Azithromycin in Post Diarrheal Haemolytic and Uremic Syndrome
Azithromycin in Post Diarrheal Haemolytic and Uremic Syndrome
Post diarrheal hemolytic and uremic syndrome (D+HUS) is the first etiology of acute renal
 failure in children less than 5 years old in France. Previous works highlighted a mortality
 rate of 2 % and a prevalence of renal sequelae at one year after D+HUS onset in 25 % of
 patients D+HUS is a consequence of a gastrointestinal infection with shiga toxin (Stx)
 producing E.coli (STEC). The most frequent straight is E.coli O157H7. The sequence of STEC
 induced HUS is now well known. Typically, digestive symptoms appear five days following STEC
 ingestion. STEC colonize the intestinal mucosa, adhere to the enterocyte and cause a typical
 attaching/effacing lesion and inflammation. Then, diarrhea and vomiting occurred. D+HUS
 occurs in about 10% of patients and is a consequence of Stx systemic absorption. Indeed, Stx
 are released in the gastrointestinal tract, then after transferred to the systemic
 circulation. At the cellular level, Stx binds the globotriosylceramide (Gb3Cer) localized at
 the surface of the endothelial and epithelial cells of target organs. Following binding to
 Gb3Cer, the A subunits of Stx are internalized and trigger the activation of the apoptotic
 program leading to cell death. In addition, Stx are also able to enhance the production and
 the release of pro inflammatory factor (IL-1, TNFα, IL-6). Cytokines locally produced by
 Stx-stimulated cells can amplify the inflammatory processes and the prothrombotic state
 leading to the constitution of the microangiopathic lesions of HUS. To this day, management
 of D+HUS involves supportive care mainly based on fluid management, dialysis and red blood
 cells transfusions. Specific therapies used in D+HUS (plasma infusion, antithrombotic and
 anti inflammatory agents) failed to improve the course of D+HUS. The use of antibiotics
 remains not recommended while meta-analysis clearly showed that the use of bactericidal
 antibiotics could worse the course of D+HUS. In vitro experimentations highlighted that some
 classes of antibiotics like fluoroquinolones dramatically increase the production and the
 release of Stx before bacterial lysis and worsen the outcome of D+HUS in animal models. By
 contrast, azythromycin, a bacteriostatic antibiotic of the macrolides family blocking the
 protein synthesis in bacteria, has a strong inhibitory effect on Stx production and release
 by STEC as well as it inhibits the in vitro growth of STEC strains. In addition, azithromycin
 is able to inhibit the Stx-induced production of inflammatory cytokines which are considered
 to be essential for the development of D+HUS. Consistently the use of azithromycin in animal
 models of D+HUS dramatically improved the survival rate. Preliminary data on humans with
 D+HUS treated with azithromycin highlighted a lower prevalence of severe gastrointestinal
 involvement than in control patients. All these data supported the hypothesis that
 azithromycin should have a beneficial effect on D+HUS and should improve the short and long
 term outcome and deserves to be formally demonstrated in human with D+HUS.
Hemolytic uremic syndrome (HUS) is defined by the combination of microangiopathic hemolytic
 anemia, thrombocytopenia and acute renal failure. The underlying lesion is thrombotic
 microangiopathy (TMA) affecting arteriole and capillary walls, with endothelial cells
 swelling and detachment and thrombi obstructing vascular lumina. Post- diarrheal HUS (D+HUS)
 is induced by an infection due to E. coli (STEC) producing a Shiga -like toxin (Stx) which is
 responsible of TMA. Serotype O157:H7 represents 63-97 % of STEC causing D+HUS. Other
 serotypes frequently implicated in outbreaks are O26:H11, O103:H2, O111:H8, O145:H28 (Mariani
 - Kurkdjian P et al, 2001) (Espié et al, 2008).

 The D+HUS mainly affects children under 3 years and occurs after a prodromal bloody diarrhea.
 The onset of HUS surrounds 3 to 5 days after the onset of diarrhea. D+HUS is the leading
 cause of acute renal failure in children under 5 years (Decludt et al. 2000).

 In France, 100 to 120 pediatric cases of D+HUS are reported each year. STEC infection was
 demonstrated in 85 % of cases. In 50 % of cases, one or more persons of the same family have
 STEC diarrhea without HUS. The death rate in the acute phase is about 2% and 50% of D+HUS
 requires dialysis. The predictive factors of a poor outcome are an anuria for more than 8
 days a neutrophil count>20000/mm3, a severe bowel disease or central nervous system (CNS)
 involvement (Scheiring et al. 2008). In 65-80 % of cases, a seemingly ad integrum recovery of
 renal function was observed within the first year after D + HUS onset. However, D+HUS is
 responsible for a nephron loss that could lead to hypertension, proteinuria and chronic renal
 failure in the long term. A study performed on 218 patients undergoing D+HUS in the chidhood,
 highlighted the presence of renal sequelae in nearly 65 % of them with a median follow-up of
 nearly 20 years. (Fila et al, submitted).

