Role of Urine dipstick and Azithromycin for the Management of an Outbreak of Shiga toxin-producing Escherichia coli Infection

Role of Urine dipstick and Azithromycin for the Management of an Outbreak of Shiga toxin-producing Escherichia coli Infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of Urine dipstick and Azithromycin for the Management of an Outbreak of Shiga toxin-producing Escherichia coli Infection Gianluigi Ardissino, Maria Grazia Nanni, Mario Luini, Maria Cristina Mancuso, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9216605/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen accounting for approximately 5–6% of cases of acute bloody diarrhea (ABD) in Europe. Herein, we report how an outbreak of STEC infection involving a group of scouts was managed with the pivotal role of urine dipstick testing for hemoglobinuria (uHb) to identify patients with ongoing hemolytic uremic syndrome (HUS). We also describe the potential benefit of azithromycin treatment to prevent the development of the most severe complication of STEC infection: hemolytic uremic syndrome (HUS). Methods All subjects involved in the outbreak were invited to provide a stool sample for Stx-encoding genes testing. Symptomatic patients underwent urine dipstick for uHb and started azithromycin (10 mg/kg/day) treatment. Patients who tested positive at urine dipstick (uHb ≥ 2+) were further investigated with blood tests to rule in or out the diagnosis of HUS. Results Out of 31 exposed subjects, 26 were symptomatic and 13 tested positive for genes encoding Stx1, Stx2 and O157. All symptomatic patients were prescribed oral azithromycin treatment for 5 days and were monitored for uHb. Two patients showed a transient positivity for uHb but none progressed to HUS. Conclusion Urine dipstick is a simple, but effective tool to monitor patients with STEC infection for the possible development of STEC-HUS. Azithromycin treatment was safe, thus can be an useful resource to manage a STEC outbreak. Outbreak Shiga toxin Escherichia coli hemolytic uremic syndrome urine dipstick azithromycin Figures Figure 1 Introduction Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen accounting for approximately 5–6% of cases of acute bloody diarrhea (ABD) in Europe, increasing to 15–20% during late summer and early fall [1]. The disease is transmitted by undercooked contaminated meat, unpasteurized dairy products, contaminated water, or contaminated vegetables. Direct contact with ruminants and person-to-person spread can also occur, being ruminants the main natural reservoir. Approximately 10–20% of infected children develop hemolytic uremic syndrome (STEC-HUS), which represents the most severe systemic complication of STEC infection, with an estimated annual incidence in Italy of 6–7 cases per million age-related population [2,3]. The risk of progression to STEC-HUS is influenced by the virulence profile of the pathogen, being highest (23%) in case of positivity for Shiga toxin (Stx) 2 alone. Infections with EC producing both Stx1 and Stx2 are less frequently associated with STEC-HUS development (12%), and the risk is negligible for Stx1 alone (< 1%) [4]. In July 2025, a group of scouts consisting of 22 children, 7 accompanying and 2 non-accompanying adults, participating in a camping activity in Northern Italy, experienced an outbreak of gastrointestinal (GI) symptoms (diarrhea, abdominal cramps, nausea, vomiting, and low-grade fever). On day 3 following the onset of symptoms in the index case, three patients with more severe gastroenteritis underwent molecular microbiology stool testing that revealed a positivity for genes encoding Stx1, Stx2, and O157. The pivotal role of urine dipstick testing for hemoglobinuria (uHb) to identify patients with ongoing hemolytic uremic syndrome (HUS), as well as the potential benefit of azithromycin treatment to prevent the development of the renal complication, are described and discussed. Methods Given the rapid spreading of symptoms among many of the participants, the activity was interrupted and all subjects returned home. In view of the potentially severe consequences of the ongoing epidemic, subjects attending the camping were traced back by the local Center for Disease Prevention and Control in order to inform them and assess their clinical conditions. All participants were invited to provide a stool sample for Stx-encoding genes testing, and health surveillance measures were promptly initiated. Symptomatic patients were referred to the nearest hospital to undergo urine dipstick for hemoglobinuria (uHb) and identify those who might already have had an ongoing HUS. Patients who tested positive (uHb ≥ 2+) on urine dipstick were further investigated to rule in or out the diagnosis of HUS with platelet count, hemoglobin, plasma albumin, and lactate dehydrogenase levels. Furthermore, all symptomatic patients were prescribed oral azithromycin at the dosage of 10 mg/kg once daily with a maximum of 500 mg/dose for 5 days. In an attempt to identify the source of the infection, detailed information was collected on the foods consumed by the group and on the activities carried out during the days preceding the onset of symptoms. During the inspection, several samples were collected from environmental and food matrices, as well as tap water from various sampling points (n: 4). The sampling included leftovers from the last meal consumed before the outbreak, which consisted of chicken, pasta with tomato