Validation of a point-of-need diagnostic tool for rapid diagnosis of norovirus gastroenteritis.

BACKGROUND: The genogroups GI and GII of norovirus (NoV) ribonucleic acid (RNA) genetic variants are the most prevalent cause of acute gastroenteritis outbreaks, especially in children, worldwide. A fast, accurate and convenient tool for diagnosis of NoV may be preferable to the more complicated performance of real-time reverse transcription-polymerase chain reaction (RT-PCR). METHODS: In this study, we developed and evaluated a tool using insulated isothermal PCR (iiPCR)-mediated POCKIT Central NoV GI and NoV GII assay systems for diagnosis of NoV infection in pediatric patients suspected with gastroenteritis. RESULTS: Performance of POCKIT Central Norovirus GI and GII assays using RT-iiPCR, compared to regular real-time RT-PCR showed the same diagnosis rate to NoV GI (100% of total percent agreement and 1.0 of Cohen's kappa value) and a similar detection rate to norovirus GII (96.3% of total percent agreement and 0.92 of Cohen's kappa value). In exclusivity tests, the POCKIT Central NoV GI and GII assays showed negative results to other viruses, indicating that the assays may be a NoV-specific detection tool. CONCLUSION: POCKIT Central NoV GI and GII Assay systems can provide a simple, rapid, sensitive, and specific point-of-need diagnostic tool for the detection of NoV GI and GII RNAs in clinical specimens from children with acute gastroenteritis.

Clinical and virological characteristics of viral shedding in children with norovirus gastroenteritis.

BACKGROUND: The correlation between the clinical manifestations and fecal viral load of norovirus (NoV) infection remains unknown. METHODS: We established a SYBR® Green-based real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to quantify NoV and then sequenced its genomes from the feces of patients admitted at the Chang Gung Memorial Hospital from 2017 to 2018. RESULTS: NoV GII.4 Sydney (n = 21, 36.2%) and GII.P16-GII.2 (n = 19, 32.8%), the two predominant genotypes found among 58 isolates, were closely related to the Taiwan variant 2012a cluster in the VP1 region and genotypes of China strain. An increase in viral load could be observed on Day 3 following the onset of NoV infection. The viral load then declined rapidly from days 10-15 but remained high for >1 month in a severe combined immunodeficiency patient. Significantly longer shedding was found in patients with fever (p = 0.03) or infected by the GII.4 Sydney strain (p < 0.01). CONCLUSION: The qRT-PCR-mediated method proposed in this work could quantify the viral load in patients with NoV infection. Significant viral shedding over a period of 2 weeks in children with acute gastroenteritis and >1 month in an immunodeficient patient was observed. Significantly longer shedding could be correlated with infection by the GII.4 Sydney strain and febrile patients.

Viral shedding in gastroenteritis in children caused by variants and novel recombinant norovirus infections.

ABSTRACT: Human norovirus (NoV) is the leading cause of acute gastroenteritis and the rapid transmission of NoV renders infection control problematic. Our study aimed to investigate viral shedding in gastroenteritis in children caused by variants of emerging norovirus strains infections.We used RNA-dependent RNA polymerase (RdRp) sequencing to measure NoV genome copies in stool to understand the relationship between the clinical manifestations and viral shedding in hospitalized patients. The near full-length NoV genome sequence was amplified via reverse transcription-polymerase chain reaction (RT-PCR) and NoV recombination was analyzed using the Recombination Analysis Tool (RAT).From January 2015 to March 2018, 77 fecal specimens were collected from hospitalized pediatric patients with confirmed NoV gastroenteritis. The NoV genotypes were GII.4 (n = 22), non-GII.4 (n = 14), GII.4 Sydney (n = 21), and GII.P16-GII.2 (n = 20). Viral load increased from days 2 to 9 from the illness onset, resulting in an irregular plateau without peaks. After day 9, the viral load declined gradually and most viral shedding in feces ceased by day 15. The average viral load was highest in GII.4 Sydney followed by GII.P16-GII.2 infections and lowest in non-GII.4 infections. GII.4 unclassified infections showed the longest viral shedding time, followed by GII.4 Sydney infections, GII.P16-GII.2 recombinant infection resulted in the shortest duration. NoVs evolved to form a group of GII.P16-GII.2 variants during the 2017 to 2018 period.The viral load and shedding period and was different in variants of NoV infections in children. High mutation rate of emerging and re-emerging variants was observed to an enhanced epidemic risk rendering continuous surveillance.

