Serotonin syndrome (SS) is a clinical toxidrome with high variability in clinical practice. It develops due to increased serotonin levels in the central nervous system. With an underestimated frequency, SS can develop following an overdose, a therapeutic dose increase, or drug to drug interaction of at least one serotonergic agent. It can present with autonomic signs, neuromuscular changes and an altered mental status. However, history and clinical examination are key features to formulate the diagnosis. Treatment options consist of supportive measures, discontinuation of the offending agent and certain therapeutic agents previously reported to improve outcomes. Physicians have limited experience with SS, partially due to the lack of its identification in clinical practice. Therefore, we have integrated, in a narrative review, the case of a young male with SS following an atypical antipsychotic overdose superimposed on chronic treatment with agents previously known to produce SS. Serotonin syndrome (SS) is the result of increased serotonin levels in the central nervous system . It can 3) and raised arterial lactate (37 mg/dL). A urinary toxicology screen was negative. The patient was subsequently assessed by the Intensive Care Unit (ICU) team, who decided to intubate and mechanically ventilate the patient due to a persistent altered consciousness and inability to maintain the airways. He was transferred to our Toxicology ICU, where, after 24 h, he developed pyrexia (40.2 degrees Celsius), persistent tachycardia and hypertension (190/80 mmHg). The patient underwent supportive treatment with sedation and intravenous fluid hydration, with a good clinical response within the next 36 h. After 3 days in the ICU, he was transferred to our Toxicology Department, observed for the next 24 h and further discharged with a follow-up by the psychiatry service.A 21-year-old male patient was brought in by ambulance after he was found by the family in an unconscious state, lying on the floor. He had taken an overdose of Olanzapine 5 mg (7 tablets) and Lorazepam 1 mg (2 tablets) from the chronic medication of another family member. The patient’s past medical history was significant for severe depression with previous suicidal attempts; the patient had been under chronic treatment with Trazodone, Aripiprazole and Sodium valproate for the past two years. The social history was remarkable for gambling dependence without the associated use of illicit drugs. In the emergency department (ED), the patient presented altered consciousness, agitation, and a Glasgow coma score (GCS) of 8, raised heart rate (120 beats per minute) and blood pressure (160/80 mmHg), tachypnoea (respiratory rate 22 per minute), and normal temperature. Clinical examination showed bullous fixed drug eruptions (similar to ’’barb burns’’ lesions) in the right cheek, chin, both knees, anterior right thigh and 3rd, 4th and 5th finger of the left hand, and 5th finger of the right hand. The neurological examination was remarkable for spontaneous lower limb bilateral clonus. A limited cranial nerve examination showed no significant abnormalities. The pupils were equal bilaterally, round, of around 5 mm, and reactive to light. The tonus was increased with lower limb hyperreflexia. There were no peripheral focal neurological findings, and meningeal maneuvers were negative. Head computer tomography (CT) ruled out an organic cause, a chest X-ray showed no pneumonia, an abdominal ultrasound was normal, and an electrocardiogram (ECG) showed sinus tachycardia. Blood tests performed in the emergency department were unremarkable, except for leukocytosis  were unaware of SS when questioned via a questionnaire for a study conducted in 1999 [% were unThe literature has described SS in different ages, from new-borns to the elderly population, with a mean age of 30 to 40 years in the adult population . In receBetween 2002 and 2016, 54,410 individuals in the United States were exposed to an overdose of SSRIs, with 102 deaths, representing the 9th most frequent cause of mortality by drug overdose . An esti1 receptors are mediated by adenylyl cyclase and represent the main target for anti-migraine drugs, such as sumatriptan/ zomitriptan as they have a high density in the limbic system. 5-HT2 receptors stimulate phosphoinositide-specific phospholipase, and their antagonism leads to the therapeutic effect of antipsychotic drugs such as risperidone. The 5-HT3 receptor is a ligand-gated cation channel predominantly expressed by neurons, and represents the pharmacological site for anesthetic and antiemetic agents. The 5-HT4 family influences gastric motility, while the 5-HT5 family is less known. 