sauce, carrots, and peas. Results Out of 31 exposed subjects, 26 were symptomatic (13 females; median age 11.2 years, IQR 9.8–18.7). All underwent the recommended clinical procedures. Two patients tested positive for uHb on initial urine dipstick testing; however, blood tests ruled out ongoing HUS. Molecular biology investigations revealed Stx-encoding genes in 10 additional patients, exhibiting a molecular pattern consistent with that of initial patients. Urine dipstick was repeated at the end of azithromycin treatment to ensure that the thrombotic microangiopathy (TMA) had not occurred in the meantime. None of the patients tested positive for uHb, and all of them recovered from GI symptoms without progressing to HUS. The scouts reported that tap water at the accommodation facility was brownish, and investigations carried out on drinking water revealed the presence of high levels of Escherichia coli in all tested samples, but unfortunately no coltural or molecular test for STEC has been performed. Examined food residues tested negative for STEC. Based on these findings the source of the infection remains undetermined, but suspicion falls on the water supply that was microbiologically and visually altered. Discussion STEC-HUS is a severe, endemo-epidemic TMA that arises as a complication of a GI infection caused by STEC. The management of outbreaks can be very challenging (occasionally dramatic) for both public health (PH) professionals and clinicians, as clearly emerged during the Japanese outbreak in 1996 [5] and the Northern European one (mainly affecting Germany) in 2011 [6]. STEC outbreaks require the integrated coordination of multiple disciplines and expertise, encompassing healthcare providers with potentially different perspectives and objectives: public health vs. clinical care, community-based vs. hospital-based services, pediatricians vs. adult physicians, human vs. veterinary health, and food safety vs. environmental health. Timely intervention is critical as STEC infections can spreads rapidly and may lead to severe and life-threatening complications. It is essential for health authorities (HA) to have a clear pre-established response plan with well-defined priorities and actions aimed at protecting both individuals and community. Based on the experience gained during previous STEC-HUS outbreaks that have taken place in our area, but also in view of new therapeutic opportunities supported by an easier access to molecular microbiology, we have developed a structured approach to manage STEC outbreaks (Fig. 1 ). This approach was successfully implemented during the epidemic herein described. We believe that this experience is worth being shared with the scientific community because it includes two innovative elements: Urine dipstick testing for early detection of HUS infected patients. The use of urine dipstick testing enables rapid and effective identification of subjects who may already have developed HUS and thus require immediate referral to hospital care. The disease severely disrupts glomerular structure due to microvascular thrombosis caused by endothelial damage induced by Stx. The described pathogenic sequence is invariably associated with hematuria – sometimes gross hematuria – making urine dipstick detection of hemoglobinuria highly sensitive (100%), although less specific (84%) [7]. Obviously, a positive dipstick test will require a full set of blood tests to rule in or out the diagnosis of HUS. In STEC-infected patients with negative urine dipstick, it should be regularly repeated to monitor for the potential onset of HUS until diarrhea has fully resolved. Azithromycin treatment for the secondary prevention of STEC-HUS in infected subjects. Antibiotic use has historically been contraindicated in STEC infections [8,9]. Antibiotic treatment seems to increase the risk of STEC-HUS development by inducing prophages containing Stx-encoding genes through the bacterial SOS response, thereby promoting Stx production, switching from a lysogenic state to a lytic cycle, and leading to Stx release from dead bacteria via phage-mediated lysis [10,11]. However, current recommendations are primarily based on clinical studies that do not distinguish between different classes of antibiotics, combining bacteriostatic and bactericidal drugs. Several in vitro studies have demonstrated that the effect of antibiotics on toxin release depends on the type and concentration of the antibiotic used, and differs among STEC strains [12,13]. Indeed, the available literature shows that bactericidal antibiotics, such as quinolones, beta-lactams, and cephalosporins, actually increase Stx production in vitro and therefore may increase the risk of progression to HUS [14,15]. In contrast, emerging evidence supports the benefits of the use of bacteriostatic agents, such as transcriptional and translational inhibitors. In particular, azithromycin has been shown to reduce Stx production in vitro [15–18] and to exert protective effects against neurological and gastrointestinal complications associated with STEC infection in animal models [19]. Moreover, a recent retrospective study evaluating the effect of the use of different antimicrobial classes during the first week of STEC-related diarrhea showed that none of the patients treated with azithromycin developed HUS [20]. In Denmark, azithromycin is currently recommended for STEC infection under specific conditions, primarily for decolonization purposes [21]. Finally, a