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ALL LIFE 2023, VOL. 16, NO. 1, 2218582 https://doi.org/10.1080/26895293.2023.2218582 REVIEW ARTICLE New approaches for severity intervention and rapid diagnosis of enterohemorrhagic Escherichia coli: a review a b Degisew Yinur Mengistu and Yizengaw Mengesha a b Department of Medical Biotechnology, Institute of Biotechnology, University of Gondar, Gondar, Ethiopia; Department of Agriculture Biotechnology, Institute of Biotechnology, University of Gondar, Gondar, Ethiopia ABSTRACT ARTICLE HISTORY Received 29 September 2021 Enterohemorrhagic Escherichia coli (EHEC) are important pathogenic E. coli characterized by the Accepted 16 April 2023 production of Shiga toxin, intimin, and enterohemolysin, which are important virulence factors for pathogenesis. These virulence factors are the cause of enterocyte extinction and affect both intesti- KEYWORDS nal cells and various extraintestinal cells such as lymphocytes, granulocytes, erythrocytes, and renal Diagnosis; EHEC; hemolytic cells. The resultant severe bloody diarrhea and colonic infections are associated with life-threatening syndrome; Shiga toxin; extraintestinal complications, leading to high morbidity and mortality worldwide. Although EHEC severity; treatment has been responsible for large outbreaks around the world, research into effective therapies to pre- vent and treat the infection in a timely manner and affordable diagnostic tests remains a challenge due to the complicated nature of the bacteria. This review addresses previous studies, the current status, and prospects regarding the severity, treatment, and diagnosis of EHEC with a focus on a new finding on the use of simple point of care diagnostic procedures. Introduction producing bloody diarrhea (Cabrera-Sosa and Ochoa 2020). EHEC strains release a very potent toxin called Several pathogenic Escherichia coli strains can cause Shiga toxin (Stx), with the O157 (O157:H7) antigen- infections in the intestine and a variety of extrain- producing E. coli bacteria being the most common. testinal sites such as the urinary tract, meninges, In particular, O157:H7 serotype has been associated and bloodstream in animals and humans (Ruci- aka 2017;Sarowskaetal. 2019;Govindarajanand with global outbreaks of food and waterborne illness Kandaswamy 2022). These strains are evolved by (Katani 2016;Ubeda et al. 2017;Hwang et al. 2021; obtaining virulence genes from plasmids, transposons, Shumi et al. 2021), both in developed and develop- bacteriophages or pathogenicity islands (Javadi et al. ing countries including: Germany and France in 2011 (Köckerling et al. 2017); EU/EEA from 2012 to 2017 2017). According to their serogroups, pathogenicity (Biohaz Panel 2020); Sweden in 2005 (Söderström et al. mechanisms, clinical symptoms, and virulence char- 2005); Canada in 1995 (Welinder-Olsson et al. 2004); acteristics, pathogenic E. coli strains are divided into Japan from 2006 to 2010 (Watahiki et al. 2014); Korea numerous groups (Ori et al. 2019;Govindarajanetal. in 2004 (Kwun and Lee 2007); and South Africa in 2003 2020). Among the pathogenic E. coli bacteria, entero- (Hunter 2003). hemorrhagic E. coli (EHEC) are important bacte- The incidence and type of toxins in pathogenic ria that cause life-threatening hemolytic uremic syn- strains acquired from a bacteriophage are determined drome (HUS) and hemorrhagic colitis (HC) (Mühlen by the Stx1 and Stx2 genes (Onuoha 2017;Daryaei et al. 2020). These bacteria are emerging zoonotic et al. 2018;Karmali 2018;Akindolire 2019;Fitzger- pathogens and are transmitted through contaminated ald et al. 2019;Manihaand Noor 2020). The sever- food and water (Igwaran et al. 2018). They primarily ity of EHEC is caused by the production of Stx colonize the human large and small intestine, causing whichinducesaninflammatory response in thegut illness through attaching and effacing (A/E) lesions in and translocates to the epithelial barrier of the gut. the intestinal epithelium (Slater and Frankel 2020)and CONTACT Degisew Yinur Mengistu degisew12@gmail.com Department of Medical Biotechnology, Institute of Biotechnology, University of Gondar, Maraki196 Gondar, 000, Ethiopia © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. 