assembled to form empty virus-like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme-linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV-like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.

Molecular cloning, expression, and antigenicity of Seto virus belonging to genogroup I Norwalk-like viruses.

The viral capsid protein of the Seto virus (SeV), a Japanese strain of genogroup I Norwalk-like viruses (NLVs), was expressed as virus-like particles using a baculovirus expression system. An antigen detection enzyme-linked immunosorbent assay based on hyperimmune antisera to recombinant SeV was highly specific to homologous SeV-like strains but not heterologous strains in stools, allowing us type-specific detection of NLVs.

Detection of TT virus sequences in children with liver disease of unknown etiology.

DNA sequences of TT virus (TTV) in 55 serum samples taken from 20 children with liver disease of unknown etiology (16 with acute liver disease, 2 with fulminant hepatitis, and 2 with chronic liver disease) and from 35 healthy children as controls were examined by using the hemi-nested polymerase chain reaction (PCR). The PCR was carried out using the established primers (NG059, NG061, NG063) to amplify TTV DNA sequences. The sequences were detected in 6 of the 20 patients (30.0%) with liver disease and in 5 of the 35 healthy children (14.2%) by direct gel analysis. There was no significant difference between the prevalence of liver disease patients and controls. However, both patients with fulminant hepatitis and both patients with chronic hepatitis had TTV DNA sequences. Four of the six TTV isolates from liver disease patients were genotype 1a, whereas only one of the five TTV isolates from controls was genotype 1a. Although the study population was small, it is possible that genotype 1a of TTV might be more pathogenic than other genotypes in children.

[Antiviral effect of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine on adenovirus].

PURPOSE: Adenovirus is the most frequent causative virus of conjunctivitis in Japan. Recently (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC) has been promoted as a new drug against adenoviral conjunctivitis. So we examined the antiviral activity of HPMPC against adenoviruses in vitro. METHOD: The antiviral activity of HPMPC against adenovirus (Ad) type 3, type 4, type 19, and type 37 isolated from conjunctivial scrapings in Japan and the prototype of adenovirus type 5 was examined by plaque reduction assay using A 549 cells in vitro. RESULTS: The 50% inhibitory dose (ID50) of HP-MPC was 3.50 (1.44-4.79) micrograms/ml for Ad type 3, 4.50 (4.17-4.92) micrograms/ml for Ad type 4, 2.11 (1.03-3.13) micrograms/ml for Ad type 5, 1.64 (1.40-2.02) micrograms/ml for Ad type 19, and 2.02 (1.17-2.73) micrograms/ml for type 37. The 50% cytotoxic dose of HPMPC for A 549 cells was 205 micrograms/ml by the deoxythimidine uptake inhibition test, and 537 micrograms/ml by the trypan blue exclusion inhibition test. CONCLUSIONS: HPMPC proved to be highly effective in inhibiting replication of adenoviruses at lower concentrations than the cytotoxic level in vitro.

Acute follicular conjunctivitis caused by adenovirus type 34.

PURPOSE: Adenovirus type 34 belongs to adenovirus subgenus B. The prototype virus of adenovirus 34 was isolated from a renal transplant recipient. However, no case of acute conjunctivitis caused by adenovirus 34 has been reported. Recently, we encountered two cases of acute follicular conjunctivitis in which adenovirus 34 was isolated. METHODS: The clinical isolates were identified by the standard neutralization test. The sequences of seven hypervariable regions in the hexon protein of these cases were compared with those of several prototype strains of adenovirus subgenus B. RESULTS: The cases were middle-aged, 34 and 41 years old, and male, and they exhibited moderate conjunctivitis with upper respiratory tract symptoms. Isolates from cell culture were identified as adenovirus 34 by NT. The mean homology rate (percentage of total number of coincident amino acids in the total length of amino acids in seven hypervariable regions) between clinical isolates and the adenovirus 34 prototype was 96.5%; in contrast, those between clinical isolates and the prototypes of adenovirus 11, adenovirus 14, and adenovirus 35 were 55.6%, 66.7%, and 57.9%, respectively. The results of conventional serotyping by neutralization test were confirmed by these values. CONCLUSIONS: These results indicate that adenovirus 34 may induce acute conjunctivitis in immunocompetent subjects and that special attention should be paid to adenovirus 34 as a causative agent for adenoviral conjunctivitis.