recent retrospective analysis conducted at our Center [22], including 89 patients treated with oral azithromycin at a dosage of 10 mg/kg once daily for 5 days, reported that only four developed HUS, whereas the expected number was eleven. This corresponds to a conversion rate of 4.5%, compared to a historical rate of 13.2% (p: 0.02). Conclusion Based on this and on previous experiences, the key procedures for the management of a STEC infection outbreak (2 or more cases of STEC infection or STEC-HUS temporally and spatially related) can be summarized in the following 10 “hints and tips”: 1. In case of a confirmed or suspected STEC outbreak, the event must be immediately reported to relevant HA. 2. HA must promptly inform all physicians operating in the involved area (including emergency departments) to ensure that all patients presenting with bloody or watery diarrhea are tested for: a. Presence of Stx-encoding genes in stool samples via PCR, to identify subjects at risk of developing HUS; b. Presence of uHb using urine dipstick testing to actively identify patients with already ongoing HUS. 3. Stx-positive patients should be prescribed oral azithromycin at a dosage of 10 mg/kg once daily (with a maximum of 500 mg/dose) until diarrhea resolution, for up to 5 days. 4. Stx-positive patients, including those receiving azithromycin, should undergo daily urine dipstick testing until diarrhea resolution, to enable early detection of HUS onset. 5. Stx-positive patients with uHb ≥2+ on urine dipstick should be immediately addressed to blood tests to rule in or out signs of HUS (platelet consumption, hemolysis in addition to renal damage). 6. If stool samples test positive for Stx or HUS is confirmed, samples must be sent to the appropriate laboratory for STEC isolation and characterization. 7. Stx-positive patients should not be admitted to community settings (kindergarten, school, summer-camp, etc.) until full symptom recovery. In presence of multiple cases, temporary closure of the institution should be considered. 8. Family members and household contacts of Stx-positive patients should be tested for Stx regardless of symptoms. 9. HA should establish a permanent team of experts conveying in case of outbreaks to coordinate, process, merge and discuss information and laboratory findings collected from different sources. 10. The source of the infection should be thoroughly investigated, with special regard to undercooked meat, unpasteurized dairy products, and raw vegetables. Possible sources also include stools from farm animals and pets, as well as freshwater sources such as pools, wells, lakes, and rivers. Recent travel history to areas with poor food chain control should also be explored. In our opinion, the described approach to the management of STEC outbreaks provides the best opportunity for the early identification of STEC-HUS and for protecting both individuals and community from the possibly devastating consequences of infection spreading, while also increasing the likelihood of identifying the source of the infection. Abbreviations STEC: Shiga toxin-producing Escherichia coli ABD: acute bloody diarrhea HUS: hemolytic uremic syndrome Stx: Shiga toxin GI: gastrointestinal uHb hemoglobinuria TMA: thrombotic microangiopathy PH: Public Health HA: health authorities Declarations Conflict of interest: Authors have no conflicts of interest to disclose. Ethical statement Given its observational nature, the study was approved by the Institutional Review Board (internal board of the Dept. of Pediatrics). All patients and/or parents (as applicable) gave their informed consent to use their relevant clinical information for research purposes. The study was conducted in accordance with the Declaration of Helsinki. Use of artificial intelligence tools The authors declare that no artificial intelligence tools were used during the preparation of this manuscript. Funding statement: The authors declare that no funding, grants, or other support were received during the preparation of this manuscript. Author Contribution G.A. conceptualized and designed the study, coordinated and supervised data collection, drafted the initial manuscript and critically reviewed and revised the manuscript.M. G. N., M. C. M., G.T.,L.D., D.R., M.T.,E.P. and D.D.C enrolled patients in the study and critically reviewed and revised the manuscript.G.A, L.D. and D.R. carried out statistical analysis.L.D., A.G., M.L. and M.G.N. managed the laboratory procedures and carried out the bacteriological investigations.All authors approved the final manuscript as submitted and agree to be accountable for all aspectsof the work. Acknowledgement We wish to express our sincere gratitude to the following members of the ItalKid-HUS Network for their valuable collaboration and their commitment to the screening of patients presenting with acute bloody diarrhea:Fiorella Acquotta, Paolo Adamoli, Nicola Altamura, Angela Amoroso, Stefano Andreoni, Massimo Andreotti, Maddalena Antolini, Milana Arghittu, Francesca Atzeri, Carlo Baldioli, Barbara Balduzzi, Irene Benini, Simone Benvenuto, Francesco Beretta, Cristina Bertulli, Valeria Besutti, Lorenzo Biscardi, Annalisa Bonazza, Cristina Bonetti, Annalisa Bosco, Grazia Bossi, Marta Brambilla, Maria Francesca Brambillasca, Valentina Burzio, Anna Elisabetta Bussolini, Elena Cama, Patrizia Carlucci, Maria Carrabba, Maria Luisa Casciana, Daniela Casnaghi, Eleonora Castellone, Valeria Castorani, Marco Cazzaniga, Claudio