2 D. Y. MENGISTU AND Y. MENGESHA The bacterial virulence factors intimin and Stx, as well intervention and diagnostic methods, their potential as a type III secretion system (T3SS), are associated benefits and challenges for application. with symptoms and intestinal damage leading to HUS, thrombocytopenia, microangiopathic hemolytic ane- Mechanisms of pathogenesis mia and renal damage (Delahoy et al. 2018; D’Accolti et al. 2021). According to Jubelin et al. (2018), the pathogenesis of To date, several studies have identified differ- EHEC is a multistep process that involves interactions ent interventional mechanisms, including dialysis between a number of dieff rent bacterial and host com- (hemodialysis or peritoneal dialysis), or a com- ponents. In humans, it encodes many virulence genes parison of two or more interventions were exam- that cause disease (Baranzoni et al. 2016). EHEC are ined. Interventions examined have included heparin, bacteria that cause bloody and watery diarrhea, as well aspirin/dipyridamole, prostanoids, ticlopidine, vin- as HC, HUS, and thrombotic thrombocytopenic pur- cristine, fresh frozen plasma (FFP) infusion, plasma- pura (TTP) in humans (Shahreza et al. 2017). EHEC pheresis with FFP or cryosupernatant plasma, systemic O157 has been identified as one of the major causative corticosteroids, Stx-binding agents, or immunosup- agents of thesediseasesinhumans. In thepasttwo pressive agents of HUS in humans, but specific treat- decades, E. coli O157:H7 has been one of the most mentoptionsarenotyetavailable(Michaeletal. 2009). prominent foodborne pathogens, causing enormous Intravenous u fl ids and volume expansion are non- losses in the human population (Yang et al. 2017). The specific interventions used to some extent to treat this production of Stx causing HUS, and colonization of disease. Antibiotics are generally used as therapeutics the intestinal epithelium through the formation of A/E for bacterial infections. However, antibiotic therapy is lesions are the main features of EHEC pathogenesis not recommended for patients with EHEC due to the (Shanmugasundarasamy et al. 2022). lack of benefit, and even harm that may result. When O157:H7 infection is suspected or confirmed, the use Attaching and effacing adherence of antibiotics is contraindicated because of the pos- sibility of increased risk of HUS due to induction of The significant attaching and effacing that causes the Stx-encoding bacteriophages (Garzón et al. 2020). formation of a histopathological lesion occurs at the Variousapproachestopathogendetection,such time of colonization of the large bowel and intestinal as selective culture media, enzyme immunoassays, epithelial cells by EHEC (Estrada-Garcia et al. 2013; microuidic fl systems and PCR assays, have been devel- Xue-Han et al. 2013). EHEC exploits a pedestal-like oped (Rajapaksha et al. 2019; Sivaramakrishnan et al. structure or ‘actin pedestal’ on the apical surface of the 2020). To promptly report outbreaks and prevent fur- intestinal epithelium for adhesion and colonization, ther cases, design of rapid EHEC detection meth- and then remains there to cause infection, microvil- ods with high sensitivity and specificity for pathogen lus disruption at the brush border, and polymerized identification is of utmost importance and benefit. actin buildup. For virulence and the emergence of A/E The current recommendation for EHEC diagnosis lesions, the locus of enterocyte effacement (LEE), a in humans by clinical laboratories is culture isola- chromosomal pathogenicity island present in EHEC, tion, which remains the gold standard for identi- is required (Desvaux et al. 2020; Hentschel et al. 