Detection of gastroenteritis viruses among pediatric patients in Hiroshima Prefecture, Japan, between 2006 and 2013 using multiplex reverse transcription PCR-based assays involving fluorescent dye-labeled primers.

Multiplex reverse transcription (RT)-polymerase chain reaction (PCR)-based assays involving fluorescent dye-labeled primers were modified to detect 10 types of gastroenteritis viruses by adding two further assays to a previously developed assay. Then, these assays were applied to clinical samples, which were collected between January 2006 and December 2013. All 10 types of viruses were effectively detected in the multiplex RT-PCR-based assays. In addition, various viral parameters, such as the detection rates and age distributions of each viral type, were examined. The frequency and types of mixed infections were also investigated. Among the 186 virus-positive samples, genogroup II noroviruses were found to be the most common type of virus (32.7%), followed by group A rotaviruses (10.6%) and parechoviruses (10.3%). Mixed infections were observed in 37 samples, and many of them were detected in patients who were less than 2 years old. These observations showed that the multiplex RT-PCR-based assays involving fluorescent dye-labeled primers were able to effectively detect the viruses circulating among pediatric acute gastroenteritis patients and contributed to the highly specific and sensitive diagnosis of gastroenteritis. J. Med. Virol. 89:791-800, 2017. © 2016 Wiley Periodicals, Inc.

Clinical evaluation of a bioluminescent enzyme immunoassay for detecting norovirus in fecal specimens from patients with acute gastroenteritis.

Noroviruses (NoVs), which belong to the family Caliciviridae, are major causative agents of acute gastroenteritis worldwide. Thus, rapid and highly sensitive assays for detecting NoVs are required. Recently, a bioluminescent enzyme immunoassay (BLEIA) for detecting NoVs in fecal specimens was developed. This new assay was evaluated using fecal specimens obtained from acute gastroenteritis patients. Of the 107 specimens that were found to be NoV-positive by RT-PCR or RT-LAMP, 104 specimens produced positive results in the BLEIA (sensitivity: 96.3%). On the other hand, no false-positive results were observed during the testing of 176 NoV-negative specimens containing group A or C rotaviruses, astroviruses, sapoviruses, adenovirus type 41, bocaviruses, or parechoviruses. Furthermore, the BLEIA was able to detect many NoV genotypes in the tested specimens, including three genotypes from genogroup I (genotypes 1, 4, and 8) and ten genotypes belonging to genogroup II (genotypes 1, 2, 3, 4, 5, 6, 12, 13, 16, and 19). By quantifying the number of NoV genome copies in the clinical specimens tested with the BLEIA, its detection limit was estimated to be 10(6) genome copies per gram of stool and below. Furthermore, as the BLEIA can be performed with an automated device and does not involve complicated procedures it can be used to rapidly test many samples. Therefore, the BLEIA is a rapid and highly sensitive method and could be used as a diagnostic tool at hospitals and clinical laboratories that deal with large numbers of clinical specimens from acute gastroenteritis patients or food handlers. J. Med. Virol. 86:1219-1225, 2014. © 2013 Wiley Periodicals, Inc.

Influenza viral load and rapid influenza diagnostic tests in children and adults.

We compared children and adults with regard to rapid influenza test sensitivity and viral load. Specimen volumes were measured, rapid tests were conducted, and viral load was determined. There was no difference between children and adults in test sensitivity or viral load, but children had higher specimen volumes.

Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers.

We applied a multiplex reverse transcription-PCR with fluorescent dye-labeled primers (fluorescent multiplex RT-PCR) for noroviruses (NoV), sapovirus (SaV), and human astrovirus (HAstV) to diagnose 71 outbreaks of acute gastroenteritis during July 2007 and May 2010 in Hiroshima prefecture. In this assay, the green, red, yellow, and blue fluorescence for NoV genogroup I, NoV genogroup II, SaV, and HAstV, respectively, were indicated on an agarose gel under ultraviolet light. In 61 virus-positive outbreaks confirmed by fluorescent multiplex RT-PCR, detection rates of outbreaks for NoVs, SaV, and HAstV were 96.7%, 3.3%, and 0%, respectively.

Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining.

[Rapid detection of novel influenza A virus and seasonal influenza A (H1N1, H3N2) viruses by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)].

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

Transition of genotypes associated with norovirus gastroenteritis outbreaks in a limited area of Japan, Hiroshima Prefecture, during eight epidemic seasons.

The transition of genotypes implicated in 102 NoV gastroenteritis outbreaks in Hiroshima Prefecture, Japan, during eight epidemic seasons was investigated. Eighteen genotypes were implicated in the outbreaks, with the chronological characteristics as in GII.3, GII.4, GII.5 and GII.12. In GII.4 variants, amino acid changes and positively selected sites were of note and significantly concentrated in the surface-exposed P2 subdomain of the VP1 protein. Notably, variant-specific epitopes at which positively selected sites are located may be significant for distinguishing a new GII.4 variant. The interaction of these genetic changes with developing immunity seems to influence NoV epidemics.