Cavalli, Lisa Lucia Chenal, Rosaria Celano, Rossella Ceruti, Marta Cerutti, Giulia Cesano, Giulia Chiopris, Anna Cogliardi, Giacomo Colella, Lucia Collini, Rosaria Colombo, Dario Consonni, Crescenzo Coppola, Angela Corna, Annalisa Corti, Giorgia Daffunchio, Simona De Franco, Elena Lucia De Rose, Laura Dell’Era, Giulia Dilio, Clelia Di Mari, Sandra Esposito, Diana Fanti, Alessandra Ferrari, Ilaria Frugnoli, Maria Forestieri, Gloria Fumagalli, Maria Rita Gallina, Miriam Chiara Gatto, Claudio Giacomazzi, Stefano Grossi, Silvia Grosso, Chiara Gualeni, Elisa La Barba, Anna Maria La Pusata, Sara La Rosa, Annalisa Lastrico, Alberto Lepre, Francesca Lizzoli, Rosa Maria Maccarone, Anna Madera, Laura Martelli, Elena Mazzali, Elisabetta Mazzola, Marcella Mercuri, Elisa Milanesi, Chiara Minini, Paola Mirri, Sonia Monticone, Alice Monzani, Vincenza Morinello, Arianna Moroni, Maria Carmela Musolino, Maria Amata Negri, Stefano Nespoli, Bianca Osnaghi, Elisabetta Pagani, Franca Pagani, Cristina Partenope, Palmiro Pedroni, Giovanni Raimondo Pieri, Maria Antonietta Piscopo, Stefano Poli, Ilaria Possenti, Giulia Ramponi, Agrippino Reciputo, Barbara Roman, Barbara Ronchi, Chiara Rosazza, Elena Rossi, Claudia Ruggieri, Maria Lorena Ruzza, Filippo Salvini, Letizia Sardella, Martina Scali, Chiara Sciuto, Alessandra Scolari, Micaela Silvestri, Daniela Simoncini, Giulia Smylie, Rosa Maria Taibi, Francesca Tel, Sara Testa, Valentina Todescato, Elena Tommasoni, Paola Tommasi, Gaia Vanzù, Antonio Vergori, Federica Vianello, Chiara Vismara, Chiara Zambetti. 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1","display":"","copyAsset":false,"role":"figure","size":44210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed approach to STEC outbreak management.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eLegend. \u003c/strong\u003eED: Emergency Department; PCR: polymerase chain reaction; Stx: Shiga toxin; HA: Health Authority; STEC: Stx-producing \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e; uHb: hemoglobinuria; PTL: platelet; STEC-HUS: Shiga toxin-producing\u003cem\u003eEscherichia coli\u003c/em\u003e-related hemolytic uremic syndrome; §: Isotonic solutions with target weight +5-10%% of usual weight; *: Complete cell blood count, serum creatinine, urea, lactate dehydrogenase, albumin, liver enzymes, C reactive protein; °at a dosage of 10 mg/kg (max. 500 mg) per day until diarrhea resolution (max. 5 days).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9216605/v1/9c3c43e3ee526c867fc2e762.png"},{"id":108977662,"identity":"27db3cdc-6f8c-4d67-942c-9928c4663332","added_by":"auto","created_at":"2026-05-11 11:32:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":207409,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9216605/v1/e397d497-4b24-4acb-91b3-33b0241b974d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of Urine dipstick and Azithromycin for the Management of an Outbreak of Shiga toxin-producing Escherichia coli Infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eShiga toxin-producing \u003cem\u003eEscherichia coli\u003c/em\u003e (STEC) is a foodborne pathogen accounting for approximately 5\u0026ndash;6% of cases of acute bloody diarrhea (ABD) in Europe, increasing to 15\u0026ndash;20% during late summer and early fall [1]. The disease is transmitted by undercooked contaminated meat, unpasteurized dairy products, contaminated water, or contaminated vegetables. Direct contact with ruminants and person-to-person spread can also occur, being ruminants the main natural reservoir. Approximately 10\u0026ndash;20% of infected children develop hemolytic uremic syndrome (STEC-HUS), which represents the most severe systemic complication of STEC infection, with an estimated annual incidence in Italy of 6\u0026ndash;7 cases per million age-related population [2,3]. The risk of progression to STEC-HUS is influenced by the virulence profile of the pathogen, being highest (23%) in case of positivity for Shiga toxin (Stx) 2 alone. Infections with EC producing both Stx1 and Stx2 are less frequently associated with STEC-HUS development (12%), and the risk is negligible for Stx1 alone (\u0026lt;\u0026thinsp;1%) [4].\u003c/p\u003e \u003cp\u003eIn July 2025, a group of scouts consisting of 22 children, 7 accompanying and 2 non-accompanying adults, participating in a camping activity in Northern Italy, experienced an outbreak of gastrointestinal (GI) symptoms (diarrhea, abdominal cramps, nausea, vomiting, and low-grade fever). On day 3 following the onset of symptoms in the index case, three patients with more severe gastroenteritis underwent molecular microbiology stool testing that revealed a positivity for genes encoding Stx1, Stx2, and O157.\u003c/p\u003e \u003cp\u003eThe pivotal role of urine dipstick testing for hemoglobinuria (uHb) to identify patients with ongoing hemolytic uremic syndrome (HUS), as well as the potential benefit of azithromycin treatment to prevent the development of the renal complication, are described and discussed.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eGiven the rapid spreading of symptoms among many of the participants, the activity was interrupted and all subjects returned home. In view of the potentially severe consequences of the ongoing epidemic, subjects attending the camping were traced back by the local Center for Disease Prevention and Control in order to inform them and assess their clinical conditions. All participants were invited to provide a stool sample for Stx-encoding genes testing, and health surveillance measures were promptly initiated.