2021). fication. Nevertheless, to reduce the analysis time, An important bacterial structure that promotes variouscosteeff ctiveand rapidimmunological and adherence is the fimbriae, thread-like structures that molecular detection systems have been developed extend from the surface of bacteria. There are and used. Effective detection of Stx has been per- three types of fimbriae responsible for adherence formed using PCR-ELISA, loop-mediated isother- (Lukaszczyk et al. 2019). This includes type I fim- mal amplification (LAMP), monoclonal and poly- briae that cause intimate adherence and A/E lesion clonal immunoassays, qPCR, multiplex-PCR, PCR- formation; P fimbriae that recognize a disaccharide less DNA co-polymerization and with DNA linked from uroepithelial cells and human erythrocytes; and gold nanoparticles (AuNPs) and magnetic micropar- S fimbriae that mediate adherence to endothelial cells, ticles (Saeedi et al. 2017).Moreover, this review dis- urinary tract epithelial cells, and brain endothelial cells cusses in detail various advanced and novel EHEC (Pal and Ayele 2017). The virulence factor produced by ALL LIFE 3 this bacterium gets transmitted to the host through a to endothelial cells that express Gb3, allowing it to T3SS. enter the bloodstream and spread to other organs (Lee and Tesh 2019), while cleaving a single adenine residue from the eukaryotic ribosomal 60S subunit’s 28S rRNA Virulence of Shiga toxin component (Hall et al. 2017). Moreover, according to The ability to produce Stx is the most important Bowen (2020), thetoxin causes diseaseby: (1)anintes- pathogenic feature of EHEC. Stx (Stx1 and Stx2) is tine epithelial barrier-crossing mechanism; (2) causing the prototype toxin of Shigella dysenteriae serotype a gut inflammatory infiltration that is neutrophil-rich; 1(Palettaetal. 2020), and consists of two major and (3) being transported by neutrophils through the subunits, the A subunit and the B subunit. The A circulation, which mediates the transport to tissues subunit suppresses cell relevant protein synthesis in with high-prevalence of Stx (Khadem 2019; Pien- the gene expression process and promotes apoptosis aar 2019;Jia andZhang 2020;Panwaretal. 2020) by acting as an RNA N-glycosidase against the 28S (Figure 1). rRNA, resulting in removal of a single adenine base from this rRNA (Menge 2020). The B subunit of Stx Clinical severity causes disease by creating a pentamer that binds to the globotriaosylceramide-3 molecule (Gb3), located EHEC outbreaks have been recorded worldwide in andexpressed in theouter layerofthe cell membrane. both developing and developed countries (Pal et al. After EHEC colonizes the human large intestine and 2019). Some people recover without treatment, but establishes pedestal-like structures on intestine epithe- others develop HC in a few days (Grantina-Ievina and lial cells (Nkiiri 2017), Stxisthenreleasedasitbinds Rodze 2020). HC is characterized by profuse bloody Figure 1. Sequences of events leading to diseases of EHEC (Source: Gerba 2009). 4 D. Y. MENGISTU AND Y. MENGESHA diarrhea, abdominal soreness, and, in most cases, Antibiotic treatment and potential preventive agents severe stomach cramps, as well as nausea, vomiting, and dehydration to some extent. Sometimes, individ- HUS includes acute renal failure causing u fl id and elec- uals may experience a low-grade fever (Kumar et al. trolyte imbalance, hemolysis, and stroke risk, as well as 2017; Travert et al. 2021). Many occurrences of HC the formation of toxins and complement complexes. are self-limiting, with patients healing in a week or To counteract this, a multidrug approach is used. less, but severe cases might cause intestinal necro- Antiplatelet and thrombolytic medicines, as well as sis, perforation, or the formation of colonic strictures thrombin inhibitors, antimicrobials, probiotics, toxin (Lianou et al. 2017;Surendran Nair 2017;Bhunia 2018; neutralizers, and antibodies against critical elements Akindolire 2019;Diasetal. 