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.

Detection, quantitation, and phylogenetic analysis of noroviruses in Japanese oysters.

Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 10(2) copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.

Isolation of influenza A H1N2 viruses from an outbreak in Yokohama City during the 2001-2002 influenza season in Japan.

Emergence of Influenza A H1N2 viruses was documented worldwide during the 2001-2002 influenza season. In Japan, H1N2 viruses were isolated from two students of a junior high school in an influenza outbreak in Yokohama City, February 2001. Genetic and antigenic analyses demonstrated that the H1N2 viruses isolated in Japan shared common features with those isolated in other countries.

[Study of a respiratory syncytial virus diagnostic test kit based on immunochromatography].

We carried out clinical and basic studies of the Directigen Lateral Flow RSV (Becton, Dickinson and Company, USA), a rapid test kit that detects respiratory syncytial virus (hereinafter referred to as "RSV") antigens based on immunochromatography. For the clinical study, 103 nasopharyngeal aspirates from patients with acute respiratory infections were used to evaluate the kit. Compared to the cell culture method, the Directigen Lateral Flow RSV showed a sensitivity of 100% (16/16) and a specificity of 94.3% (82/87), and an agreement rate of 95.1% (98/103). When compared to conventional testing kits, we found that the total agreement rate with the Directigen RS (Nippon Becton Dickinson and Company) was 88.3% (91/103) and with RSV TestPack (Dainabot Co., Ltd.) was 91.3% (94/103). The detection limit of the Directigen Lateral Flow RSV was 2 x 10(3) PFU/ml for both RSV subgroups A and B. In the crossreactivity test, only RSV was found positive. No other microorganisms were crossreactive. We also studied storage stability of nasopharyngeal aspirates and found that stability was not affected by storage at room and refrigerator temperatures for 14 days. Taken all together, the Directigen Lateral Flow RSV is useful for the diagnosis of RSV infection in a clinical setting because its performance is equivalent to conventional testing kits and is easy to use.