Currently, STEC-HUS treatment relies heavily on supportive care, which includes fluid resuscitation, the correction of electrolyte abnormalities, and the control of hypertension [34]. Blood or platelet transfusions and renal replacement therapy (RRT) are often required [102,103]. Other treatments include antibiotics, plasma exchange, and eculizumab [104]. Even after active supportive treatment, around 30% of patients still display different long-term sequelae after acute onset, including renal sequelae (proteinuria, chronic kidney disease, etc.) and neurological complications (tetraplegia, cognitive impairment, etc.) [105,106]. In recent years, multiple alternative therapies targeting Stxs, such as antibodies, toxin receptor Gb3Cer inhibitors and Retro-2, have been developed and evaluated, bringing new hope for the prevention and treatment of STEC-HUS [107,108,109,110] (Figure 1 and Figure 3). Some of the most promising treatments are already in clinical trials (Table 1). 4.2. Renal Replacement TherapyAround 40~71% of STEC-HUS patients require RRT [129,130,131]. When patients develop oliguria AKI, fluid overload, refractory hyperkalemia, or uremia, RRT is required [104]. It includes peritoneal dialysis (PD), hemodialysis (HD), and continuous renal replacement therapy (CRRT). The selection of dialysis methods should be based on a comprehensive analysis of the characteristics of patients, the performance of dialysis methods (indications/contraindications and advantages/disadvantages), institutional resources, and local practices [132,133]. Current evidence indicates that there is no significant difference in mortality for PD, HD, and CRRT in AKI [133,134]. Although the application of PD has decreased significantly in the past few decades, overall, the application of PD in AKI has gradually increased recently in certain regions [135,136]. In Argentina, the country with the highest incidence of STEC-HUS in the world, PD is the most commonly used method, and has always been the main RRT method for the pediatric treatment of AKI [135]. The benefits of acute PD include the relatively low cost, simple technology, lack of requirement for anticoagulation or a central venous catheter, and better tolerance for patients with hemodynamic instability. It is especially applicable for patients with hypoglycemia or fluid restriction, such as neonates [133]. However, PD also has some potential disadvantages. The most common complications include catheter dysfunction, liquid leakage, hyperglycemia, and peritonitis. In addition, the following factors should also be considered in practice: the unpredictability of solute clearance and water ultrafiltration; the fact that PD is ineffective for patients in a highly catabolic state; the potential risk of hyperglycemia; the fact that PD is contraindicated in patients with recent abdominal surgery or active abdominal disease; the increased risk of pulmonary disease progression; and the higher nursing workload [133].A multicenter retrospective study in Argentina involving 389 children with STEC-HUS requiring PD treatment showed that acute PD is a safe and effective treatment for AKI in STEC-HUS children. Although PD showed more complications compared to HD or CRRT, none of the patients needed to change to another form of RRT due to the ineffectiveness of the technique. Complications related to catheter implantation should be taken into consideration during PD, and the prophylactic use of antibiotics should be considered before placing a PD catheter, to reduce the incidence of peritonitis [132].The main advantage of HD is rapid fluid and solute removal, making it suitable for children with congenital metabolic disorders and severe hyperammonemia who show no response to drug therapy [137,138]. Similar to PD, HD can be performed outside the ICU. The drawback is the need for well-functioning vascular access and hemodynamic stability. Anticoagulation with heparin, which is often necessary for patients undergoing HD, increases the risk of hemorrhage [133].CRRT is the most appropriate treatment for critically ill patients with multiorgan dysfunction and hemodynamic instability [133,138,139,140]. Its significant advantages are better hemodynamic stability and reduced cross cell solute migration, avoiding the increased intracranial pressure that may be caused by HD. CRRT provides more efficient solute removal, liquid ultrafiltration, and easier fluid balance control than PD [133,140]. The disadvantages include the higher cost, the need for continuous anticoagulation, and the risk of circuit clotting, as well as the greater nursing workload and experience needed due to its technically challenging system [133]. As such, the use of CRRT required for AKI varies widely around the world and it is less widely used in low-income countries than in high-income countries [141] (Figure 1 and Figure 3). 