5-HT6 and 5-HT7 receptors are still under investigation since many antipsychotics and antidepressants act through antagonism [Serotonin  is a monoamine neurotransmitter synthetized from the amino acid tryptophan. The metabolic pathway uses multiple mechanisms, feed-back loops, and enzymes. Tryptophan hydroxylase 1 and 2, are essential for the synthesis of serotonin in the gastrointestinal (GI) tract and the central nervous system (CNS), respectively. Serotonin is stored in the presynaptic vesicles and released into the synaptic cleft, following axonal depolarization . Throughtagonism ,23. 1A and 5-HT2A are the most often involved in SS. Genetic susceptibility may be a culprit for SS development and variability, especially in the context of therapeutic doses [2A receptor subtype and homozygous carriers of the C102 allele of the same receptor are considered to provide a higher risk of serotonin induced adverse drug reactions [1A receptors have higher serotonin affinity, which is associated with milder presentation, while 5-HT2A activation has been associated with life threatening SS. Other factors may influence the pharmacology of serotonergic drug, such as age, sex, nutrition, health status [Increased levels of serotonin in the CNS represent the foundation for SS development . SS is tic doses . Single eactions ,29,30. Peactions ,32. Addih status  or hormoh status . The rol1A, and serotonin reuptake inhibitors as chronic treatment led to increased levels of serotonin the CNS. The superimposed overdose with an atypical antipsychotic, with an affinity for 5-HT2A/2C, 5-HT3 and 5-HT6 receptors, escalated serotonin concentrations and precipitated the clinical syndrome. Other factors, such as the patient’s poor general health status and nutritional deficiencies, might have contributed.Multiple pathophysiological pathways are probable in our case. The use of serotonin receptor agonists, namely 5-HTA variety of drug classes have been associated with SS, such as antidepressants, monoaminoxidase inhibitors, antiemetics, antimigraine agents, illicit drugs, over the counter drugs or herbal supplements . They caAn association of MAOIs and SSRIs or SRNIs can cause life-threatening SS . Other c2A/2C, 5-HT3, 5-HT6 receptors, antihistamine H1 receptors, dopamine D1, D3, D4, D5 receptors, as well as cholinergic muscarinic receptors. Studies have demonstrated a higher affinity for 5-HT2 receptors compared to dopaminergic D2 receptors. It undergoes first-pass metabolism through cytochrome P450 2D6 with different pharmacokinetic properties and two-phase elimination when taken in overdose [2 and 5-HT1A agonist and antagonist of serotonin 5-HT2A receptors. It demonstrated high affinity for dopamine D2, D3, serotonin 5-HT1A, 5-HT2A, histamine H1 and alpha-1 adrenergic receptors when studied in vitro. Aripiprazole has been associated with serotonin syndrome [2A antagonism, there is enhanced 5-HTA1 agonism, which increases serotonin levels, potentiates the effects of other drugs, and increases receptor sensitivity to serotonin; this explains the development of SS [Atypical antipsychotics (AA) are a relatively new class of medication acting on a wide range of receptors. They can be divided into two main categories depending on their capacity for dopamine receptor antagonism. AA have been increasingly used in a variety of conditions such as schizophrenia, depression, bipolar disorder, or agitation in geriatric patients . Olanzapoverdose . Case reoverdose , but moroverdose , clomiproverdose , metoclooverdose  or duloxoverdose . It is int of SS . Trazodont of SS . Valproint of SS .The triad of autonomic signs, altered mental status, and neuromuscular changes characterizes SS . NeuromuSS is diagnosed based on clinical presentation. A thorough examination of the patient’s medical history, looking for chronic medication for depression or long-term pain, use of illicit drugs, such as ecstasy, amphetamine, or cocaine, and over the counter medication such as St John’s wort, appetite suppressants or tryptophan, should be performed. There should be careful consideration of whether a recent increase in dose, addition of a second drug, or resumption of an agent with prolonged half-life has been performed in a patient’s chronic medication . The onsIn our case, the medical history provided by the family characterized a patient with a history of depression and previous suicidal attempts, for which he was taking three antidepressants previously reported to determine SS. We do not have a