Divergence of group a rotavirus with genetic variations before and after introduction of rotavirus vaccines in northern Taiwan.

Despite the development of vaccines in 2006, rotavirus is still a major cause of acute gastroenteritis worldwide. This study was performed to analyze the presence of circulating rotaviruses before and after the introduction of rotavirus vaccines to allow phylogenetic comparisons of vaccine strains in northern Taiwan.Rotavirus genotyping and sequencing of rotavirus VP7 and VP4 PCR products were performed by Reverse Transcriptase Polymerase Chain Reaction and DNA autosequencing. Phylogenies were constructed by the neighbor-joining and maximum-likelihood methods using CLUSTAL W software included in the MEGA software package (version 6.0).Between April 2004 and December 2012, a total of 101 rotavirus specimens from pediatric patients with acute gastroenteritis hospitalized in Chang Gung Children's Hospital were amplified, and their VP4 and VP7 sequences were determined. These 101 specimens consisted of 55 pre-vaccine strains (G1 [13, 23.6%], G2 [12, 21.8%], G3 [16, 29.1%], and G9 [14, 25.5%]) and 46 post-vaccine strains (G1 [25, 54.3%], G2 [12, 26.1%], G3 [5, 10.9%], and G9 [4, 8.7%]). The most common combination of the G and P types was G2P[4], accounting for 36% cases, followed by G9P[8] (25%), G1P[8] (20%), G3P[4] (15%), G3P[8] (10%), G1P[4] (5%), and G2P[8] (5%). Phylogenetic analysis showed that only the G1 and P[8] genotypes clustered in the same lineages with the rotavirus vaccine strains.Based on our results, the inclusion of G9, modified G2 and G3 with target lineages, and the combination G2P[4] and G9P[8] in the rotavirus vaccines in Taiwan is warranted as a vaccination strategy.

In search of the vector(s) of Babesia rossi in Nigeria: molecular detection of B. rossi DNA in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) ticks collected from dogs, circumstantial evidence worth exploring.

The brown dog tick Rhipicephalus sanguineus (sensu lato) (Acari: Ixodidae) has a cosmopolitan distribution, is a proven vector of a host of pathogens with emerging evidence incriminating it in the transmission of some others. Specifically it is reputed as the main vector of Babesia vogeli whereas the southern African yellow dog tick Haemaphysalis elliptica, long considered to be H. leachi, is apparently the only proven vector of B. rossi, since the resurrection of the separate species H. elliptica as a member of the leachi-group by Apanaskevich et al. However, recent epidemiological surveys conducted in Nigeria show higher prevalence of B. rossi than B. vogeli infection in dogs most of whom were infested with R. sanguineus and rarely with ticks of the H. leachi group. The discrepancy between tick distribution and Babesia spp. prevalent in dogs stimulated us to investigate the possible role of R. sanguineus (s.l.) in the natural transmission of B. rossi. Out of a total of 66 tick samples identified morphologically and molecularly as R. sanguineus collected from dogs manifesting clinical signs of tick-borne diseases, eight (12%) were positive in nested PCR for Babesia sp. DNA. Sequencing results for these amplified products showed that all of the 18S rDNA sequences (693 bp) were identical to each other, and bore 99.3-99.9% identities with those from other B. rossi isolates accessible in GenBank. None of the ticks harbored the DNA of B. vogeli or B. canis. The possible implications for the detection of B. rossi DNA in R. sanguineus (s.l.) ticks collected from dogs in the epidemiology of B. rossi infection of dogs in Nigeria is highlighted.

Characterization of the role of global regulator FliA in the pathophysiology of Pseudomonas aeruginosa infection.

