Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

Molecular epidemiology and genotype distribution of noroviruses in children in Thailand from 2004 to 2010: a multi-site study.

This study identified norovirus in children presenting with acute gastroenteritis and determined the capsid genotypes of the circulating norovirus strains in multiple regions in Thailand during October 2004 to December 2006 and March 2008 to August 2010. A total of 7,420 stool samples were collected from both cases (3621) and controls (3799). The stool samples were screened by two real-time RT-PCR assays to detect genogroup I and genogroup II noroviruses. Norovirus-positive samples were identified in 516 cases (14.3%) and 181 controls (4.8%) with more than half of norovirus positive samples from 7-24 months old children. Positive samples were sequenced and genotyped for the capsid gene. GII.4 was the genotype observed most frequently (56.4%) followed by GII.3 (28.2%). Five peaks of infection were observed, with predominant capsid genotypes that alternated during the surveillance periods between GII.4 and GII.3. Analyses of positive samples showed variation in genotype from each region as well as from different study periods. This emphasizes the importance of multi-site studies to investigate norovirus epidemiology. Additionally, the observed regional and temporal variations suggest that a systematic nation-wide surveillance effort in Thailand is needed to track the continually changing norovirus epidemiology.

Antibiotic-Resistant Bacteria in Green Turtle (Chelonia mydas) Rearing Seawater.

Antibiotic resistance of microorganisms is a serious health problem for both humans and animals. Infection of these bacteria may result in therapy failure, leading to high mortality rates. During an early intervention program process, the Sea Turtle Conservation Center of Thailand (STCCT) has faced high mortality rates due to bacterial infection. Previously, investigation of juvenile turtle carcasses found etiological agents in tissue lesions. Further determination of sea water in the turtle holding tanks revealed a prevalence of these causative agents in water samples, implying association of bacterial isolates in rearing water and infection in captive turtles. In this study, we examined the antibiotic resistance of bacteria in seawater from the turtle holding tank for a management plan of juvenile turtles with bacterial infection. The examination was carried out in three periods: 2015 to 2016, 2018, and 2019. The highest isolate numbers were resistant to beta-lactam, whilst low aminoglycoside resistance rates were observed. No gentamicin-resistant isolate was detected. Seventy-nine isolates (71.17%) were resistant to at least one antibiotic. Consideration of resistant bacterial and antibiotic numbers over three sampling periods indicated increased risk of antibiotic-resistant bacteria to sea turtle health. Essentially, this study emphasizes the importance of antibiotic-resistant bacterial assessment in rearing seawater for sea turtle husbandry.

Molecular Epidemiology and Genetic Diversity of Norovirus in Young Children in Phnom Penh, Cambodia.

This study investigated the genetic diversity of noroviruses identified from a previous surveillance study conducted at the National Pediatric Hospital in Phnom Penh, Cambodia, from 2004 to 2006. In the previous study, 926 stool samples were collected from children aged 3-60 months with acute diarrhea (cases) and without diarrhea (controls) with reported 6.7% of cases and 3.2% of controls being positive for norovirus. The initial norovirus diagnostic assay was performed with real-time reverse transcription-polymerase chain reaction (real-time RT PCR) which also distinguished between genogroups I and II (GI and GII). Norovirus infection was most commonly detected in children aged 12-23 months in both cases and controls. Norovirus Genotyping Tool and phylogenetic analysis of partial sequences of the 3' end of the RNA-dependent RNA Polymerase (RdRp) and the capsid domain region were employed to assign genotypes of the norovirus strains. GII.4 was the most predominant capsid genotype detected at 39.5% followed by GII.6 at 14.9%. The GII.4 Hunter 2004 variant was the predominant strain detected. Six RdRP/capsid recombinants including GII.P7/GII.6, GII.P7/GII.14, GII.P7/GII.20, GII.P12/GII.13, GII.P17/GII.16, and GII.P21/GII.3 were also identified. This study of norovirus infection in young children in Cambodia suggests genetic diversity of norovirus as reported worldwide.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