4.3. AntibioticsAntibiotics use in STEC-HUS is controversial and not currently recommended [142,143,144]. Opponents argue that the use of antibiotics may lead to an increase in Stx release from dead bacteria or to alterations in the intestinal flora that are conducive to the further attachment of STEC to the intestinal wall, the induction of phage production, and the expression of stx genes, which may lead to disease progression and deterioration [104,145,146]. Proponents argue that antibiotic use in the early stages of HUS reduces STEC, thereby improving HUS outcomes [104]. A prospective cohort study of 71 children under 10 years of age with diarrhea caused by E. coli O157:H7 was conducted to assess the impact of antibiotic treatment on the risk of developing HUS. The results showed that, among the 71 children, 10 (14%) developed HUS, of which 5 were treated with antibiotics. A higher initial white blood cell count, stool culture evaluation soon after onset, and antibiotic treatment were significantly associated with HUS. Their data confirm that the administration of sulfa-containing antibiotics to children infected with E. coli O157:H7 increases their risk of developing HUS and suggest that β-lactam antibiotics are associated with a similar risk. Although the confounders for disease severity in this study cohort have been questioned, the guidelines do not recommend antibiotics treatment for STEC-HUS considering the potential risk [145].However, most of the studies illustrating a relationship between antibiotics and HUS are prospective cohort studies, case-control studies, or retrospective studies, with a small sample size and different antibiotic treatment regimentations in different periods, and many results are contradictory [147,148,149]. A meta-analysis showed that antibiotic administration was not associated with a higher risk of HUS. A similar conclusion was drawn in another meta-analysis; however, antibiotic use was significantly associated with the occurrence of HUS after excluding studies with a high risk of bias and those without an acceptable definition of HUS. Therefore, antibiotics are not recommended for STEC infected individuals [143,144]. The relationship between HUS and antibiotic use is confounded by the fact that patients who become more unwell are more likely to develop HUS and receive antibiotic treatment. A single-center prospective randomized controlled trial examined the effect of trimethoprim-sulfamethoxazole on symptom duration, the fecal excretion of the pathogen, and the risk of developing HUS in children with E. coli O157:H7 enteritis. The results showed that antibiotic treatment had no statistically significant effect on symptom progression, the excretion of fecal pathogens, or the incidence of HUS [150]. Therefore, multicenter-randomized clinical trials with sufficient power to study this topic are needed to further determine whether it increases the risk of HUS [150]. Rapid diagnostic methods to permit early randomization and group statistical analysis according to the severity of the disease are required in the study [144].Some of the determinants of the progression to HUS are the infectious STEC strain, the type of toxin that it produces, and the types of antibiotics used to treat the infection. One plausible mechanism by which antibiotics increase the risk of HUS is increasing the production and/or release of Stx by inducing a precursor phage containing the Stx-encoding gene, thereby enhancing Stx transcription, phage-mediated lysis, and bacterial cytotoxic release [151,152]. An in vitro study investigated the role of different antibiotic combinations in inducing Stx2-containing phages and correspondingly affecting Stx2 transcription and production during the EHEC O104: H4 outbreak in Germany [153]. The effects of antibiotics on Stx2-harboring phage induction and Stx2 under 1/4 MIC conditions were investigated. The results demonstrated that several antibiotics, including chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline, significantly reduced the baseline levels of phage induction, Stx2 transcription, and Stx2 production in an EHEC O104: H4 outbreak strain producing Stx2.As one of the fluoroquinolone antibiotics, ciprofloxacin is often used to treat diarrhea and suspected gastrointestinal infections due to its inhibitory effect on bacterial DNA synthesis, however, its use for STEC-infected patients with diarrhea remains controversial. It has previously been shown to induce Stx-harboring phages containing Stx2 outbreak isolates in EHEC O157:H7 by inhibiting DNA replication and triggering a bacterial SOS response, significantly increased phage induction, and Stx2 production [152,153]. So far, in vitro studies on the use of fluoroquinolones have generally shown mostly unfavorable outcomes after administration. A number of studies found that ciprofloxacin can induce Stx production [142,154,155,156,157,158,159], however, only a few studies found that it can inhibit Stx production [157,158]. Most of these studies used the O157:H7 STEC strain. The results of the two studies that focused on the effect of antibiotics on STEC O104:H4 toxin production contradicted each other with regard to ciprofloxacin. In one study, ciprofloxacin induced toxin production [153], and in the other, it inhibited toxin production [158]. Similarly, results from in vivo studies of fluoroquinolones have varied widely, as some studies have found improved survival of STEC-infected animals after the administration of fluoroquinolones [160,161,162], while others found they caused HUS in