clear timeline about the onset of symptoms as he was found lying on the floor with empty blisters in his hand. It should be noted that another family member was taking treatment with Olanzapine. The patient exhibited clonus, one of the most important signs associated with SS. Altered mental status, together with hyperthermia, tachycardia and hypertension, led us to consider SS as the main diagnosis. There is no specific laboratory test to diagnose SS. Only in severe cases, metabolic acidosis, rhabdomyolysis, increased levels of liver enzymes, kidney function tests or coagulation tests abnormalities can be determined. Imaging, such as head CT, lumbar puncture, and ECG, should be performed to exclude other organic causes . We perfDifferential diagnosis should be made with other toxin-induced hyperthermic syndromes. Neuroleptic malignant syndrome (NMS), anticholinergic/antimuscarinic syndrome, and malignant hyperthermia are the main conditions to consider. A broad differential diagnosis should also be considered for conditions such as sepsis, febrile neurologic illness , nonconvulsive status epilepticus, hyperthyroidism, and rhabdomyolysis , or stifSS should primarily be differentiated from NMS. The latter is a toxidrome induced by dopamine antagonists characterized by hyperthermia, rigidity, altered mental status and autonomic dysfunction . The twoAnticholinergic toxicity is characterized by central and peripheral features. It is frequently encountered in the ED as there are over 600 identified compounds with anticholinergic action. The syndrome can present with altered mental status, such as anxiety, agitation, coma, or seizures, secondary to a reduced cholinergic activity in the CNS. The peripheral findings provide features distinguished from SS: normal reflexes, dry oral mucosa, erythematous skin, and urinary retention . Malignant hyperthermia is a life-threatening pharmacogenomic condition affecting skeletal muscle. It arises in susceptible individuals after the exposure to volatile anesthetic drugs, e.g., halothane, isoflurane, or succinylcholine . The genHyperactive delirium shares multiple common findings with SS. This is particularly important in elderly patients where specific drug agents causing SS are used, but delirium is frequently encountered . AlteredThe sudden discontinuation of antidepressants may have features of SS. Symptoms consist of malaise, nausea, changes in sleep, mood, or appetite, but the duration is less than a week, with spontaneous resolution . The mainstay treatment consists of supportive measures and the discontinuation of the offending agent. Where indicated, the patient should be admitted to a high dependency unit and close vital sign monitoring should be ensured . If possMild cases can benefit from fluid replacement and sedation where necessary. Benzodiazepines are indicated in cases of agitation, regardless of the severity of SS. Diazepam or lorazepam improve survival due to the added benefit of lowering blood pressure, heart rate and temperature, by controlling the hyperadrenergic state . If persModerate cases should be admitted for cardiac monitoring and may require the addition of a serotonin antagonist if there is no response to benzodiazepines . Butyrop2A action, but cyproheptadine has an additional 5-HT1A receptor antagonist mechanism. It may be more effective than chlorpromazine as it has been shown to prevent life-threatening hyperthermia, provide symptomatic relief, and even complete symptom resolution in some cases [Chlorpromazine and cyproheptadine have been extensively used. Both agents have 5-HTme cases . The inime cases ,71.Severe or life-threatening cases should be admitted to the ICU where orotracheal intubation, active cooling, management of autonomic instability and the use of Cyproheptadine might be required. Hyperthermia control is critical as it is often responsible for the severity of serotonin syndrome . It deveSS remains a challenging condition due its wide variety of symptoms, lack of objective standardized diagnostic tests and limited research in the literature. When facing such an emergency, the most important step is to carefully evaluate the patient’s drug history, present complaints, and perform a detailed clinical examination. The combination of multiple agents targeting serotonin receptors is a common strategy for treatment-resistant depression. This strategy, however, creates the perfect environment for the potential development of SS, especially in the context of an overdose. In our case, the patient was taking chronic treatment with strong serotoninergic agents, and the overdose of Olanzapine led to a serotonin receptor blockage, increased serotonin levels and the development of SS. The clinical judgement becomes crucial when there is limited available data, or the patient has severe consciousness impairment. Clinicians should therefore bear in mind that SS can be considered as a differential diagnosis for a broad range of conditions and more important when there is relevant drug history. Moreover, prior to initiating a medication acting on serotonin receptors, careful assessment and monitoring should be conducted.


