Characterization of genome sequences and clinical features of coxsackievirus A6 strains collected in Hyogo, Japan in 1999-2013.

Coxsackievirus A6 (CV-A6) is an enterovirus, which is known to cause herpangina. However, since 2009 it has frequently been isolated from children with hand, foot, and mouth disease (HFMD). In Japan, CV-A6 has been linked to HFMD outbreaks in 2011 and 2013. In this study, the full-length genome sequencing of CV-A6 strains were analyzed to identify the association with clinical manifestations. Five thousand six hundred and twelve children with suspected enterovirus infection (0-17 years old) between 1999 and 2013 in Hyogo Prefecture, Japan, were enrolled. Enterovirus infection was confirmed with reverse transcriptase-PCR in 753 children (791 samples), 127 of whom (133 samples) were positive for CV-A6 based on the direct sequencing of the VP4 region. The complete genomes of CV-A6 from 22 positive patients with different clinical manifestations were investigated. A phylogenetic analysis divided these 22 strains into two clusters based on the VP1 region; cluster I contained strains collected in 1999-2009 and mostly related to herpangina, and cluster II contained strains collected in 2011-2013 and related to HFMD outbreak. Based on the full-length polyprotein analysis, the amino acid differences between the strains in cluster I and II were 97.7 ± 0.28%. Amino acid differences were detected in 17 positions within the polyprotein. Strains collected in 1999-2009 and those in 2011-2013 were separately clustered by phylogenetic analysis based on 5'UTR and 3Dpol region, as well as VP1 region. In conclusion, HFMD outbreaks by CV-A6 were recently frequent in Japan and the accumulation of genomic change might be associated with the clinical course.

Shiga Toxin Subtypes and Virulence Genes in Escherichia coli Isolated from Cattle.

Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H- strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfAO113 were detected in more than 12 serotypes.

Comprehensive Study of Pathogenic Genes Distributed in Escherichia coli Isolated from Cattle.

Pathogenic genes such as stx1, stx2, STh gene, STp gene, LT gene, invE, eae, aggR, afaD, astA, cdt and cnf were investigated in Escherichia coli isolated from cattle during Nov. 2012 and Aug. 2013. Plural pathogenic genes were concurrently detected by multiplex PCR, and screen-positive genes were confirmed and sub-classified by PCR. Among 100 cattle investigated, 180 E. coli strains with diarrheic genes (DEC) were detected in 79 cattle, and 45 of them, isolated from 32 cattle, were Shiga toxin-producing E. coli (STEC). More than 30% of cattle carried astA, cdt, cnf and stx2 in descending order. STh gene, LT gene, invE, aggR and afaD were not detected in this study. Both stx1 and stx2 were concurrently detected from 6 of 45 STEC strains and stx2 alone was detected from 19. Seventeen STEC strains carried STp gene, astA, or cdt along with stx1 or stx2. Additionally, 135 remaining DEC were classified into 18 enterotoxigenic E. coli with STp gene, 25 enteropathogenic E. coli with eae, and 92 other DEC with astA, cdt and cnf. Both O and H serotypes were identified in 48 strains, including O157 : H7, O1H7 and so on. O157 : H7 were identified in 3 strains that carried stx2 and eae together, as found in human pathogenic strains isolated from patients with gastroenteritis and hemolytic-uremic syndrome.

Low total IgM values and high cytomegalovirus loads in the blood of newborns with symptomatic congenital cytomegalovirus infection.

AIMS: Neurological outcomes differ considerably between symptomatic and asymptomatic infants with congenital cytomegalovirus (CMV) infection. Our objective was to characterize laboratory markers in symptomatic newborns in comparison with asymptomatic newborns with congenital CMV infection. METHODS: Ten newborns with symptomatic and 13 newborns with asymptomatic congenital CMV infection were included in this 3-year prospective cohort study. Total immunoglobulin M (IgM), CMV-IgM, CMV antigenemia, and CMV-DNA in blood and urine were measured and their positive rates and quantitative values compared between the symptomatic and asymptomatic groups. RESULTS: Fifty percent of newborns in the symptomatic group were positive based on total IgM; this was significantly lower than in the asymptomatic group (100%). Quantitative total IgM values were significantly lower, and there were significantly more copies of CMV-DNA in the blood of symptomatic newborns than in asymptomatic newborns (median values for total IgM: 14 vs. 43 mg/dL and blood CMV-DNA: 3.2×102 vs. 3.5×101 copies/106 white blood cells). CMV-IgM, CMV antigenemia, and urine CMV-DNA did not differ significantly between groups. CONCLUSION: Low total IgM values and high blood CMV loads were associated with the presence of symptoms in newborns with congenital CMV infection.

Dried umbilical cord is a potential material for retrospective diagnosis of intrauterine enterovirus infection.