animals, with no difference in survival compared to controls. On the contrary, results from clinical studies have shown surprisingly positive effects of fluoroquinolone use. A retrospective cohort study of 3323 symptomatic O157 STEC infections in the UK found no association between fluoroquinolone use and the development of HUS [163]. During the 2011 O104:H4 STEC outbreak, a small clinical study in Germany showed that ciprofloxacin reduced the risk of developing HUS [164]. In studies conducted in Japan, fluoroquinolones were found not to affect and may have even reduced, the incidence of HUS [147,165]. A clinical study has found that combination therapy with meropenem and ciprofloxacin (or rifaximin) can eradicate E. coli O157 in patients eight days earlier than in patients with no antibiotics administered with a lower epileptic seizure rate and mortality rate [166]. This indicates that the combination of suppressive antibiotics with antibiotics that rapidly eradicate infection may be beneficial [146]. However, given the current mixed and contradictory results and conclusions, there is still insufficient evidence that the administration of ciprofloxacin is better than no antibiotic use [146,167].Studies have also reported that subinhibitory concentrations of meropenem, azithromycin, and gentamicin do not increase Stx production in 12 different serotypes of highly virulent STEC [168]. Gentamicin, which blocks ribosomal protein synthesis and is not absorbed by the intestinal wall to achieve high intestinal concentrations, may be a potential treatment for STEC infection, however, its potential renal injury side effects may limit its use in STEC-HUS. Azithromycin inhibits RNA-dependent protein synthesis, thereby inhibiting Stx production. Azithromycin treatment resulted in the lowest toxin production from 12 highly virulent STEC strains [168]. Furthermore, studies have reported that sub-inhibitory doses of azithromycin have no effect on toxin production in vitro, and azithromycin does not induce Stx2a transcription in STEC O104:H4 [155,168]. Some researchers have suggested that STEC infections can be treated with an oral protein synthesis inhibitor for three days, followed by a wall synthesis inhibitor for seven days [142]. RCT studies of azithromycin in the treatment of diarrhea-associated HUS are currently being conducted in France, and the results will further reveal whether azithromycin can be used to treat STEC-HUS (Table 1 and Figure 3).In a recent review of antibiotics and HUS, Tarr and Freeman recommended against the use of antibiotics in patients with a confirmed or suspected STEC infection based on evidence from multiple retrospective cohort studies. The reason is that there is still no data that convincingly show that antibiotics are superior to no antibiotic treatment at all, and many studies have shown that antibiotics increase the risk of developing HUS [167]. As such, antibiotic administration is performed on a case-by-case basis in consideration of the necessity of antibiotic use, the STEC strain, the timing of treatment, and the type of antibiotics to be used. The necessity of antibiotic use includes whether infections of other systems exist or the need to prevent potential infection risks, such as the need for peritoneal dialysis catheter implantation and the combined use of eculizumab, among others [132,146]. 4.5. EculizumabEculizumab is a humanized anti-C5 monoclonal antibody with a high affinity for the human C5 complement protein. It inhibits the activation of the complement factor C5 and prevents the formation of the C5b9 membrane attack complex. This biological agent was approved by the FDA in September 2011 for the treatment of aHUS [177,178]. Although the role of the complement protein in STEC-HUS has not been fully understood, eculizumab has been used as an off-label treatment for STEC-HUS patients with severe complications of the nervous system, such as those with neurological or multiple-organ dysfunction. A positive clinical improvement after treatment has been reported, however, the overall quality of evidence is low [179,180]. For example, three children with STEC-HUS and progressive central nervous system involvement reported significant neurologic improvements within 24 h after the first eculizumab infusion. Screening for mutations in the genes encoding complement regulatory proteins and testing for anti-CFH antibodies were negative in these patients. This suggests that a complement activation blockade may provide potential benefits in patients with STEC-HUS [181].In a retrospective single-center matched cohort study in France, the renal outcomes were compared in 18 and 36 matched children treated with or without eculizumab for STEC-HUS, respectively. There was no statistically significant difference in the evolution of hematological and renal parameters, the incidence of a decreased glomerular filtration rate, proteinuria, or hypertension between these two groups. Children treated with eculizumab frequently displayed neurological sequelae during follow-up, which may reflect the involvement of more severe neurological complications at the onset of HUS in the eculizumab group [182]. Notably, eculizumab increases the risk of