BACKGROUND: Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. METHODS: In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. FINDINGS: TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected. Results from TAC matched 5 out of 6 pathogen specific real-time PCR assays. TAC required a total of 5-6 hours to complete all the procedures from nucleic acid extraction and data analysis, whereas a minimum of 18 hours and 4 hours are required for conventional bacteriology and enzyme-linked immunosorbent assay, respectively, per pathogen. DISCUSSION: With TAC, pathogen load can be estimated from the amount of nucleic acid present for each pathogen, which can be analyzed further to better determine pathogen attribution and to compare pathogen load between case and control samples. Unfortunately, such correlative analysis was not possible because of the limited sample size available in this study. A larger sample size is needed for further evaluation of TAC on a specific population set, including military personnel. Regardless, TAC was found to be a useful and functional diagnostic platform that is less time-consuming, easy to use with high reproducibility, and costs less per sample compared to the current typically employed methods. The successful application of TAC in acute diarrhea stool samples from a US military population in the Philippines demonstrates its versatility as a potential candidate for a next-generation diagnostics platform.

A Field-Expedient Method for Direct Detection of Enterotoxigenic E Coli and Shigella from Stool.

We describe a field-expedient analytic system that fills a unique and critical public health role and potentially provides a valuable aid in diagnostics. Dual-fluorigenic, hydrolysis probe (TaqMan), PCR assays for detection of causative agents of enterotoxigenic Escherichia coli (ETEC) disease and shigellosis/bacillary dysentery were prepared in a thermal-stable, hydrolytic enzyme resistant format. The assays were packaged as a kit for use with a portable, ruggedized, qRT-PCR thermocycler. The analytical limit of detection of each q RT-PCR: ETEC-STIa, ETEC-STIb, and ETEC-LT assay was 30 colony forming units (CFU) and Shigella/enteroinvasive E coli assay was 3 CFU. During field evaluation, testing was conducted using a blind-panel of 138 stored stool samples previously obtained from enterotoxigenic E coli disease (n=91) and shigellosis (n=47) patients. Sample processing and analyses were completed in 3 days. Test results of the qRT-PCR assays showed promise as aid in pathogen identification when compared to culture, digoxigenin-labeled probe (ETEC), and serotyping (Shigella) the qRT-PCR. The sensitivity of each of the 4 qRT-PCR assays was 100% and specificity was ETEC-STIa (92.4%), ETEC-STIb (92.6%), ETEC-LT (79.6%), and Shigella/enteroinvasive E coli (81.6%). Sequencing of qRT-PCR amplicon indicated that the sensitivity and specificity of each qRT-PCR assay exceeded the comparator methods. The system shows promise as a rapid method for direct detection of ETEC and Shigella from stool and is applicable for use in clinical diagnostics and biosurveillance as an extension of temporary field laboratories or as a part of fixed reference laboratory facilities.

Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand.

Noroviruses (NoVs) are an important human pathogen associated with acute viral gastroenteritis worldwide. NoVs display a significant amount of genetic heterogeneity, making it difficult to develop comprehensive detection assays. In this study, primer sets and probes were designed for a TaqMan(®)-based real-time reverse transcription-polymerase chain reaction (RT-PCR) for norovirus detection purposes. The assay was optimized and utilized as a multiplex real-time RT-PCR assay for genogroup I (GI) detection, and a singleplex real-time RT-PCR assay for genogroup II (GII) detection. The assays showed high specificity for NoV detection and no cross-reactivity was observed between GI and GII. The detection limit of the assay was as low as 10 and 50 RNA copies per reaction for GI and GII, respectively. The optimized protocol was employed to assess the presence of NoV strains in clinical samples collected throughout Thailand during December 2005 to November 2006. The percentage of NoV infections among children with acute gastroenteritis (case) was 23.8% (119/500) and for children without acute gastroenteritis (control) it was 6.8% (30/441). The frequency of NoV infections varied geographically, with the highest frequency observed in the central region and the lowest frequency in the northern region (P>0.0001). Of the 149 positive case and control specimens, GII was found to be the predominant genogroup (98.6%). Partial capsid sequences were successfully obtained from 67 NoV-positive specimens and a phylogenetic analysis was performed to genotype the viral strains. GII.4 was the most common genotype detected.