2019). of the pathogenetic pathway, are used to halt patho- HUS causes significant kidney damage, anemia, logical processes necessary for disease elimination and and thrombocytopenia in immunocompromised indi- treatment (Charnaya and Moudgil 2017;Songetal. viduals. It develops within two weeks of the onset 2019). of diarrhea, but occasionally cases without prodro- Antibiotic therapy is not usually used in EHEC to mal diarrhea do occur (Swelam et al. 2017). While avoid release of toxic substances by dead pathogenic some HUS patients have significant renal disease but bacterial cells. Monitoring hemoglobin, hematocrit, no thrombocytopenia or little hemolysis, other HUS and platelet count is also important to control hema- patients have hemolytic anemia and/or thrombocy- tologic problems (Garzón et al. 2020). Fluid and elec- topenia with little to no renal disease. trolyte imbalances are corrected with intravenous u fl id Lethargy,irritability,convulsions,paresis,stroke, administration. Renal replacement therapy, apheresis, cerebral edema, or coma are only a few examples of andantihypertensives canbeusedtotreat acuterenal extrarenal symptoms; others include respiratory syn- failure. A new EHEC treatment method involves bind- dromes, elevated pancreatic enzymes or pancreatitis, ing Stx in the gastrointestinal system using ligands deep abscesses or myocardial involvement (Bateman such as Synsorb Pk, a synthetic analog of the Stx recep- and Conrad 2016). TTP is the most common form tor(Gb3),tobindtoStx in thegastrointestinaltract of HUS in adults, particularly in the elderly, and kid- and prevent the spread of the toxin to extraintesti- ney damage is typically less common in TTP than nal sites (Detzner et al. 2022). Antibodies are used for in children, but neurologic symptoms are more com- therapeutic purposes by neutralizing the toxin. Stx1 mon (Kappler et al. 2017). Although most children monoclonal antibodies targeting the epitopes of the A recover from HUS without any lasting damage, some subunitofStx1havebeenshowntoneutralizethetoxin patients may experience long-term renal complica- (Mühlen et al. 2020;Mühlenand Dersch 2020). Orally tions of varying severity (Khalid and Andreoli 2019). administered immunoglobulin, such as for rotavirus, Extrarenal issues can also include pancreatic insuf- have been used for gastrointestinal infections (Jasion ficiency, temporary or persistent insulin-dependent and Burnett 2015). diabetic mellitus, and neurologic deficits such poor On the other hand, the use of recombinant secre- fine-motor coordination (Khalid and Andreoli 2019). tory immunoglobulin A (sIgA) in passive mucosal Clinical cases of EHEC can occur at any time. immunotherapy is common for pre-harvest interven- In various countries, the estimated annual inci- tion of EHEC. Customized intervention strategies will dence of EHEC O157:H7 ranges from 0.5 to 50 be required to prevent pre-harvest E. coli O157 col- cases per 100,000 people. EHEC O157:H7 infec- onization, which should lower carcass contamination tions arise in temperate climates during summer and improve public health. Specific direct-fed micro- months due to animal shading (Smith and Fratam- bial strains, vaccine technology, sodium chlorate, and ico 2018). However, not all EHEC infections follow neomycin sulfate are a few pre-harvest interventions thesameseasonaltrend andmay peak at dieff rent that have significant promise, while brown seaweed times. Mortality rates for clinical cases vary by dis- and water chlorination have little to no discernible ease. HC is self-limiting in most cases but can be fatal. benefit (Loneragan and Brashears 2005). Thus,asIgA It is estimated that EHEC-associated HUS/TTP kills directed against intimin would serve as a drug to pre- 1–10% of adolescents and up to 50% of the elders vent the interaction of luminal EHEC cells with the (Nkiiri 2017). ALL LIFE 5 host epithelium and eliminate them by entrapment in the probiotic cell