Neisseria meningitis infections, so specific vaccinations and brief antibiotic coverage are required at the start of treatment [178].In a review of STEC-HUS, 16 reports describing the use of eculizumab in STEC-HUS were reviewed (eight case reports/series, seven retrospective studies, and one prospective cohort study). All the studies described its use in severe STEC-HUS with neurological or multiorgan dysfunction; none of them were randomized or blind. Control groups were used in four studies. Despite the overall low quality of evidence, the study showed positive clinical improvements after the treatment of patients with severe progressive STEC-HUS with neurological involvement with eculizumab [180].Since the majority of patients recover with supportive treatment, the risks and benefits of eculizumab need to be fully evaluated before its use, especially for those with complement activation, neurological involvement, and a high risk of death. In the meantime, randomized controlled trials of eculizumab after the stratification of disease severity will provide more convincing evidence, and positive results are expected in some ongoing clinical trials [182,183] (Table 1). 4.6. AntibodiesAntibodies can neutralize Stxs in the serum and potentially even in the gut, making these molecules powerful weapons against toxins. In order to produce antibodies for therapeutic use, there are three main approaches: polyclonal antibody (pAb) generation by animal immunization; monoclonal antibody (mAb) production for the secretion of mAbs specific to lymphocyte immortalization; and the creation of different recombinant antibody (rAb) forms for different targets through DNA recombination techniques and heterologous expression systems [184] (Figure 1 and Figure 3). Compared with monoclonal antibodies, polyclonal antibodies (pAbs) have many advantages in antitoxin therapy, including the ability to recognize a large number of epitopes, stronger affinity than monoclonal antibodies, and the ability to recognize variants of toxins to reduce the risk of escape [118,185]. However, although polyclonal antibodies have shown promising neutralization in vitro and in vivo, animal sources of polyclonal antibodies may induce anti-antibody action that inactivates therapeutic antibodies before they can exert their toxin-neutralizing activity. In addition, the amount of animal serum is limited by the size of the immunized animal [184]. Equine polyclonal antibodies (EpAbs) are easy to manufacture and have been successfully used in a variety of diseases. In the past, serum diseases and anaphylactic shock (mainly due to the presence of Fc fragments) have discouraged the use of EpAb, however, the new-generation (third-generation serum) processed and purified EpAb contains a highly purified F(AB ‘)2 fragment and is well tolerated. Inmunova (San Martin, BA, Argentina) has developed an equine anti-Shiga toxin (NEAST, INM004). INM004 has the advantage of being broad-spectrum: it can recognize and neutralize different variants of Stx. Additionally, NEAST is designed to prevent HUS in patients with STEC infections [118]. The efficacy and potency of this antiserum against Stx1 and Stx2 have been demonstrated in different preclinical models, and it has been shown to be safe in animals [186]. A phase I clinical trial at a hospital in Buenos Aires demonstrated the product’s safety in healthy adult volunteers and evaluated its pharmacokinetics. Phase Ⅱ and phase III clinical trials were conducted in Argentina with enrolled children diagnosed with STEC infections, and the results are pending [118,187] (Table 1).Several monoclonal antibodies have been developed to neutralize the toxicity of Stxs, such as monoclonal antibodies against Stx1 and 2 (cαStx1 and cαStx2, Shigamabs®), and urtoxazumab (TMA-15, Teijin Pharma Limited, Tokyo, Japan), which is a humanized monoclonal antibody against Stx2. These monoclonal antibodies have shown promising results in preclinical studies, and their efficacy will be further verified in clinical trials (Table 1) [113,114,188,189,190]. As a chimeric mouse–human monoclonal antibody, Shigamabs® demonstrated the ability to neutralize Stxs in mice and was well tolerated in healthy human volunteers and children infected with STEC in phase Ⅱ clinical trials [114,116]. TMA-15 (urtoxazumab) was produced by combining mouse antibody complementary regions with human frame and stationary regions; it could protect mice from death 24 h after STEC infections and reduce brain damage and death in probiotic piglet models [190,191,192]. TMA-15 was found to be well tolerated in healthy adults and pediatric patients with confirmed STEC infections when tested intravenously in phase Ⅰ and Ⅱ clinical trials [188] 4.7. Gb3Cer InhibitorsAnother promising therapeutic strategy is the use of Gb3Cer inhibitors. Glucosylceramide (GlcCer) is a biosynthetic precursor of glycolipids including Gb3Cer and other sphingolipids [193]. GlcCer synthesis is catalyzed by glucosylceramide synthase (GCS, also known as UGCG). Subsequently, galactose is added to produce lactosylceramide (LacCer). Additional glucose is then added to produce Gb3Cer and other sphingolipids [13]. Different inhibitors of GCS have been identified