OBJECTIVE: To determine whether dried umbilical cords (UCs) are useful for retrospective diagnosis of intrauterine enterovirus (EV) infection. METHODS: Dried UCs in two patients with neonatal EV sepsis and 10 neonates without infectious signs were enrolled. Viral RNA was extracted from their dried UCs, and nested reverse transcription polymerase chain reaction (RT-PCR) was performed. RESULTS: Infection routes estimated by the clinical course were intrauterine infection in Case 1 and post-natal horizontal infection in Case 2. EV-RNA was detected from dried UC in Case 1, but not in Case 2 and 10 neonates. CONCLUSIONS: This report showed the potential use of dried UCs for retrospective diagnosis of intrauterine EV infection.

[Simultaneous screening method for Bordetella species by conventional PCR assay].

A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.

One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.

BACKGROUND: In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society. METHODOLOGY: To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses. RESULTS AND CONCLUSIONS: We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.

Cultivation for 21 days should be considered to isolate respiratory adenoviruses from samples containing small numbers of adenoviral genomes.

Adenovirus types 1, 2, and 3 can usually be isolated in only a short time, although occasionally it may take longer. This phenomenon has been explained empirically as being due to the viral load in the sample, although to date there has been no experimental confirmation of this. In this study we therefore tried to establish a correlation between the quantity of respiratory adenovirus genome in the clinical sample and the time required for its isolation. The correct choice of sensitive cell line is important for this purpose, thus we compared the sensitivity of three different cell lines (HeLa, A549, and RD), and found A549 to be the most sensitive to adenoviruses 1-3. Stored clinical samples (n=21) containing adenoviruses 1-3 were diluted to make solutions containing between 10 and 10(8) copies/microL of adenovirus genome (n=242). These diluted clinical samples were then inoculated into A549 cells, which were cultivated for 21 days and the results compared to the number of viral genomes in each cultivated sample. Adenoviruses could be isolated from all samples (41/41) containing >/=10(6) copies/microL within 6 days, whereas samples containing 10 and 10(2) copies/microL required cultivation for 12.6+/-3.8 and 11.2+/-3.8 days (mean+/-S.D.), respectively, before adenoviruses could be isolated. A cultivation time of 21 days should therefore be considered for the isolation of respiratory adenoviruses from samples containing <10(3) adenovirus genome copies/microL.

Usefulness of real-time reverse transcription-polymerase chain reaction for the diagnosis of echovirus aseptic meningitis using cerebrospinal fluid.

Quantitative real-time reverse transcription-polymerase chain reaction (q-RT-PCR) was used to diagnose echovirus infection and the results were compared to those obtained with the viral culture rate. Cerebrospinal fluid (CSF) from a total of 40 aseptic meningitis patients was used. Positive CSF samples, determined by viral culture (n=29), contained significantly higher echovirus genome copy numbers (mean, 329 copies/microL) than did culture-negative CSF samples (n=11) (mean, 34.2 copies/microL; P<0.05). Echoviruses were identified as echovirus serotype 9 (E-9) (n=21); E-30 (n=16); and E-5, E-7, and E-18 (n=1 each) by neutralization and/or conventional PCR-sequencing techniques. Viral culture-positive samples were collected at 1.41-/+1.27 days after the onset of illness, and culture-negative samples were collected at 4.91-/+3.34 days. Samples from which virus could be isolated were collected significantly earlier than were samples from which virus could not be isolated. These results strongly suggest the importance of early collection of CSF for echovirus isolation, and demonstrate the high sensitivity of q-RT-PCR for the detection of echoviruses in CSF.

Detection and quantification of enterovirus 71 genome from cerebrospinal fluid of an encephalitis patient by PCR applications.

Enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease (HFMD) and is known to cause encephalitis, but several reports have identified EV71 in cerebrospinal fluid (CSF). We detected EV71 in CSF from a 20-month-old infant. The patient was diagnosed with brainstem encephalitis associated with HFMD. The clinical features of the patient were high fever (39.1C) and myoclonic jerks, and magnetic resonance imaging of the brain showed a bright signal area around the 4th ventricle. From a nasopharyngeal swab and rectal swab, EV71 was detected using reverse transcription (RT)-nested polymerase chain reaction (PCR). From CSF, the EV71 genome was identified using pan-enterovirus RT-nested PCR and sequencing. By real-time PCR, the nasopharyngeal swab, rectal swab, and CSF contained 1.8 x 10(4), 9.8 x 10(4), and 1.8 x 10 copies of the EV71 genome/microL, respectively. The enterovirus could only be isolated by cell culture from the rectal swab, and it was identified by a neutralization test using EV71-specific antiserum. RT-nested PCR and real-time PCR are considered to be sensitive tools for EV71 diagnosis in CSF.

Molecular epidemiology of adenovirus type 3 detected from 1994 to 2006 in Hyogo Prefecture, Japan.