 Classically, humans are infected by contaminated food (minced meat undercooked products,
 unpasteurized milk and cheese...), by the animal environment (especially cattle) or by
 transmission from person to person. EHEC virulence is associated with the presence of toxins
 called Shigatoxins (Stx) and the presence of the eae gene, responsible for damage attachment
 - clearing in the colon and caecum. Two major types of Shigatoxin, Stx1 and Stx2, and many
 Stx1 or Stx2 variants were identified: three variants for Stx1 and at least, six variants for
 Stx2. The determination of different profiles variants Stx is considered as predictive of the
 severity of STEC infections with progression to HUS, especially Stx2d activated and Stx2c
 After ingestion of contaminated food, STEC are able to withstand the acidity of the stomach
 and colonize the digestive tract. Toxins produced by the bacteria go through the intestinal
 epithelium, join the circulatory system and reach their target organs, mainly kidneys and
 CNS. Indeed, toxins bind to a specific glycolipid receptor, globotriosylceramide (Gb3) on the
 surface of endothelial cells of target organs (digestive tract, kidney, CNS, pancreas)
 driving on the one hand, the release of pro-inflammatory factors (TNF- α, IL6, IL8) involved
 in the formation of lesions of microangiopathy and leading to apoptosis of the target cells
 (Hurley et al. 2001) (Thorpe et al, 1999) (Zoja et al.2010).

 To this day, management of D+HUS requires only supportive care. All specific therapies
 (antithrombotic and anti inflammatory agents, specific antibodies against Shigatoxin, plasma
 transfusion and plasma exchange) did not show efficacy (Scheiring et al. 2008) (Loirat et al.
 2012).

 Several retrospective and prospective studies suggest that antibiotics increase the risk of
 HUS by release of Stx during bacterial lysis but also to certain types of antibiotics,
 increased synthesis of Shiga toxin. The meta-analysis performed by Wang et al on the use of
 antibiotics in patient with STEC diarrhea and D+HUS highlighted an increased risk of
 developing D+HUS in patients treated with antibiotics (Wang et al. 2000). A second
 meta-analysis bringing together 20 studies, that compared the risk of HUS occurrence in
 patients with STEC diarrhea treated or not with antibiotics, has not concluded (Panos et al.
 2006). According to these results, antibiotherapy is actually not recommended for patients
 with diarrhea EHEC and patients with D+HUS (Scheiring et al. 2008). However, these studies
 did not distinguish between different classes of antibiotics, bringing together all the
 bacteriostatic antibiotics as well as bactericidal. But, in vitro studies have clearly shown
 that certain classes of antibiotics such as quinolones induced the production of Shigatoxin
 (multiplying production by a factor of 100 for ciprofloxacin) while others had a strong
 inhibitory action as the azithromycin (Ohara et al. 2002 ) .

 Azithromycin, an antibiotic of the macrolide family, binds to the 50S subunit of the
 bacterial ribosome and inhibits protein synthesis by blocking transpeptidation. This
 mechanism inhibits the production and the release of Shiga toxin in STEC (Ohara et al. 2002).
 Studies on animal models of D+HUS (mice, piglet) showed a drastic reduction in mortality.
 Moreover, in addition to its inhibitory action on the synthesis of Stx, azithromycin has a
 modulating effect on the inflammatory reaction on the vascular endothelium induced by the
 Shigatoxin that contribute to thrombotic microangiopathy. Indeed, azithromycin dramatically
 decreased secretion of IL6, IL1 and TNFα (Gantzhorn et al. 2009) (Ohara et al. 2002) (Zhang Q
 et al. 2009).

 Moreover, a recent work from Amran et al performed in a mouse model of D+HUS highlighted a
 protective effect of azithromycin on the occurrence of neurological inhibition of
 inflammatory mechanism induced by Shigatoxin in the vascular endothelium brain and neurons
 (Amran et al. 2013).

 On the human being, the German outbreak of D+HUS in O104H4 helped highlight the importance of
 an early treatment with azithromycin on the eradication of carriage of the bacteria as well
 as the eradication of Stx in stool (Nitschke et al. 2012). In addition, preliminary data from
 a retrospective study of D+HUS in Robert Debré hospital showed a lower incidence of severe
 digestive tract impairment in patients with D+HUS who received treatment with azithromycin
 compared to the control group (unpublished data).

 The objective of a prospective study on patients with D+HUS treated with azithromycin will be
 to prove its beneficial effects on renal, hematological, digestive tract and neurological
 impairment seen in D+HUS and to reduce the potential sequelae.
Inclusion Criteria:

 -  Patient aged from 6 months to 18 years old, residing in France, with a D+HUS according
 to the definition used by the National Institute for Public Health Surveillance (InVS)
 :

 -  Renal impairment with a serum creatinine > 60μmol/l in children less than 2 years old
 and > 75μmol/l over 2 years old

 -  AND hemolytic anemia defined by hemoglobin < 10g/dl associated with schizocytes ≥ 2%

 -  Collection of free and informed consent of the holders of parental authority

 Exclusion Criteria:

 -  Age < 6 months and > 18 years.

 -  Administration of antibiotics in the 15 days preceding the diagnosis of D+HUS.

 -  Personal or family history of atypical HUS .

 -  More than 15 days between the onset of diarrhea and the diagnosis of D+HUS.

 -  Hypersensitivity to azithromycin, erythromycin, or any macrolide

 -  patient treated with dihydroergotamine, ergotamine or cisapride

 -  Severe hepatic impairment

 -  No affiliation to a social security scheme (beneficiary or legal)

 -  Pregnancy

 -  Breastfeeding