[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.

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.

Culture-proven neonatal sepsis in Japanese neonatal care units in 2006-2008.

BACKGROUND: Recent Japanese epidemiology of neonatal sepsis and its predominant pathogens has not been reported. It is also unknown whether there are center differences in the incidence of neonatal sepsis, including early-onset sepsis (EOS) and late-onset sepsis (LOS) in Japan. OBJECTIVES: To investigate the morbidity and characteristics of neonatal sepsis in recent years and the differences in the incidence of sepsis among Japanese neonatal care units. METHODS: We retrospectively collected the data of newborn infants with culture-proven sepsis that occurred in five Japanese centers of perinatal care from 2006 to 2008. The incidence of sepsis was calculated, including EOS and LOS, and compared among centers. RESULTS: Morbidity from sepsis occurred in 51/6,894 (0.74%) infants. The incidence of EOS and LOS was 0.13 and 0.61%, respectively. The incidence of total sepsis and LOS in infants <1,000 g of birth weight was significantly higher than that in infants who weighed >1,000 g at birth, whereas there were no significant differences in the incidence of EOS between the different birth weights. Methicillin-resistant Staphylococcus aureus was the most common pathogen involved in morbidity and mortality of neonatal sepsis. Significant center differences were observed in the incidence of LOS, but not EOS. CONCLUSIONS: The majority of culture-proven neonatal sepsis is LOS, which differs among centers, especially in infants who weigh <1,000 g at birth in Japan. We consider that it is important to control nosocomial infection in newborn care units to further reduce the morbidity of neonatal sepsis in Japan.

Novel high-speed real-time PCR method (Hyper-PCR): results from its application to adenovirus diagnosis.

PCR, including real-time PCR, usually requires at least 1 h to obtain results. To shorten this time, a novel real-time PCR method (Hyper-PCR) was developed. This method utilizes high-speed DNA polymerase and a thin disc-type reaction vessel that can quickly alter the temperature of the reaction mixture in a newly developed PCR machine. Reactions capable of amplifying adenovirus (Ad) DNA can be completed within 11 min (3 temperature steps, 55 cycles). Hyper-PCR can detect 3.1-18.0 DNA copies/reaction of Ad types 1-4, 7, 8, 11, 15, 19, and 37. Hyper-PCR and conventional real-time PCR were applied to diagnose 147 clinical samples, and the results were compared. Hyper-PCR had a sensitivity of 100% (73/73) and a specificity of 100% (74/74), using conventional real-time PCR as the gold standard. Our newly developed PCR method, Hyper-PCR, was able to diagnose Ad infection within 17 min (not including the time for genome extraction). The thermocycling time of the novel PCR is faster than that of previously available PCR applications, and this method is thought to be potentially applicable to clinical and environmental diagnostics, where rapid diagnosis is important.

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.