Acute Infectious Gastroenteritis: The Causative Agents, Omics-Based Detection of Antigens and Novel Biomarkers

Acute infectious gastroenteritis (AGE) is among the leading causes of mortality in children less than 5 years of age worldwide. There are many causative agents that lead to this infection, with rotavirus being the commonest pathogen in the past decade. However, this trend is now being progressively replaced by another agent, which is the norovirus. Apart from the viruses, bacteria such as Salmonella and Escherichia coli and parasites such as Entamoeba histolytica also contribute to AGE. These agents can be recognised by their respective biological markers, which are mainly the specific antigens or genes to determine the causative pathogen. In conjunction to that, omics technologies are currently providing crucial insights into the diagnosis of acute infectious gastroenteritis at the molecular level. Recent advancement in omics technologies could be an important tool to further elucidate the potential causative agents for AGE. This review will explore the current available biomarkers and antigens available for the diagnosis and management of the different causative agents of AGE. Despite the high-priced multi-omics approaches, the idea for utilization of these technologies is to allow more robust discovery of novel antigens and biomarkers related to management AGE, which eventually can be developed using easier and cheaper detection methods for future clinical setting. Thus, prediction of prognosis, virulence and drug susceptibility for active infections can be obtained. Case management, risk prediction for hospital-acquired infections, outbreak detection, and antimicrobial accountability are aimed for further improvement by integrating these capabilities into a new clinical workflow.


Introduction
AGE is defined as a diarrhoeal disease of rapid onset presenting with the incidence of three or more soft or liquid stools, or three bouts of vomiting per 24 h, with addition of abdominal pain, or fever [1,2]. This is due to the damage at the villous brush border of the intestine, which reduces the capacity to absorb intestinal contents and subsequently results in osmotic diarrhoea. Some of the causative agents would also release their toxins which bind to the specific enterocyte receptors, releasing chloride ions into the intestinal lumen which eventually causes secretory diarrhoea [3,4].
Acute infectious gastroenteritis (AGE) often affects children aged below 5 years old and causes the second highest mortality rate globally after pneumonia [5,6]. The condition is more common in developing countries than in developed countries. There have been estimated to be about 125 million gastroenteritis cases among infants 0-11 months of age and 450 million cases in children 1-4 years of age, prominently in developing countries [7].

Causative Agents of AGE
Viruses, bacteria, and parasites are among the most common infectious agents causing AGE in children. Amongst them, viruses such as Rotavirus and Norovirus contribute to the greatest rate of infection. In addition to that, astrovirus, adenovirus, and bacteria (Salmonella and Eschrichia coli) as well as parasites (Entamoeba histolytica) are also equally prominent. These agents are transmitted to the host via various routes such as faeco-oral, person-to-person, and by fomites. Table 1 represents the causative agents from the highest prevalent to the less common (virus > bacteria > parasite) and their current methods of detection.

Norovirus
Norovirus is an emerging cause of acute gastroenteritis, responsible for approximately 17

Astrovirus
With an astrovirus-causing acute gastroenteritis' global average incidence of 11%, the highest prevalence of human astrovirus (HAstV) infections was in the group of children between 37 and 48 months old [47,48]. HAstV infection mainly occurs during the dry season in the African continent; meanwhile, the highest occurrence reported in the tropical areas is often in the rainy season and winter season in temperate climate countries [47,49]. Patients usually manifested with diarrhoea, fever, vomiting, and abdominal pain [48,50].

Enteric Adenovirus Serotypes 40 and 41
Between 2.3 and 5% of the diarrhoea cases in children were caused by adenoviruses (AdV) serotypes 40 and 41 [51]. Enteric adenovirus serotypes 40 and 41 account for both sporadic or epidemic gastroenteritis in infants and young children, which are detected throughout the year with a summer peak between May and July [51][52][53]. Those infected with this pathogen will acquire mild fever and vomiting, abdominal pain, bloody diarrhoea, respiratory symptoms such as cough, and/or secondary lactose malabsorption [53,54]. Patients with adenovirus gastroenteritis have a significantly higher CRP (mean 3.4 mg/dL) and ESR value (mean 24 mm/h) compared with other gastroenteritis pathogens, and pulmonary-associated symptoms and vomiting frequency are increased in patients with this infective gastroenteritis [55]. Intravenous cytosine nucleotide analogue (CDV) that inhibits DNA polymerase has the highest in vitro activity against adenovirus, and is the preferred therapeutic agent. Elevation in lymphocyte counts (specifically CD4) were correlated with the clearance of AdV infection and improvement in survival [56,57].