[Detection of TT virus in hemodialysis patients].

Recently a newly discovered DNA virus, transfusion transmitted virus (TTV), was introduced as a cause of post-transfusion hepatitis. We studied the frequency of TTV viremia in 60 hemodialysis patients in Yamaguchi, Japan. TTV DNA was detected by heminested PCR, using primers described by Okamoto et al. TTV DNA was detected in 18 patients (30%). There was no differences in clinical characteristics, including age, gender, history of blood transfusion, and double infection of other hepatitis viruses, between TTV DNA positive patients and negative patients. Also the frequency of TT viremia was not associated with the duration of hemodialysis. These results suggest that the routes of TTV infection may be different from those of infection by HBV, HCV, or HGV.

Detection of a novel DNA virus (TTV) sequence in peripheral blood mononuclear cells.

DNA sequences of a novel DNA virus (TTV) were examined in 81 peripheral blood mononuclear cell (PBMC) DNA samples from 48 children and 33 adults, 22 cord blood mononuclear cells (CBMC) DNA samples, and 7 autopsy liver tissue DNA samples by a hemi-nested polymerase chain reaction (PCR). The PCR was carried out using the published primers (NG059, NG061, NG063) to amplify TTV DNA sequences. The sequences were detected in 4 of 81 (5%) PBMC DNA samples, in none of 22 (0%) CBMC DNA samples, and in 2 of 7 (29%) liver tissue DNA samples by direct gel analysis. The PCR-amplified products were confirmed by direct sequencing. The sequencing showed considerable diversities, with differences of 0-55% in 6 TTV isolates, compared with the prototype sequence of TTV. These results suggest that TTV is a ubiquitous virus that produces asymptomatic infection in a large proportion of the general population without transfusion of blood-derived products. To our knowledge, this is the first report describing the detection of TTV DNA sequences in PBMCs.

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.

A case of Guillain-Barré syndrome associated with human parvovirus B19 infection.

We report a 4-year-old boy who presented with Guillain-Barré Syndrome 11 days after the onset of erythema infectiosum. The illness resolved without gamma globulin therapy.

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

[Serotype determination of enteroviruses that cause hand-foot-mouth disease; identification of enterovirus 71 and coxsackievirus A16 from clinical specimens by using specific probe].

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are known to be major causative agents of hand-foot-and-mouth disease prevalent in summer in Japan. Discrimination and identification of these viruses were often hampered by a nonneutralizable or nontypable virus. Therefore, a Southern blot hybridization that utilizes mixed probes specific to serotype was developed. Firstly, an approximately 650 bases spanning 5'-noncoding region to one third of VP2 including entire VP4 was amplified with a set of primers containing enterovirus common sequences and a genomic RNA as template. Secondary, the nucleotide sequences were determined using seven CA16 and eighteen EV71 strains including the standard strains, and the deduced amino acid sequences of VP4 were searched to find residues which are conserved in the same serotypes but diverged among different serotypes. Candidate positions for the mixed probes were defined at the carboxyl terminus of VP4. Thirdly, Southern blot analyses were carried out using thirty-nine enterovirus standard strains, seven CA16 isolates and sixty-six EV71 isolates previously identified by the neutralization test. The results revealed that each mixed probe exclusively bound to the homologous DNAs but not to the heterologous ones. In an attempt to determine serotypes without virus isolation, clinical specimens from hand-foot-and-mouth disease were examined. Of 78 throat swabs and 15 vesicular fluids, 71 (91.0%) and 13 (86.7%) specimens were clearly identified, indicating that the method described here offer advantages over the traditional neutralization assay: It is rapid, specific and less labor-consuming.