Doo Sung Cheon

A Graphene Oxide Based Immuno‐biosensor for Pathogen Detection

The development of novel biosensors for highly sensitive, selective, and rapid pathogen detection is of paramount importance for medical diagnostics, food safety screening, and environmental pollution monitoring. Graphene oxide (GO), which is well known as a promising precursor for graphene, has great potential for use in biosensors because of its unique characteristics such as facile surface modification, high mechanical strength, good water dispersibility, and photoluminescence. Recently, Lu et al., and Liu et al. demonstrated the usefulness of the fluorescence quenching properties of GO in DNA biosensing. Gold nanoparticles (AuNPs) have been shown to be excellent quenchers for organic fluorescent tags, semiconductor nanocrystals, and oxidized carbon nanotubes. Herein, we demonstrate a GO-based immuno-biosensor for detecting a rotavirus as a pathogen model. The detection occurs with high sensitivity and selectivity by GO photoluminescence quenching induced by fluorescence resonance energy transfer (FRET) between GO sheets and AuNPs. A GO-based immuno-biosensor system is shown in Figure 1. GO, which was synthesized by a modified Hummers method, was deposited on an amino-modified glass surface. The disruption of the sp structure of graphene crystals during the oxidation process results in the recombination of electron–hole pairs localized within small sp carbon domains that are embedded in an sp matrix, thus resulting in photoluminescence with a quantum yield of 70.3% relative to the fluorescein dye. A homogenous solution of GO shows a broad fluorescence emission peak around 547 nm upon excitation at 400 nm (Figure S1a,b in the Supporting Information). Since the GO has negatively charged functional groups such as carboxylic acids, hydroxy groups, and epoxides, the GO sheets are bound to the positive surface through an electrostatic interaction, which is strong enough to ensure that the GO sheets are retained during a washing step. The antibodies for rotavirus are immobilized on the GO array by a carbodiimide-assisted amidation reaction, and the rotavirus cell is captured by specific antigen–antibody interaction (Figure 1). The capture of a target cell was verified by observing the fluorescence quenching of GO by FRET between the GO and AuNPs. To realize such a novel GO immuno-biosensor, we first synthesized AuNP-linked antibodies (Ab-DNA-AuNP complexes; Figure S2 in the Supporting Information) which were bridged with 100-mer singlestranded DNA molecules. The DNA molecule was used as a mediator as the synthetic method, which is based on phosphoramidite chemistry, provides facile control of distance between Ab and AuNPs, so the AuNPs are placed close to the GO surface. The high affinity of amino functional groups of the DNA nucleotides for multiple AuNPs leads to an enhancement of the quenching efficiency of GO. When the Ab-DNA-AuNP complexes were selectively bound to the target cells that were attached to the GO arrays, a reduction in the fluorescence emission of GO by quenching was detected, thus enabling the identification of pathogenic target cells. AFM analysis was performed to obtain the topographical profile for each step during the fabrication of GO immunobiosensor (Figure 2). GO sheets ranging from 500 nm to 2 mm in lateral dimension (Figure S1b in the Supporting Information) were dispersed uniformly as monoand bilayers, which were determined by measuring the height (1.1 nm) between the two black marks in Figure 2a. The antibody-linked GO array shows brighter spots, particularly at the edges, and also folded structures, which contain many carboxylic acid groups. The measured height of 11.9 nm corresponds to the theoretical value of an antibody (10–15 nm) and approximately 15.3% area per spot was linked by antibodies. Once the target rotavirus was captured, the height of the complexes increased to approximately 81 nm. Considering that the size Figure 1. Illustration of a GO-based immuno-biosensor.