Salmonella
Salmonella has become a major foodborne pathogen across the globe, causing about 3.4 million cases and 681,316 annual deaths, with 63.7% of cases occurring in children under 5 years of age [58][59][60]. There were at least 150 non-typhoidal Salmonella serotypes that can cause gastroenteritis, with Salmonella Typhimurium and Salmonella Enteritidis being the most common serotypes [59,61,62]. A study in Greece recorded that the highest rate of infection was in August, with infants being the most vulnerable group [63]. The contributing factor of salmonellosis was mainly related to consumption of contaminated food and poor clean water supply [64,65]. Salmonellosis can cause the increase in C-reactive protein values (CRP), erythrocyte sedimentation rate (ESR) and body temperature [66]. In terms of management, the recommended empiric parenteral therapy includes cefotaxime or ceftriaxone, whereas oral therapy includes amoxicillin, trimethoprim-sulfamethoxazole, or azithromycin. Salmonella isolates have a high resistance towards at least one antimicrobial agent [61,67], especially towards clindamycin, oxacillin, penicillin, and vancomycin, thus antibiotic susceptibilities of Salmonella must be determined for the targeted antibiotic therapy [62].

Escherichia coli
The WHO Global Burden of Foodborne Diseases report estimates that more than 300 million illnesses and nearly 200,000 deaths are caused by diarrheagenic Escherichia coli (DEC) globally each year. DEC is one of the major causal agents of diarrhoea in children under 5 years of age in developing countries [68][69][70], whereas children under 2 years of age are at the highest risk of infection with Enteropathogenic Escherichia coli [71,72] and Escherichia coli O157:H7 [71,73]. However, the major cause of paediatric infections in certain areas such as Ahvaz, Iran, were non-O157:H7 E. coli [74]. The incidence of non-O157 Shiga Toxin-producing Escherichia coli has been increasing in recent years, including those caused by serotypes O26, O45, O103, O111, O121, and O145 [75]. Co-infections can occur especially between EPEC and Campylobacter spp. [76]. Interestingly, Enteropathogenic Escherichia coli cases are less frequently detected in Malaysia and its similar geographical/climatic areas, in comparison with a country such as Iran where it has been reported that acute gastroenteritis was greatly caused by this strain [77]. Most DEC are sensitive to ciprofloxacin and the empirical antibiotic of choice [78,79].

Biomarkers in the Detection of Common Aetiological Agents of AGE
There are various biomarkers utilised in the detection of aetiological agents of AGE via various platforms, with the most common being the detection of antigens via enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), and immunochromatography. Moreover, detection of genes could also be conducted via procedures such as real-time PCR (RT-PCR) and multiplex real-time PCR. These are cost-effective methods that can be implemented in the future for detection of biomarkers identified via omics technologies. The breadth of the available tests, their sensitivity, and specificity are summarized in Table 1.

Norovirus
Viral RNA and antigens are the main biomarkers for detection of norovirus infection. Real-time quantitative polymerase chain reaction, Multiplex Gastrointestinal Platforms, enzyme immunoassays (EIAs) and genotyping are the main methods used in laboratory diagnosis of norovirus [99]. Cepheid Xpert ® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) for detection and differentiation of norovirus GI and GII, which account for the majority of norovirus infections worldwide [100,101]. Another real-time PCR platform, RIDA ® GENE Norovirus, can also be used as an alternative in detecting the virus [102]. Although PCRs are highly sensitive and specific, they are expensive and require specialized techniques and equipment. Rapid diagnostic tests are usually carried out during outbreak screening and patient management, which include immunochromatographic test, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay (FIA). RIDA ® QUICK Norovirus (R-Biopharm AG, Darmstadt, Germany) is one of the most used rapid immunochromatographic tests to detect norovirus [103][104][105]. Other immunochromatographic tests that are still used include QuickNaviTM-Norovirus 2 [106,107]. RIDASCREEN ® Norovirus 3rd Generation is an example of ELISA that is still currently in use [108,109]. For FIA, the Automated Fluorescent Immunoassay System NORO (AFIAS-Noro) assays (Boditech Med Inc, Gangwon-do, South Korea) are newly developed diagnostic tests for norovirus infections [95].

Astrovirus
Stool samples can be investigated using RT-PCR to detect the presence of human astrovirus (HAstV) [47,48,55]. One of the available kits is the RT-PCR Luminex Assay, with which a portion of the ORF2 capsid region is targeted by using a set of specific reverse primers labeled with biotinTEG at 5 -ends and specific probes sequences [110]. A one-step, accelerated, real-time RT-LAMP (rRTLAMP) assay can also be used by targeting the 5 -end of the capsid gene for rapid and quantitative detection of HAstV [111,112].