rather than the host cell receptor. the mucosal layer and subsequent fecal shedding. The For instance, supernatant from cultures of Bifidobac- mechanism in the gastrointestinal tract is therefore terium longum HY8001 inhibits the action of Stx by that sIgA functions primarily to eliminate pathogens interfering with the B subunit of Stx in binding to Gb3 by immune exclusion (Saberianfar et al. 2019). Saberi- (Goldwater and Bettelheim 2012). One study showed anfar et al. (2019) produced a chimeric antibody that the probiotic Clostridium butyricum MIYAIRI (VHH10-sIgA) with sIgA functionality against three strain 588 inhibited the growth and toxin production EHEC strains: O26:H11, O111: Hnm, and O157:H7. of EHEC O157:H7 and had prophylactic and thera- Because antibiotic treatment of EHEC is controversial peutic effects on EHEC O157:H7 infections in gnoto- or has adverse effects that may aggravate the course biotic mice (Takahashi et al. 2004). Dini et al. (2016) of the disease, particularly by triggering Stx produc- isolated the podophage CA933P and the probiotic Lac- tion, currently available antibiotics cannot be used as tobacillus plantarum strain CIDCA 83114, which suc- therapy. New antibiotic drugs such as sIgA and lacto- cessfully inhibited EHEC pathogenesis in vitro,and ferrin (LF) are needed. Studies have shown that LF they are promising candidates for the biocontrol of has potential as a therapeutic agent in EHEC infec- EHEC (Dini et al. 2016). By reducing intestinal inflam- tions that can be used for treatment (Flores-Villaseñor mation, enhancing intestinal barrier function, and et al. 2012). This iron binding protein has shown broad managing the intestinal microbiota in infected female spectrum of antimicrobial activity both in vivo and in mice, Pediococcus pentosaceus IM96 protects against vitro (Farnaud and Evans 2003;Orsi 2004;Zimecki EHEC O157:H7 and may be an alternative therapy to et al. 2004). LF bactericidal activity sets in after the antibiotics for treating EHEC O157:H7 (Wang et al. host cell membrane has been penetrated and dam- 2020). Taken together, probiotics and their combina- aged (Haney et al. 2007; Flores-Villaseñor et al. 2012). tion with prebiotics, which are non-digestible nutri- Short cationic peptides with amphipathic structures, ents that are degraded by intestinal microbiota, ben- known as antimicrobial peptides (AMPs), which are eficially influencing their growth and activity , may mainly found in animals and plants, play an important be a promising alternative treatment approach to role in the defense against pathogens. For example, improve and maintain host health (Tabashsum et al. cecropin A and LL37 can trigger anti-inflammatory 2019). effects (Ishida et al. 2016);astudy usingmiceasa model of intestinal inflammation showed that a hybrid EHEC vaccines peptide of cecropin A and LL37 (C-L peptide) can provide effective therapy against intestinal inflamma- Novel alternatives for treatment include the use of tion and the impairment of epithelial barrier function attenuated live bacteria, such as Salmonella spp., as caused by EHEC O157:H7 (Wei et al. 2020). Another carriers for vaccine proteins against mucosal diseases study using a mouse model of EHEC O157:H7 infec- such as EHEC, as well as protein-based and DNA vac- tion shows that porcine-derived AMP PR39 alleviates cines (Nimmanapalli and Gupta 2020). Several vaccine inflammation levels, maintains intestinal barrier func- strategies have been demonstrated in animal mod- tion and immune regulation, and balances the gut els. The production of attenuated and recombinant microbiota, making this drug a potential treatment for vaccine against EHEC is another promising novel EHEC O157:H7 (Haiwen et al. 2019). approach to eradicate this disease (Goumari et al. 2020). Vaccines can be produced by using recombi- nant virulence proteins containing Stx, intimin, and Probiotics for the treatment of EHEC infections E. coli secreted protein A (EspA), an important part Probiotics that block host cells and the bacterial of the T3SS that is needed for EHEC pathogene- interaction are another promising option for treating sis, peptides such as Stx2 subunit A-Stx1 subunit B, EHEC. Probiotics can balance the composition of the and EHEC O157:H7 pathogenic host cells (Nesta and intestinal population and control the intestinal micro- Pizza 2018). Cattle vaccines against EHEC O157:H7 biota (Wang et al. 2020). They are harmless engineered can reduce shedding and have received full or con- bacteria that express a surface molecule resembling the ditional approval in some countries, including the host cell receptor, enticing the pathogen to attach to United States and Canada, but are not widely used 6 D. Y. MENGISTU AND Y. MENGESHA (Mesele and Abunna 2019). A recent study shows endothelial Tie-2 receptor and play a crucial role that vaccine formulated with the EHEC antigens in regulating endothelial cell function (Eklund and LomW, EscC, LpfA1, or LpfA2 and administered using Saharinen 2013). Another study shows that sensory AuNPs can elicit robust antigen-specific cellular and protein indicators can also be used as biomark- humoral responses associated with reduced EHEC col- ers for the diagnosis of a disease or the outcome onization in a mouse model (Sanchez-Villamil et al. of a therapeutic intervention (Bhar et al. 2020). 2022). Taken all together, distinguishing the different EHEC isotypes is not an easy task, and various molec- ular methods have been developed to distinguish Detection of EHEC Shigella spp. from EHEC. Nowadays, clinical labo- The identification of biomarkers or unique genes ratories use matrix-assisted laser-desorption ioniza- and antigens as biomarkers for Stx-producing strains, tion time-of-ifl ght mass spectrometry (MALDI-TOF including EHEC, is beneficial for diagnosis, epidemi- MS) in routine diagnostics to identify bacteria (Van ological surveillance, and phylogenetic investigation, den Beld et al. 2022). The other commercially avail- R R especially during outbreaks. To this end, several stud- able databases like VITEK MS, MALDI biotyper , ies have been conducted to develop a new marker and MALDI Security-Relevant (SR) library can distinguish methods to distinguish EHEC from other pathogenic Shigella spp. and E. coli from other Enterobacteriaceae, and non-pathogenic E.coli (McMahon et al. 2017). but they cannot differentiate among Shigella spp. and The current evolution of EHEC epidemiology requires E. coli, including the EHEC pathotype (Van den Beld the development of genetic markers to increase the et al. 2022). efficiency of diagnostic tests. For the detection of E.coli from environmental samples the most common Culture-dependent detection methods genetic markers such as 23S rRNA and lacZ are used with the established PCR method. Most PCR assays Over a long period of time, culture methods were amplify the virulence genes of EHEC, such as eaeA, referred to as a gold standard for the detection of Stx1,and Stx2,orphenotypicgenes,suchas rfbE (O EHEC in clinical and environmental samples. These antigen), fliC (H antigen), uidA and lacZ which are techniques often used vancomycin, cefsulodin, and commonly shared (Fagan et al. 1999;Sharma 2006; cefixime to limit the growth of Aeromonas spp. and Madic et al. 2010;Deshmukhand Roy 2021). Addi- Proteus spp., whereas selective novobiocin or acri- tionally, the orphan gene yaiO, which encodes an flavinwereemployedtoinhibit thegrowthofGram- outermembraneprotein, andthe xanQ gene can be positive organisms. However, the most common used to detect EHEC (Srivastava et al. 2018; Rajapak- biochemical detection method is sorbitol MacConkey sha et al. 2019;Ricke et al. 2019;Deshmukhand agar (SMAC). Because EHEC strains have lost their Roy 2021). However, the above genetic markers do sorbitol fermenting gene, they are unable or slow to not play a significant role in distinguishing EHEC ferment SMAC (Blackburn and