Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0-6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics.

BACKGROUND: Twenty-five percent to 50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap. METHODS: Two hundred ninety seven children ≤6 years of age, admitted to hospital in Slovenia, October 2011 to October 2012, with AGE, and 88 ≤6 years old healthy children were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites. RESULTS: At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing, and a potential viral pathogen was detected in 17 additional samples (5.8%). CONCLUSIONS: Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe.

Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.

A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1.

Newcastle disease virus (NDV), also designated avian paramyxovirus type 1 (APMV-1), is a serious pathogen of poultry, causing highly contagious Newcastle disease (ND), with high morbidity or mortality, depending on the strain. Accordingly, rapid and reliable detection of APMV-1 and differentiation between vaccine and virulent strains is of crucial importance for ND diagnosis and plays an important role in effective control of the disease. In this study, two real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays using minor groove-binding (MGB) probes were developed for broad range detection and simultaneous pathotyping of APMV-1. The two assays were evaluated for their ability to detect in allantoic fluids viral RNA of all known APMV-1 lineages. Additionally, the applicability of the developed assays was assessed by the detection and pathotype prediction of APMV-1 in swabs and organs. The assays demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation ranging from 0.2% to 3.9% and from 0.6% to 7.2% for intra-assay and inter-assay variability, respectively. The results indicated the suitability of both assays as a complementary method for rapid screening and typing of APMV-1.