and used to treat several glycosphingolipidoses, such as Fabry disease [194]. These compounds inhibit sphingolipid synthesis in cultured cells without inhibiting cell growth or increasing intracellular ceramide levels [193].Eliglustat (EG) and miglustat (MG) have been shown to inhibit Gb3Cer expression by blocking GCS; they can prevent the toxic effects of Stx2 on human colon epithelial cells, human renal tubular epithelial cells (HRTECs), human glomerular endothelial cells (HGECs), and proximal renal tubular epithelial cells (HK-2) [195,196,197]. HRTECs with 50 nmol/L EG at 24 h or 500 nmol/L EG for 6 h reduced Gb3Cer expression and completely inhibited the effects of Stx2 on cell viability, proliferation, and apoptosis. Pretreatment with MG for 24 h and especially for 48 h produced a significant protective effect with reduced Gb3Cer expression, cell death, intracellular edema, and cell detachment [195]. EG may be a potential therapeutic agent for preventing Stx2-induced AKI. EG is approved in the United States for patients diagnosed with Gaucher disease type 1 for whom enzyme replacement therapy is unsuitable, and it may be more suitable for clinical use in patients with STEC-HUS [196].C-9 (GENZ-123346), an analog of EG, is a specific inhibitor of GCS, which reduces Gb3Cer, protecting target organs from toxins [110]. Primary human renal tubular epithelial cells (HRTECs) and human glomerular endothelial cells (HGECs), preincubated with C-9, showed reduced Gb3Cer expression and were protected from Stx2 challenge [198]. Oral C-9 treatment significantly reduced mortality to 50% in Stx2-treated rats with reduced Gb3Cer expression in the kidney. It also prevented kidney and colon lesions from Stx2. However, in animal studies, it is necessary to start the inhibitor’s administration two days in advance. In addition, a significant increase in 24-h urinary albumin was reported in mice administered C-9 for 3 weeks [199], although it is unknown whether this phenotype recovers after the withdrawal of the inhibitor. However, this may limit the clinical use of C-9 in the treatment of STEC-HUS patients.Venglustat, a novel central nervous system (CNS)-active GCS inhibitor, has been shown to reduce cerebral glycolipids and prolong life in a murine model of both type 3 Gaucher disease and Sanhoff [200,201]. Venglustat is being developed as a substrate reduction therapy for a variety of diseases, including type 3 Gaucher disease and Fabry disease [202,203]. It showed good safety and tolerability in phase I clinical trials [203], and it is undergoing phase Ⅱ clinical trials for patients with Fabry disease, while patients with Gaucher type 3 are also being recruited [204] (Figure 1). 4.9. Retro-2Stxs bound to Gb3Cer on the cell membrane induce endocytosis of the toxins; they then bypass the late endocytic pathway to reach the Golgi apparatus and ER through a retrograde transport pathway (Figure 1). Blocking the retrograde transport of Stxs is another therapeutic strategy that can be considered. Stechmann et al. utilized high-throughput screening to identify Retro-1 and Retro-2, which are small-molecule inhibitors that can protect cells from ricin and Stxs by selectively blocking retrograde transport at the early endosomal-trans-Golgi interface without affecting organelle integrity. In mice, Retro-2 significantly protects against nasal exposure to the lethal dose of ricin [205]. Retro-2 was subsequently shown to have protective effects against Stxs in cells and mice [206]. Studies have shown that Retro-2 targets the ER outlet site component Sec16A and affects the downstream transport of the Golgi SNARE proteins syntaxin-5 from the ER to the Golgi apparatus [207] (Figure 1).However, the extremely poor solubility of Retro-2 at all gastrointestinal pH values limits its application. Gandhi et al. developed Retro-2-loaded self-nanoemulsifying drug delivery systems. Lauroyl arginine ethyl (LAE) is a cationic surfactant with L-arginine as a hydrophilic component and lauric acid as the hydrophilic part [208]. Due to their chemical properties, they can break down cell membranes at very low concentrations, altering their potential, affecting cell permeability, and causing bacterial cell death, and they can be used as antimicrobial agents. They can also be quickly metabolized by the body into natural dietary ingredients—lauric acid and arginine—making them safe for users [208]. In this technique, Retro-2-loaded arginine-anchored nanospheres (R-AR-NGs) were prepared by mixing solutions of LAE and Retro-2 in DMA with a solution of oil and surfactant in selected proportions. When R-AR-NG is added to the aqueous phase, positively charged nanospheres spontaneously form. AR-NG breaks down spontaneously into L-arginine and kills EHEC in the gut. AR-NG then binds to the LPS released by dead E. coli through electrostatic interaction. AR-NG is released in the gut environment and maintains Retro-2 lysis, thereby inhibiting the retrograde transport of Stx. The nanoglobule significantly increased the water solubility of Retro-2 and blocked Stx’s intestinal-to-blood transport. This technique opens up the possibility for Retro-2 to control EHEC O157:H7 infection in clinical applications [208].