\u003c/p\u003e \u003cp\u003eSymptomatic patients were referred to the nearest hospital to undergo urine dipstick for hemoglobinuria (uHb) and identify those who might already have had an ongoing HUS. Patients who tested positive (uHb\u0026thinsp;\u0026ge;\u0026thinsp;2+) on urine dipstick were further investigated to rule in or out the diagnosis of HUS with platelet count, hemoglobin, plasma albumin, and lactate dehydrogenase levels.\u003c/p\u003e \u003cp\u003eFurthermore, all symptomatic patients were prescribed oral azithromycin at the dosage of 10 mg/kg once daily with a maximum of 500 mg/dose for 5 days.\u003c/p\u003e \u003cp\u003eIn an attempt to identify the source of the infection, detailed information was collected on the foods consumed by the group and on the activities carried out during the days preceding the onset of symptoms. During the inspection, several samples were collected from environmental and food matrices, as well as tap water from various sampling points (n: 4). The sampling included leftovers from the last meal consumed before the outbreak, which consisted of chicken, pasta with tomato sauce, carrots, and peas.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOut of 31 exposed subjects, 26 were symptomatic (13 females; median age 11.2 years, IQR 9.8\u0026ndash;18.7). All underwent the recommended clinical procedures. Two patients tested positive for uHb on initial urine dipstick testing; however, blood tests ruled out ongoing HUS. Molecular biology investigations revealed Stx-encoding genes in 10 additional patients, exhibiting a molecular pattern consistent with that of initial patients.\u003c/p\u003e \u003cp\u003eUrine dipstick was repeated at the end of azithromycin treatment to ensure that the thrombotic microangiopathy (TMA) had not occurred in the meantime. None of the patients tested positive for uHb, and all of them recovered from GI symptoms without progressing to HUS.\u003c/p\u003e \u003cp\u003eThe scouts reported that tap water at the accommodation facility was brownish, and investigations carried out on drinking water revealed the presence of high levels of \u003cem\u003eEscherichia coli\u003c/em\u003e in all tested samples, but unfortunately no coltural or molecular test for STEC has been performed. Examined food residues tested negative for STEC.\u003c/p\u003e \u003cp\u003eBased on these findings the source of the infection remains undetermined, but suspicion falls on the water supply that was microbiologically and visually altered.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSTEC-HUS is a severe, endemo-epidemic TMA that arises as a complication of a GI infection caused by STEC. The management of outbreaks can be very challenging (occasionally dramatic) for both public health (PH) professionals and clinicians, as clearly emerged during the Japanese outbreak in 1996 [5] and the Northern European one (mainly affecting Germany) in 2011 [6].\u003c/p\u003e\n\u003cp\u003eSTEC outbreaks require the integrated coordination of multiple disciplines and expertise, encompassing healthcare providers with potentially different perspectives and objectives: public health vs. clinical care, community-based vs. hospital-based services, pediatricians vs. adult physicians, human vs. veterinary health, and food safety vs. environmental health. Timely intervention is critical as STEC infections can spreads rapidly and may lead to severe and life-threatening complications. It is essential for health authorities (HA) to have a clear pre-established response plan with well-defined priorities and actions aimed at protecting both individuals and community.\u003c/p\u003e\n\u003cp\u003eBased on the experience gained during previous STEC-HUS outbreaks that have taken place in our area, but also in view of new therapeutic opportunities supported by an easier access to molecular microbiology, we have developed a structured approach to manage STEC outbreaks (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This approach was successfully implemented during the epidemic herein described.\u003c/p\u003e\n\u003cp\u003eWe believe that this experience is worth being shared with the scientific community because it includes two innovative elements:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eUrine dipstick testing for early detection of HUS infected patients.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe use of urine dipstick testing enables rapid and effective identification of subjects who may already have developed HUS and thus require immediate referral to hospital care. The disease severely disrupts glomerular structure due to microvascular thrombosis caused by endothelial damage induced by Stx. The described pathogenic sequence is invariably associated with hematuria \u0026ndash; sometimes gross hematuria \u0026ndash; making urine dipstick detection of hemoglobinuria highly sensitive (100%), although less specific (84%) [7]. Obviously, a positive dipstick test will require a full set of blood tests to rule in or out the diagnosis of HUS. In STEC-infected patients with negative urine dipstick, it should be regularly repeated to monitor for the potential onset of HUS until diarrhea has fully resolved.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cstrong\u003eAzithromycin treatment for the secondary prevention of STEC-HUS in infected subjects.