McCarthy 2000;Perry pathogenic E. coli from Shigella spp. Recently, z3276 2017). Similarly, because of lack of β-glucuronidase and Stx genes were identified to specifically detect E. or 4-methylumbelliferyl-b-D-glucuronidase activity coli O157:H7 and screen for non-O157 Stx-producing of EHEC strains, selective culture media called chro- TM E. coli (STEC) (Stx1 and Stx2) (Deshmukh and mogenic media (CHROMagar O157 or Colorex Roy 2021). O157 Agar) exploit a substrate for β-glucuronidase to In addition to the unique gene markers and anti- detect E.coli as common urinary pathogens (Ebrahimi gen markers of EHEC, cytokines and chemokines et al. 2016;Perry 2017). Theadventofnumerouschro- could play an important role as biomarkers in diag- mogenic agars and antibody-bead retrieval systems nosing EHEC pathogenesis (Alakrea et al. 2022). Stud- has improved the diagnosis of EHEC, and they are still ies have shown that 174 serum cytokines have been the method of choice for many diagnostic laboratories detected, and vfi e serum biomarkers have been iden- (Jia et al. 2021). Even though many modified EHEC tified to predict the severity of EHEC-induced HUS methods have been investigated, the culture method (Shimizu et al. 2012). Among them are, angiopoi- hasdisadvantages,suchasthe inabilitytodetectnon- etin (Ang-1 and -2), which competitively bind to the viable organisms, the possibility that normal flora ALL LIFE 7 may mask STEC growth, the relatively low detection and accurate pathogen detection tests with maximum limit, the length of time (24–48 h) for a definitive sensitivity. diagnosis, and the additional procedures required to obtain serotype, virulotyping or antimicrobial DNA amplification detection methods resistance (AMR) information (Naylor et al. 2007; Newell and La Ragione 2018;Bizot et al. 2021). More- Various methods have been developed worldwide for over, because the culture method is labor intensive, the detection O157:H7 (e.g. PCR, selective expensive and time-consuming, there is an urgent culture, immunological techniques, LAMP, and molec- need for rapid, economical testing for ular approaches) (Zhao et al. 2019). The methods for EHEC. detecting EHEC O157:H7 do not distinguish between EHEC O157:H7 and non-EHEC O157, which are bio- chemically like other E. coli and ferment sorbitol (Mer- Immunology based detection methods canoglu Taban and Aytac 2019). Stx production is Immunology based detection methods have advan- frequently used to identify non-O157 EHEC, which tages for clinical laboratories because they have high is then sent to a reference lab for additional char- ecffi iency (specificity and sensitivity), straightforward acterization. Additionally, traditional biochemically execution, and can be performed quickly (Moges et al. based selective culture techniques are time-consuming 2022). There are several commercial immunoassays, and typically take up to 72 h. Therefore, the cre- TM such as ELISA immunoassay (EIA): ProSpecT Shiga ation of quick detection methods based on genetic R R Toxin E. coli, Premier EHEC, Ridascreen Vero- and immunological targets has drawn attention (Baker TM toxin test; Shiga toxin Check and Shiga Toxins, EIA 2015). Rapid immunological tests are exploited to with Reflex to E coli O157; lateral o fl w assay (LFA): detect O and H antigens or various genes associated R R Duopath Verotoxins, ImmunoCard STAT! EHEC, with EHEC. These tests are utilized with human clin- Ridascreen Quick Verotoxin/O157 and Shiga toxin ical or food samples, but some validated kits are not TM Quick Check ; immunomagnetic separation, such sensitive enough to test fecal samples from animals R TM as the Rapid Check Confirm STEC; and an opti- (Newell and La Ragione 2018). Because of their lack cal immunoassay, such as the Biostar OIA Shigatoxin of specificity, approaches based on the detection of (Silva et al. 2019). However, the aforementioned com- somatic O157 and flagellar H7 antigens are also inad- mercially