Enteroviral meningitis without pleocytosis in children

Objectives This study aims to describe the clinical characteristics of enteroviral meningitis in association with the absence of cerebrospinal fluid (CSF) pleocytosis. Design This was a retrospective analysis of databases of patients diagnosed with enteroviral meningitis by CSF reverse transcription-PCR testing. Presence of CSF non-pleocytosis at each age group was analysed by use of the two criteria. Clinical variables were compared with regard to the presence of CSF pleocytosis. Multiple logistic regression analysis was used to identify factors that were associated with CSF pleocytosis. Setting Two hospitals in South Korea, between January 2008 and August 2011. Patients 390 infants and children with enteroviral meningitis. Interventions None. Main outcome measures Proportion of enteroviral meningitis without CSF pleocytosis. Results Among the 390 patients with enteroviral meningitis, 16–18% did not have CSF pleocytosis. In particular, CSF pleocytosis was not present in 68–77% of the neonates with enteroviral meningitis, demonstrating that the proportion of CSF pleocytosis decreased significantly with age (p<0.001). In multivariate models, younger age (adjusted OR 0.981; 95% CI 0.973 to 0.989), lower peripheral white blood cell count (adjusted OR 0.843; 95% CI 0.791 to 0.899), and shorter interval between onset and lumbar puncture (adjusted OR 0.527; 95% CI 0.315 to 0.882) were associated with the absence of CSF pleocytosis in enteroviral meningitis. Conclusions This study demonstrated high proportion of non-pleocytic enteroviral meningitis in young infants and identified several clinical factors that contributed to the absence of CSF pleocytosis. We suggest that CSF enterovirus PCR testing is likely to detect more cases of enteroviral meningitis, especially in young infants.

Norovirus: A Possible Cause of Pneumatosis Intestinalis

Objective: Pneumatosis intestinalis (PI) in children is associated with immunosuppression, mucosal disruption from trauma, obstructive pulmonary disease, congenital heart disease, and gastrointestinal infections. Our study is the first report of norovirus infection–associated PI. Patients and Methods: A retrospective review was performed in pediatric patients (older than 30 days) with PI from March 2005 to April 2009. Since December 2008, in addition to routine stool examinations, reverse-transcriptase polymerase chain reaction testing for calicivirus (norovirus and sapovirus), adenovirus, astrovirus, and enterovirus has been performed. Results: Twenty-seven patients with PI were identified. The median age was 1.4 (range 0.2–14.8 years). Seventeen patients (63.0%) were immunocompromised hosts. Pathogens were identified in 5 immunocompromised patients (5/27 and 5/8 since December 2008). Of note, norovirus was identified in 4 patients (80%, 4/5) during the cold weather season. The genotype of noroviruses in these patients was GII-4. Among 27 patients with PI, 10 patients (37.0%) developed PI in the spring and 11 (40.7%) in the winter. Twenty-four patients survived (88.9%, 24/27). None of the patients with norovirus or rotavirus infection died. Conclusions: Our data suggest that norovirus infection may contribute to the development of PI in immunocompromised hosts.

Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines

Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations. Graphical Abstract

[Application of a diagnostic method using reverse transcription-PCR ELISA for the diagnosis of enteroviral infections].

BACKGROUNDnEnteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections.nnnMETHODSnReverse transcription (RT)-PCR ELISA kit for targeting 5 non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them.nnnRESULTSnThe detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5NCR (P=0.016).nnnCONCLUSIONSnOur results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.

The Prevalence and Distribution of the P and G Genotypes of a Group A Rotavirus Detected in Acute Gastroenteritis Patients from Incheon

Rotavirus is the main cause of severe diarrhea in infants and young children of the world. However, the frequency of genetic alterations makes it hard to control the prophylaxis. Therefore, continuous monitoring of the rotavirus’s genetic change is inevitable to prevent disease prevalence and is useful in inventing an efficient vaccine. From January 2005 to December 2010, we investigated 11,607 stool samples of acute gastroenteritis patients in the Incheon metropolitan area. About 13.18% (1,530 stool samples) of all samples had a positive reaction against rotavirus using an antigen capture enzyme- linked immunosorbent assay (ELISA). Then, the 160 stool samples were searched for subtypes of group A rotavirus by using a reverse transcription polymerase chain reaction (RT-PCR) and a nested multiplex RCR. In P sub-typing, P8 (56.3%) was an extremely prevalent genotype, followed by P6 (21.3%), and P1A (10.0%). G1 (39.4%) was most widespread in the G subtype, followed by G4 (25.0%) and G3 (18.8%). G1P8 (35.5%) was the most common G and P subtype combination, followed by G4P6 (19.3%) and G3P8 (13.1%). These results might be useful data for understanding the epidemiological status of group A-rotavirus dispersion in the Incheon metropolitan area.