\u003c/strong\u003e Antibiotic use has historically been contraindicated in STEC infections [8,9]. Antibiotic treatment seems to increase the risk of STEC-HUS development by inducing prophages containing Stx-encoding genes through the bacterial SOS response, thereby promoting Stx production, switching from a lysogenic state to a lytic cycle, and leading to Stx release from dead bacteria via phage-mediated lysis [10,11]. However, current recommendations are primarily based on clinical studies that do not distinguish between different classes of antibiotics, combining bacteriostatic and bactericidal drugs.\u003c/p\u003e\n \u003cp\u003eSeveral \u003cem\u003ein vitro\u003c/em\u003e studies have demonstrated that the effect of antibiotics on toxin release depends on the type and concentration of the antibiotic used, and differs among STEC strains [12,13]. Indeed, the available literature shows that bactericidal antibiotics, such as quinolones, beta-lactams, and cephalosporins, actually increase Stx production in vitro and therefore may increase the risk of progression to HUS [14,15]. In contrast, emerging evidence supports the benefits of the use of bacteriostatic agents, such as transcriptional and translational inhibitors. In particular, azithromycin has been shown to reduce Stx production \u003cem\u003ein vitro\u003c/em\u003e [15\u0026ndash;18] and to exert protective effects against neurological and gastrointestinal complications associated with STEC infection in animal models [19].\u003c/p\u003e\n \u003cp\u003eMoreover, a recent retrospective study evaluating the effect of the use of different antimicrobial classes during the first week of STEC-related diarrhea showed that none of the patients treated with azithromycin developed HUS [20]. In Denmark, azithromycin is currently recommended for STEC infection under specific conditions, primarily for decolonization purposes [21].\u003c/p\u003e\n \u003cp\u003eFinally, a recent retrospective analysis conducted at our Center [22], including 89 patients treated with oral azithromycin at a dosage of 10 mg/kg once daily for 5 days, reported that only four developed HUS, whereas the expected number was eleven. This corresponds to a conversion rate of 4.5%, compared to a historical rate of 13.2% (p: 0.02).\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on this and on previous experiences, the key procedures for the management of a STEC infection outbreak (2 or more cases of STEC infection or STEC-HUS temporally and spatially related) can be summarized in the following 10 \u0026ldquo;hints and tips\u0026rdquo;:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e In case of a confirmed or suspected STEC outbreak, the event must be immediately reported to relevant HA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e HA must promptly inform all physicians operating in the involved area (including emergency departments) to ensure that all patients presenting with bloody or watery diarrhea are tested for:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Presence of Stx-encoding genes in stool samples via PCR, to identify subjects at risk of developing HUS;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Presence of uHb using urine dipstick testing to actively identify patients with already ongoing HUS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u0026nbsp; Stx-positive patients should be prescribed oral azithromycin at a dosage of 10 mg/kg once daily (with a maximum of 500 mg/dose) until diarrhea resolution, for up to 5 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u0026nbsp; Stx-positive patients, including those receiving azithromycin, should undergo daily urine dipstick testing until diarrhea resolution, to enable early detection of HUS onset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e Stx-positive patients with uHb \u0026ge;2+ on urine dipstick should be immediately addressed to blood tests to rule in or out signs of HUS (platelet consumption, hemolysis in addition to renal damage).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.\u003c/strong\u003e If stool samples test positive for Stx or HUS is confirmed, samples must be sent to the appropriate laboratory for STEC isolation and characterization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.\u003c/strong\u003e Stx-positive patients should not be admitted to community settings (kindergarten, school, summer-camp, etc.) until full symptom recovery. In presence of multiple cases, temporary closure of the institution should be considered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eFamily members and household contacts of Stx-positive patients should be tested for Stx regardless of symptoms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9.\u003c/strong\u003e\u0026nbsp; HA should establish a permanent team of experts conveying in case of outbreaks to coordinate, process, merge and discuss information and laboratory findings collected from different sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10.\u003c/strong\u003e The source of the infection should be thoroughly investigated, with special regard to undercooked meat, unpasteurized dairy products, and raw vegetables. Possible sources also include stools from farm animals and pets, as well as freshwater sources such as pools, wells, lakes, and rivers. Recent travel history to areas with poor food chain control should also be explored.