FliA is known to be a sigma factor that regulates bacterial flagella gene expression. Accumulating evidence suggests that FliA is involved in bacterial behavior other than motility. To elucidate the contribution of FliA to Pseudomonas aeruginosa pathophysiology, we analyzed the biological properties and gene expression profiles of a ΔfliA mutant. Transcriptome analysis results demonstrated that the expression levels of flagella biogenesis genes decreased dramatically in the mutant; consequently, the ΔfliA mutant failed to synthesize flagella and exhibited reduced motility. The ΔfliA mutant displayed stronger hemolytic and caseinolytic activities, as well as pyocyanin production. The expression of type 6 secretion system-II genes and interbacterial competition activity was decreased in the ΔfliA mutant. Direct evidence of fliA participation in virulence was obtained from analysis of hypervirulent strain B136-33. Adhesion to and cytotoxicity toward mammalian cells and penetration through cell layers were noted; furthermore, the colonization ability of the fliA::Tn5 mutant in the intestines of laboratory mice was compromised. Notably, the fliA-overexpressing strain displayed phenotypes similar to that of the fliA-defective strain, indicating that optimal FliA levels are critical to bacterial physiology. Our findings indicate that FliA plays diverse roles in P. aeruginosa, not only in flagella biosynthesis, but also in pathophysiology.

Genome Sequence Analysis of a G2P[4] Group A Rotavirus Strain with an Unusual Canine-Origin NSP1 A15 Genotype.

Here, we disclose the complete genomic sequence of a rare rotavirus group A G2P[4]-I2-R2-C2-M2-A15-N2-T2-E2-H2 strain detected in a fecal specimen from a rotaviral acute gastroenteritis patient who had previously received the Rotarix vaccine.

First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria.

The present study aimed to detect the presence of Ehrlichia canis in naturally infected dogs in Nigeria, using a combination of PCR and sequence analysis of the 16S rRNA gene and two genes encoding the tandem repeat-containing proteins (TRPs), TRP19 and TRP36. Out of a total of 100 blood samples collected from domestic dogs presented to veterinary hospitals in Jos, the capital city of Plateau State of Nigeria, 11 were positive in nested PCR for E. canis. Sequencing results for these amplicons showed that all of the 16S rDNA sequences (1623 bp) or the TRP19 coding sequences (414 bp) were identical to each other and had very high similarities (99.3-100%) with those from other E. canis strains accessible in GenBank. The TRP36 gene sequences derived from the 11 Nigerian isolates were identical to each other except for the number of the 27-bp repeat unit in a tandem repeat region, which was found to be 8, 12 or 18. Without considering the number of tandem repeats, these sequences had 100% identity to that of the reported Cameroon 71 isolate, but distinctly differed from those obtained from other geographically distant E. canis strains previously published. A phylogenetic tree of E. canis based on the TRP36 amino acid sequences showed that the Nigerian isolates and the Cameroon 71 isolate fell into a separate clade, indicating that they may share a common ancestor. Overall, this study not only provides the first molecular evidence of E. canis infections in dogs from Nigeria but also highlights the value of the TRP36 gene as a tool to classify E. canis isolates and to elucidate their phylogeographic relationships.

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan.

The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.

Detection and characterization of four novel genotypes of Ehrlichia canis from dogs.

The genetic diversity of Ehrlichia canis strains worldwide is currently poorly defined. The present study aimed to characterize E. canis strains in naturally infected dogs in Taiwan, using a combination of PCR and sequence analysis of the 16S rDNA and two antigen-encoding genes, gp19 and gp36. Genomic DNA was extracted from 34 parasitemic dogs and the genes of the pathogen were separately amplified, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rDNA sequences (1623 bp) amplified from the Taiwanese isolates were identical and had very high similarity (99.4-100%) with previously reported E. canis sequences. Nevertheless, most of the gp19 gene sequences (414 bp) from the Taiwanese isolates had three specific nucleotide substitutions at positions 9, 323 and 371 that resulted in three amino acid changes. The gp36 gene of the Taiwanese isolates consists of three regions: a 5' end pre-repeat region (426 bp), a tandem repeat region with variable numbers of the 27-bp repeat unit depending on the isolate, and a 3' end region (87 bp). The nucleotide sequences of the 5' end region of gp36 from Taiwanese isolates were identical to each other, but unexpectedly, quite distinct from the sequences of eleven other E. canis strains previously published, with 86.7-87.2% identities only. A phylogenetic tree of E. canis strains based on the gp36 amino acid sequences showed that the Taiwanese isolates fell into a separate clade, indicating the presence of a novel strain that had not yet been characterized.