Enteric Adenovirus Serotypes 40 and 41
Adenovirus antigen and hexon-coding gene in enteric adenovirus serotypes 40 and 41 can be recognised and used for screening and detection methods of this pathogenic agent infection. BioNexia RotaAdeno and RIDA Quick Rota-Adeno-Combi R-Biopharm, which are the immunochromatographic tests (ICT) and LIAISON Adenovirus chemiluminescence immunoassays (CLIA) can be utilised to detect enteric adenovirus antigen [113,114]. The samples from ICT and LIAISON CLIA can subsequently undergo RT-PCR for genotyping of hexon-coding genes by using a specific TaqMan Array Card, which is a 384-well singleplex real-time PCR format that has been recognised to detect multiple infection targets [115,116].

Salmonella
As for Salmonella infection, the virulence genes include flagellin (filcC), invA, invF, sitC, hilAgene, sipC, sipF genes as well as heat stable enterotoxin gene, parE [117]. Multiplex real-time PCR such as RIDA GENE-gastrointestinal kits, EntericBio real-time Gastro Panel I, and Seeplex Diarrhea ACE detection has allowed a more rapid detection of multiple targets in a short period of time and some of the tests was followed by hybridisation to microarray/macroarray to achieve multiparametric detection of AGE aetiological agents [118]. In terms of sensitivity and specificity, RIDA GENE-gastrointestinal kits were reported to have 25% sensitivity and 99.7% specificity [119][120][121]. As for EntericBio real-time Gastro Panel I, its sensitivity and specificity were much higher, which were 100% and 97.8%, respectively [120]. Seeplex Diarrhea ACE has a sensitivity of 40-100% and specificity of 96-100% [119,122,123].

Escherichia coli
Most of the current omics approaches were focused on the detection of Shiga Toxinproducing Escherichia coli (STEC) infections. This is due to the fact that accurate diagnosis of STEC infection is very crucial because appropriate early treatment decreases the risk of serious complications and improves overall patient outcome, especially in children [124,125]. Although non-O157 serotypes account for the majority of STEC infections, they are significantly under-reported because frontline microbiology laboratories mainly focus on the detection of O157 STEC using specific agar-based methods [126]. CHROMagar STEC is a new chromogenic medium invented to improve detection of STEC, which detects O157 and non-O157 STEC through a chromogenic substrate [126,127]. However, PCR is a more sensitive test than culture [126,127]. RIDA ® GENE real-time PCR kits EAEC, EHEC/EPEC, and ETEC/EIEC (R-Biopharm, Darmstadt, Germany) all can be used to detect the aatA and aggR, eae, and elt and estA genes of Enteroaggregative, Enteropathogenic, and Enterotoxigenic Escherichia coli, respectively [128]. These will ensure that different pathotypes of diarrheagenic Escherichia coli can be detected and differentiated successfully.

Entamoeba histolytica
Entamoeba histolytica infection can cause a significant decrease in serum leptin and has been suggested to be a potential biomarker for this pathogenic infection [129], which can be detected using the ELISA technique (kit supplied by RayBiotech.Inc, Guangzhou, China). Moreover, there was also a marked increase in HDL, obestatin, calprotectin and SIgA concentration level with a concurrent decrease in cholesterol, triglyceride, LDL and VLDL concentration levels. All of these serums can be analysed and measured using ELISA techniques [130]. ELISA (Techlab II Entamoeba histolytica) can also be used to detect Entamoeba histolytica antigen presented in stool with a specificity of 100% but with a low sensitivity of 19.2% [131]. Thus, multiplex PCR is a more favourable option as it has sensitivity of 100% with specificity of 95.8% [132]. During progression of amoebiasis, a small non-coding RNA known as microRNA (miRNA) is involved in promoting apoptosis in epithelial colon cells and comprehensive profiling of miRNA using Taqman Low-Density Arrays showed a significant interaction between miRNA and parasite presented [133]. Taqman Low-Density Arrays has a sensitivity of 92% and a specificity of 100% in diagnosis of Entamoeba histolytica infection [134].