\u003c/p\u003e\n\u003cp\u003eIn our opinion, the described approach to the management of STEC outbreaks provides the best opportunity for the early identification of STEC-HUS and for protecting both individuals and community from the possibly devastating consequences of infection spreading, while also increasing the likelihood of identifying the source of the infection.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSTEC: Shiga toxin-producing \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eABD: acute bloody diarrhea\u003c/p\u003e\n\u003cp\u003eHUS: hemolytic uremic syndrome\u003c/p\u003e\n\u003cp\u003eStx: Shiga toxin\u003c/p\u003e\n\u003cp\u003eGI: gastrointestinal\u003c/p\u003e\n\u003cp\u003euHb hemoglobinuria\u003c/p\u003e\n\u003cp\u003eTMA: thrombotic microangiopathy\u003c/p\u003e\n\u003cp\u003ePH: Public Health\u003c/p\u003e\n\u003cp\u003eHA: health authorities\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eAuthors have no conflicts of interest to disclose.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical statement\u003c/strong\u003e \u003cp\u003e Given its observational nature, the study was approved by the Institutional Review Board (internal board of the Dept. of Pediatrics). All patients and/or parents (as applicable) gave their informed consent to use their relevant clinical information for research purposes. The study was conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eUse of artificial intelligence tools\u003c/strong\u003e \u003cp\u003eThe authors declare that no artificial intelligence tools were used during the preparation of this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding statement:\u003c/h2\u003e \u003cp\u003eThe authors declare that no funding, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eG.A. conceptualized and designed the study, coordinated and supervised data collection, drafted the initial manuscript and critically reviewed and revised the manuscript.M. G. N., M. C. M., G.T.,L.D., D.R., M.T.,E.P. and D.D.C enrolled patients in the study and critically reviewed and revised the manuscript.G.A, L.D. and D.R. carried out statistical analysis.L.D., A.G., M.L. and M.G.N. managed the laboratory procedures and carried out the bacteriological investigations.All authors approved the final manuscript as submitted and agree to be accountable for all aspectsof the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe wish to express our sincere gratitude to the following members of the ItalKid-HUS Network for their valuable collaboration and their commitment to the screening of patients presenting with acute bloody diarrhea:Fiorella Acquotta, Paolo Adamoli, Nicola Altamura, Angela Amoroso, Stefano Andreoni, Massimo Andreotti, Maddalena Antolini, Milana Arghittu, Francesca Atzeri, Carlo Baldioli, Barbara Balduzzi, Irene Benini, Simone Benvenuto, Francesco Beretta, Cristina Bertulli, Valeria Besutti, Lorenzo Biscardi, Annalisa Bonazza, Cristina Bonetti, Annalisa Bosco, Grazia Bossi, Marta Brambilla, Maria Francesca Brambillasca, Valentina Burzio, Anna Elisabetta Bussolini, Elena Cama, Patrizia Carlucci, Maria Carrabba, Maria Luisa Casciana, Daniela Casnaghi, Eleonora Castellone, Valeria Castorani, Marco Cazzaniga, Claudio Cavalli, Lisa Lucia Chenal, Rosaria Celano, Rossella Ceruti, Marta Cerutti, Giulia Cesano, Giulia Chiopris, Anna Cogliardi, Giacomo Colella, Lucia Collini, Rosaria Colombo, Dario Consonni, Crescenzo Coppola, Angela Corna, Annalisa Corti, Giorgia Daffunchio, Simona De Franco, Elena Lucia De Rose, Laura Dell\u0026rsquo;Era, Giulia Dilio, Clelia Di Mari, Sandra Esposito, Diana Fanti, Alessandra Ferrari, Ilaria Frugnoli, Maria Forestieri, Gloria Fumagalli, Maria Rita Gallina, Miriam Chiara Gatto, Claudio Giacomazzi, Stefano Grossi, Silvia Grosso, Chiara Gualeni, Elisa La Barba, Anna Maria La Pusata, Sara La Rosa, Annalisa Lastrico, Alberto Lepre, Francesca Lizzoli, Rosa Maria Maccarone, Anna Madera, Laura Martelli, Elena Mazzali, Elisabetta Mazzola, Marcella Mercuri, Elisa Milanesi, Chiara Minini, Paola Mirri, Sonia Monticone, Alice Monzani, Vincenza Morinello, Arianna Moroni, Maria Carmela Musolino, Maria Amata Negri, Stefano Nespoli, Bianca Osnaghi, Elisabetta Pagani, Franca Pagani, Cristina Partenope, Palmiro Pedroni, Giovanni Raimondo Pieri, Maria Antonietta Piscopo, Stefano Poli, Ilaria Possenti, Giulia Ramponi, Agrippino Reciputo, Barbara Roman, Barbara Ronchi, Chiara Rosazza, Elena Rossi, Claudia Ruggieri, Maria Lorena Ruzza, Filippo Salvini, Letizia Sardella, Martina Scali, Chiara Sciuto, Alessandra Scolari, Micaela Silvestri, Daniela Simoncini, Giulia Smylie, Rosa Maria Taibi, Francesca Tel, Sara Testa, Valentina Todescato, Elena Tommasoni, Paola Tommasi, Gaia Vanz\u0026ugrave;, Antonio Vergori, Federica Vianello, Chiara Vismara, Chiara Zambetti.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eThe database supporting the conclusions of this article is available if necessary.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTerajima J, Izumiya H, Hara-Kudo Y, Ohnishi M. 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Shiga toxin-induced haemolytic uraemic syndrome and the role of antibiotics: a global overview. J Infect. 2019;79:75\u0026ndash;94. https://doi.org/10. 1016/j.jinf.2019.05.018\u003c/li\u003e\n \u003cli\u003eMcGannon CM, Fuller CA, Weiss AA. Different classes of antibiotics differentially influence Shiga toxin production. Antimicrob Agents Chemother. 