available assays are not used as point of care equate (Jyoti and Tomar 2018; Umesha and Manuku- tests (POCT), i.e. they are neither laboratory diagnos- mar 2018). tics performed close to the patient, nor affordable to Nowadays DNA-based techniques are used to iden- implement in routine clinical laboratories in low and tify whether EHEC is present in a fecal specimen or middle-income countries. Therefore, several studies food sample (Forsythe 2020). Molecular methods such have been conducted to find a new immunological tar- as real-time polymerase chain reaction (PCR) testing get for the development of a low cost EHEC diagnostic have been used to detect foodborne diseases (such test. Shiga et al. (2020) developed a latex agglutination as E. coli O157:H7) over the past decade by ampli- test (LAT), lateral o fl w assay (LFA), and capture ELISA fying many key genes (Liu et al. 2017). For instance, (cEIA) using Stx1 and Stx2 monoclonal antibodies multiplex PCR, CRISPR-directed real-time PCR, com- with high efficiency (96%), which could robustly diag- mercial real-time PCR kits, as well as droplet dig- nose EHEC. Latex agglutination test using recombi- ital PCR (ddPCR) technology and the geneFields nant single-chain antibody fragments (scFvStx1 and EHEC/SS kit using marker genes (Stx1,Stx2, eaeA, scFvStx2) coupled to nanoparticles like latex particles and EHEC hlyA gene, iCh7, fl rfbE )weredeveloped canbeusedasaroutinelaboratoryorPOCT, with low to advance the specificity and sensitivity, rapidity, cost and short execution time (Shiga et al. 2020). The affordability and reliability of this diagnostic method rabbit antiserum specific for STEC strain VT3 ( Stx1, (Fagan et al. 1999;Sharmaand Dean-Nystrom 2003; Stx2 and eae) prepared by Hajra et al. (2007)and used Madic et al. 2010;Delannoyetal. 2012;Sadhu et al. for the latex agglutination test, was approximately 98% 2018; Mercanoglu Taban and Aytac 2019;Nihayati efficient. Therefore, efforts should be directed toward et al. 2020). Despite the need for trained personnel, the development of affordable POCTs to provide rapid operating space, and sophisticated instrumentation, 8 D. Y. MENGISTU AND Y. MENGESHA post-amplification procedures such as electrophoresis writing, writing review and editing; YM was involved in writing review and editing, revising critically, data curating, validating. take longer (3–4 h) to determine the result, require Both authorshavecommented,read, andapprovedthe final extracted DNA from the sample, and are sensitive to manuscript as well as agreed to be accountable for all aspects contaminants or PCR inhibitors (World Health Orga- of the work. nization 2020). As a result, the demand for simple and cost-eeff ctive molecular testing assay is increasing. LAMP is a Disclosure statement ground-breaking nucleic acid amplification technique No potential conflict of interest was reported by the author(s). based on auto-cycling strand displacement DNA syn- thesis using Bst DNA polymerase (Srividya et al. 2019; Data availability statement Yinur, 2019). Since the beginning of molecular biol- ogy, technologies such as multiplex PCR, real-time Data sharingisnot applicable to this articleasnonew data were PCR, reverse transcription PCR, quantitative PCR, createdoranalyzedinthisstudy. immunocapture PCR, PCR-ELISA, and other molec- ular detection methods have been developed (Dhama Funding et al. 2014;Indraswarietal. 2021). Each of these meth- The author(s) reported there is no funding associated with the ods has its own set of drawbacks. Of the above tech- work featured in this article. niques, LAMP is most likely superior because it is efficient, rapid, cheap and easy for the detection of EHEC (Dhama et al. 2014;Ravan et al. 2020). 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All Life – Taylor & Francis
Published: Dec 31, 2023
Keywords: Diagnosis; EHEC; hemolytic syndrome; Shiga toxin; severity; treatment
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