The molecular epidemiology of 126 adenovirus type 3 (AdV3) isolates obtained in Hyogo Prefecture (population: 5.5 million) from 1994 to 2006 was studied. The hexon-coding region, including 7 hypervariable regions (HVRs) (1,419 bp), was sequenced. We found 5 nonsynonymous nucleotide substitutions in the HVRs. The results are strongly suggestive of positive Darwinian selection. We classified the AdV3 strains analyzed here into 3 genome types: AdV3x (n=44), AdV3y (n=46), and AdV3z (n=36). AdV3x first appeared in 2001 in Hyogo Prefecture, and was detected predominantly during a large outbreak of AdV3 in 2003-2005. AdV3x was identical to a Korean strain responsible for a large outbreak of AdV3 in Korea in 1998-1999. We conclude that at least 3 genome types of AdV3 have circulated in Hyogo Prefecture, Japan, during the past 13 years (1994-2006). The findings also suggest that AdV3x was imported from Korea to Hyogo Prefecture in 2001.

Detection of dual-infected cases of adenoviruses and coxsackieviruses type B by real-time PCR but not by the conventional viral culture technique.

The aim of this study was to evaluate the applicability of diagnostic methods for dual-infected cases of human adenoviruses (AdVs) and coxsackieviruses type B (CBs). For this purpose, 100 nasopharyngeal samples from patients with acute exudative tonsillitis and clinically suspected AdV infection were analyzed. Using PCR and real-time PCR techniques for AdVs and CBs, we found 86 AdVs-only positive samples; we also found five dual-infected samples containing 5.4 x 10(5) to 7.1 x 10(8) copies/mL of AdV genomes and 1.4x104 to 1.3 x 10(9) copies/mL of CB genomes. By viral culture using A549 cells, two co-infected samples, which contained over 10(8) copies/mL of AdV genomes and <10(5) copies/mL of CB genomes, became AdV dominant, while three samples with less than 2.0 x 10(6) copies/mL of AdV genomes became CB dominant. An immunochromatography kit for diagnosing AdVs at the bedside was positive for 3/5 dual-infected patients, and PCR techniques for AdVs and CBs were both positive for 5/5. Viral culture is usually considered to be the gold standard for AdV diagnosis, but our results demonstrate the importance of PCR applications for the detection of AdV and CB genomes, particularly in clinical cases of suspected AdV infection. Even though the sample size of dual infection (n=5) is small, our results show the existence of dual infection cases which were difficult to diagnose by viral culture alone.

Evaluation of a bedside immunochromatographic test for detection of adenovirus in respiratory samples, by comparison to virus isolation, PCR, and real-time PCR.

An immunochromatography (IC) kit for human adenovirus (HAdV) was evaluated with 138 patient nasopharyngeal samples. The samples were collected at a sentinel clinic in Japan from January through June 2003. Patients were diagnosed by clinical manifestation of pharyngoconjunctival fever (n = 38) or exudative tonsillitis (n = 100). The IC kit was positive for 84% (116 of 138) of patients diagnosed at bedside. The remaining extract solution of the IC kit test was transferred into maintenance medium and tested via laboratory diagnoses. The IC kit had 95% sensitivity (116 of 122 patients) with HAdV isolation (isolation) as the standard and 91% sensitivity (116 of 128 patients) with PCR as the standard. All of the IC kit-positive samples were isolation and PCR positive. Similarly, all the isolation-positive samples were PCR positive. Twenty-two IC kit-negative samples were evaluated by real-time PCR. Six samples were IC kit negative and isolation positive and contained 3.8 x 10(7) to 2.5 x 10(9) copies of the HAdV genome/ml. Five samples that were only PCR positive contained 3.0 x 10(4) to 3.8 x 10(5) copies of the HAdV genome/ml, but one sample was real-time PCR negative. We conclude that the IC kit is a useful bedside diagnostic tool for HAdV infections because it has 95% sensitivity (compared to isolation), but a negative result does not always rule out HAdV infection.

Outbreak of central nervous system disease associated with hand, foot, and mouth disease in Japan during the summer of 2000: detection and molecular epidemiology of enterovirus 71.

Few outbreaks of the serious enterovirus 71 (EV71) infections, which affect the central nervous system (CNS), had been reported in Japan before 2000. During June through August 2000, a patient died of pulmonary edema caused by brainstem encephalitis accompanied by EV71-induced hand, foot, and mouth disease (HFMD), and many patients complicated by serious CNS disease, including paralysis, were hospitalized in a restricted area in Hyogo Prefecture, Japan (K-area). During the same period, endemics of HFMD were reported in other areas in Hyogo Prefecture, where EV71 was isolated from HFMD patients, but few patients developed aseptic meningitis. The isolations of EV71 from K-area patients were difficult with the use of Vero cells, so the strains were isolated by use of GL37 cells; Vero cells, however, could isolate EV71 strains from other areas in Hyogo Prefecture. We sequenced VP4 coding regions of these EV71 isolates and found that the isolates from K-area had the same sequence, which, except for one isolate, was different from the sequences of EV71 strains isolated from other areas of Hyogo Prefecture. Although these results were not enough to state that EV71 from K-area was a virulent strain, it seemed reasonable to conclude that serious CNS diseases in K-area were caused by EV71 because it was the only infectious agent detected in the inpatients of K-area.