2010;54:3790\u0026ndash;3798. https://doi.org/10.1128/ AAC.01783-09\u003c/li\u003e\n \u003cli\u003eCorogeanu D, Willmes R, Wolke M, Plum G, Utermöhlen O, Krönke M. Therapeutic concentrations of antibiotics inhibit Shiga toxin release from enterohemorrhagic \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eO104:H4 from the 2011 German outbreak. BMC Microbiol 2012;12:160. https://doi. org/10.1186/1471-2180-12-160\u003c/li\u003e\n \u003cli\u003eKimmitt PT, Harwood CR, Barer MR. Toxin gene expression by Shiga toxin-producing \u003cem\u003eEscherichia coli\u003c/em\u003e: the role of anti- biotics and the bacterial SOS response. Emerg Infect Dis. 2000;6:458\u0026ndash; 465.\u0026nbsp;https://doi.org/10.3201/eid0605.000503\u003c/li\u003e\n \u003cli\u003eBielaszewska M, Idelevich EA, Zhang W, Bauwens A, Schaumburg F, Mellmann A, et al. Effects of antibiotics on Shiga toxin 2 production and bacteriophage induction by epidemic \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eO104:H4 strain. Antimicrob Agents Chemother. 2012;56:3277\u0026ndash;3282.\u0026nbsp;https://doi.org/10.1128/AAC.06315-11\u003c/li\u003e\n \u003cli\u003eOhara T, Kojio S, Taneike I, Nakagawa S, Gondaira F, Tamura Y, et al. Effects of azithromycin on Shiga toxin production by \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eand subsequent host inflammatory response. Antimicrob Agents Chemother. 2002;46:3478\u0026ndash; 3483. https://doi.org/10.1128/AAC.46.11.3478-3483.2002\u003c/li\u003e\n \u003cli\u003eBerger M, Aijaz I, Berger P, Dobrindt U, Koudelka G. 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J Infect Dis. 2009;199:486\u0026ndash;493. https://doi.org/10. 1086/596509\u003c/li\u003e\n \u003cli\u003eMody RK, Hoekstra RM, Scott MK, Dunn J, Smith K, Tobin D\u0026rsquo;Angelo M, et al. Risk of hemolytic uremic syndrome related to treatment of \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eO157 infection with different antimicrobial classes. Microorganisms. 2021;9:1997.\u0026nbsp;https://doi.org/10.3390/microorganisms9091997\u003c/li\u003e\n \u003cli\u003eAgger M, Scheutz F, Villumsen S, M\u0026oslash;lbak K, Petersen AM. Antibiotic treatment of verocytotoxin-producing \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(VTEC) infection: a systematic review and a proposal. J Antimicrob Chemother. 2015;70:2440\u0026ndash;2446.\u0026nbsp;https://doi.org/10.1093/jac/dkv162\u003c/li\u003e\n \u003cli\u003eArdissino G, Dato L, Mancuso MC, Ria T, Daprai L, Gazzola A, et al. Azithromycin for the prevention of hemolytic uremic syndrome in Shiga toxin-positive diarrhea: a proof of concept. J Am Soc Nephrol. 2024;35(10S):10.1681/ASN.2024fmfgwrv7. https://doi.org/ 10.1681/ASN.2024fmfgwrv7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Outbreak, Shiga toxin, Escherichia coli, hemolytic uremic syndrome, urine dipstick, azithromycin","lastPublishedDoi":"10.21203/rs.3.rs-9216605/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9216605/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eShiga toxin-producing \u003cem\u003eEscherichia coli\u003c/em\u003e (STEC) is a foodborne pathogen accounting for approximately 5\u0026ndash;6% of cases of acute bloody diarrhea (ABD) in Europe. Herein, we report how an outbreak of STEC infection involving a group of scouts was managed with the pivotal role of urine dipstick testing for hemoglobinuria (uHb) to identify patients with ongoing hemolytic uremic syndrome (HUS). We also describe the potential benefit of azithromycin treatment to prevent the development of the most severe complication of STEC infection: hemolytic uremic syndrome (HUS).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAll subjects involved in the outbreak were invited to provide a stool sample for Stx-encoding genes testing. Symptomatic patients underwent urine dipstick for uHb and started azithromycin (10 mg/kg/day) treatment. Patients who tested positive at urine dipstick (uHb\u0026thinsp;\u0026ge;\u0026thinsp;2+) were further investigated with blood tests to rule in or out the diagnosis of HUS.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOut of 31 exposed subjects, 26 were symptomatic and 13 tested positive for genes encoding Stx1, Stx2 and O157. All symptomatic patients were prescribed oral azithromycin treatment for 5 days and were monitored for uHb. Two patients showed a transient positivity for uHb but none progressed to HUS.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eUrine dipstick is a simple, but effective tool to monitor patients with STEC infection for the possible development of STEC-HUS. Azithromycin treatment was safe, thus can be an useful resource to manage a STEC outbreak.\u003c/p\u003e","manuscriptTitle":"Role of Urine dipstick and Azithromycin for the Management of an Outbreak of Shiga toxin-producing Escherichia coli Infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-26 17:13:43","doi":"10.21203/rs.3.rs-9216605/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bbba8a22-49ea-4a24-a25c-41b449195135","owner":[],"postedDate":"April 26th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-10T18:31:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T17:56:05+00:00","index":25,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T10:14:06+00:00","index":21,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T18:39:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-26 17:13:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9216605","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9216605","identity":"rs-9216605","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}