Omics-Based Technologies for AGE Diagnosis and Management
Recent advancement in omics technologies has enabled researchers to further elucidate the potential causative agents for AGE, as only known pathogens can be detected and identified by the current molecular target-dependent techniques. Metagenomics based on next-generation sequencing (NGS) platform, is a target-independent assay that allows concurrent detection and genomic characterization of all microorganisms present in a sample [136]. The latest NGS platform was first applied in AGE studies to look at the most prevalent genotype in rotavirus. The method was conducted on rotavirus positive specimens to further elucidate the most prevalent genotype in various geographical areas [135,[137][138][139][140]. Metagenomic approach using NGS can also be used to analyze norovirus strain diversity [141][142][143]. Additionally, next generation whole genome sequencing performed on fecal samples is proposed as a powerful tool for the diagnosis and genetic analysis of norovirus infection [144,145].
Similarly, astrovirus infection can be detected using the NGS platform via metagenomic analysis [136,146]. Amino acid sequence of the capsid region (ORF2) classified the identified HAstV infection into the "classical" genotypes (Mamastrovirus 1 (MAstV-1) species) or the "novel" recombinant genotypes (MAstV-6, 8, and 9 species) [147]. The other astrovirus genotypes that can be detected using NGS are also listed in Table 2 [146]. By using the similar approach, enteric adenovirus serotypes 40 and 41 can be specifically detected, in addition it is able to correctly identify and differentiate the different genotypes of human adenovirus (HAdV) A-G [136,148]. Furthermore, Entamoeba histolytica has also been analysed for its respective strains using NGS platform [149] (Table 2).
Single-cell transcriptomics is the latest omics approach to unravel the dynamics of cellular reconstruction by measuring the concentration of mRNA to thousands of genes in each cell, which can be applied in the study of gastrointestinal pathogens [150]. Transcriptomics analysis using Serial Analysis and Cap Analysis of Gene Expression (SAGE/CAGE), and microarrays also have been used to identify the present of nadA in detection of Salmonella enterica [151][152][153] and EHEC EDL933 in detection of Escherichia coli [154].
Proteomics is specially dedicated to protein analysis, by looking at the different composition of amino acids. This platform has been growing steadily and widely utilized with the supplementation of advances in both mass spectrometry and bioinformatics [155]. In detection of Salmonella Enteritidis, proteomic approaches have been used to detect isogenic Type Three Secretion System-I (TTSS-I) known as M1511 and its wild-type isolate, P125109 using a high-resolution Fourier transform mass spectrometer [156]. For Escherichia coli identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) can also be used because it is a rapid, low cost, reliable and accurate proteomic method for E. coli identification up to the subspecies level compared with classical culture microbiological techniques [157,158].
Metabolomics belongs to the 'omics' family that deals with the characterization of the set of metabolites in a given biological system [159]. As such, metabolomics analysis can be used to detect Salmonella enterica serovar Typhimurium. The most widely used techniques in metabolomics analysis are gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis coupled to mass spectrometry (CE-MS) [154,160,161]. Liquid chromatography-mass spectrometry (LC-MS) is also the metabolomic analysis technique used to detect changes in gut microbiota in children with diarrhoea caused by diarrhoeagenic Escherichia coli (DEC), as DEC-infected children and healthy children have a statistically different composition of microbiota [162].
Transcriptomics • Serial Analysis and Cap Analysis of Gene expression (SAGE/CAGE) • Microarray Salmonella enterica nadA gene [155][156][157] Enterohaemorrhagic Escherichia coli EHEC EDL933 [146][147][148]  Higher levels of histamine and lower levels of ornithine in DEC samples than in the healthy group. [156] Emerging omics technologies have evoked the elucidation of significant infectious agents of AGE. These multi-omics approaches can be used as the basis for the detection of the biomarkers which can be elucidated in the future through much cheaper methods such as PCR and ELISA in the clinical setting. Therefore, the technology in deciphering the role of causative agents of AGE should be explored and made accessible. Since multidrug resistance and healthcare-associated infections pose threats to current epidemic, this demand revolution in management strategies, hence multi-omics approaches can be one of the potential solutions in detecting potential biomarkers. Ability to manage infections can be transformed with routine access of pathogen genomic data, but only if we can amalgamate with other data for critical outcomes. Extensive clinical data related to pathogen genotypes in the long term will enable prediction of prognosis, virulence, and drug susceptibility for active infections. Integrating these capabilities into a new clinical approach in detecting biomarkers should improve case management, risk prediction, outbreak detection, and antimicrobial accountability of AGE.

Conclusions
Although rotavirus remains the main pathogen causing acute gastroenteritis in children, other pathogenic agents such as norovirus is evolving to emerge as one of the important AGE-causative agents and requires attention in the future studies. This is without ignoring the fact that bacteria and parasite pathogens also have a continuous and very relevant role in causing acute infectious gastroenteritis especially among children. With the emergence of other causative pathogens, different numerous mutations resulted in more pathogenic strains, as well as lack of reliable prognostic parameters, more robust diagnostic and predictive biomarkers need to be considered. Assuming the cost as the barrier, NGS and other omics-based approaches can be used as the fundamental methodologies for novel biomarker detection in clinical microbiology. With the wider and hopefully in the near future more accessible utilization of different omics-based approaches, these modalities should be readily considered and integrated for a more holistic, and comprehensive management of patients with AGE.