Introduction: Leptospirosis is a neglected and re-emerging zoonotic disease that may cause ocular involvement, uveitis being the main complication of the systemic disease. Aim: The purpose of this review was to raise awareness and update on uveitis caused by leptospirosis, which is a challenging pathology because it can mimic other types of uveitis. Materials and methods: An article review, was conducted by searching PubMed (MEDLINE), Scielo, Cochrane Library databases using the following MeSH and DeCS terms: “leptospirosis”, “uveitis”, “ocular”, “eye” and “human”. The inclusion criteria were articles between 2000 and 2021 in English, Spanish, French or Portuguese. Results and Discussion: A total of 49 articles were obtained with the mentioned inclusion criteria, and additionally 5 articles before the year 2000, which were considered due to their relevance and scarcity of articles on the pathology. Afterwards, a description of the disease was made.Conclusion: This literature review was steered to raise awareness and to apprise physicians and ophthalmologists about a pathology that is becoming increasingly relevant, but underdiagnosed, even in developed countries.Abbreviations: REU = recurrent equine uveitis, MAT = microagglutination  Leptospira interrogans and is a public health issue because it is a neglected [1] and re-emerging disease affecting humans .Leptospirosis is a zoonosis of worldwide distribution, more endemic in some regions of the world. It is caused by 4].In 1803, William Wittmen was the first to perform a clinical report of leptospirosis and later, in 1886, Weil described the jaundice component of the disease, and the entity was coined with his name. Later, in 1917, Noguchi first isolated the organism from a Norwegian rat. Leptospirosis uveitis was initially reported in an original paper by Weil [5].The systemic disease has an extremely broad spectrum, ranging from an influenza syndrome to the presence of Weil’s disease including, fever, haemorrhage, acute liver and renal failure. Laboratory tests are required to confirm the diagnosis [6]. The ocular involvement in humans can range from non-specific congestion to scleral jaundice and uveitis, which is a complication of systemic disease [7]. In addition to causing systemic infection, after a period of latency, leptospirosis may result in an immune disorder [8]. REU is one of the main causes of vision loss and is usually bilateral and has a major impact on financial costs, owners and animal health, often resulting in euthanasia [9]. REU has been used as a natural model for the study of autoimmune uveitis in humans, and has provided insight into endogenous factors of how the blood-ocular barrier is destroyed and initiates cellular and humoral processes that lead to vision loss [8].Leptospirosis can affect animals varying from domestic to wild. In animals, however, leptospirosis-associated uveitis only occurs in horses, and is known as recurrent equine uveitis (REU)  and Abela-Ridder et al. suggest that it accounts for 20-40% of cases of fever of unknown origin [12]. Regarding leptospirosis, it has been estimated that there are 1,000,000 cases per year worldwide and some authors, such as Wunder et al. [12].The disease is endemic in Asia, Oceania, and South America; however, cases are increasingly found in Europe and North America [2].This is a re-emerging disease and therefore was not followed up in countries such as the United States, in which the incidence is unknown. However, it is known that there were 0.6 hospitalizations/ 1,000,000 people (95% CI 0.5-0.6) between 1998 and 2009 [3].In China, Zhang et al. have reported an incidence of 0.7/ 100,000 people per year during the period 1991-2010. It was more frequent in men - 66.8%, compared to women - 33.2%, and especially in rural workers [5]. Leptospirosis is one of the most frequently neglected diseases, as a consequence of under-reporting and lack of awareness of its existence [13]. In other Asian countries, such as Malaysia, it is considered to be endemic [14].Conversely, Mancel et al. have described an incidence of leptospirosis as high as 180/ 100,000 inhabitants per year, in the islands of New Caledonia in the South Pacific [15]. In the Netherlands, an incidence of 0.25 cases/ 100,000 inhabitants per year has been found in the period from 1925 to 2008 [16]. Meanwhile, in Germany, Jansen A et al. describe an incidence of 0.06 per 100,000 inhabitants during the period 1998-2003 [17]. The annual incidence in Spain was 0.86 cases per million inhabitants/ year during the period from 2009 to 2012 [18].In European Union countries the incidence has been estimated at 1-4 cases/ million inhabitants depending on the country [19]. The main risk factors that have been associated with outbreaks of leptospirosis are flooding, the rainy season and hot weather [20]. In Colombia, the incidence has been estimated at between 1.1 and 5.4 cases per 100,000 inhabitants in the past decade [21]. In other countries such as Brazil, both urban and rural incidence is 1.9/ 100,000 people [22].In Latin America, leptospirosis in humans in countries such as Argentina was estimated at 0.32/ 100,000 people in 2014 [23]. Similarly, middle-aged men are more commonly affected. The main risk factor for acquiring the disease is exposure to water or soil contaminated with leptospires . Other risk factors that have been associated with the pathology have been identified as areas with higher rainfall, forested areas especially in overpopulated regions, population in poverty, low educational level, vulnerable conditions and lack of sanitary structures .The vectors of leptospirosis are rodents, especially rats in urban environments. The bacterium usually remains in the kidneys of rodents for months and is then passed to humans via the urine of infected animals that contaminate water. It is most common in rubbish collectors, soldiers, milkmen, farm workers and travelers. The most common source of infection is urine from livestock, rodent, dogs or contaminated freshwater reservoirs. Cases have also occurred through infected blood. The disease is most prevalent in the tropics and subtropical areas [6].The precise incidence of uveitis in systemic leptospirosis is unknown, but has been estimated to be between 3% and 92% depending on endemic or non-endemic areas .The pathogenesis of leptospirosis is not well documented. Ninety percent of cases are asymptomatic. These are the initial cases or asymptomatic, or mild cases, in which a balance between inflammatory response and anti-inflammatory mechanisms will be observed, i.e., homeostasis will be maintained. In the other 10%, an imbalance between inflammatory response and anti-inflammatory mechanisms will be noted, leading to a large release of cytokines that will ultimately paralyze the immune response and lead to sepsis, multi-organ failure and death [26].Its pathogenesis is not well known at the ocular level either, it is believed to be produced by the migration of antibodies to the anterior chamber, but there is a rapid decrease in these and the microorganism takes advantage of this to multiply . It is also considered capable of releasing enzymes or bacterial toxins that injure tissues. Elevated levels of lipopolysaccharides have been found in aqueous humour in patients with leptospirosis uveitis, inducing congestion and increased vascular permeability. It has also been found that at a systemic level, high titers of TNF, Interleukin 6, 8, 10 can lead to immune-mediated vasculopathy due to activation and deposition of immunocomplexes. This leads to vascular endothelial damage and tissue injury [29]. Leptospiral lipoproteins coded by LruA and LruB in patients with leptospiral uveitis have been discovered in humans and LruA and LruB type IgG and IgA antibodies have also been found, which could have a cross-reaction against ocular components such as the retina, lens and ciliary body, although these antibodies can be seen in other types of uveitis such as Bechet’s . Rapid progression to cataract has also been found in patients with uveitis due to leptospirosis and is believed to be due to leptospira that are cross-reactive against proteins that keep lens transparency [Systemic clinic31].The incubation period for leptospirosis is 3 to 30 days. The onset of symptoms is sudden-onset, biphasic fever. There is a septicaemia phase and an immune phase. The first period is a fever or septicaemia phase, followed by a fever-free period. This is followed by a second period of fever with or without jaundice or immune phase. In addition, other symptoms may be present: headache, retro-ocular pain, chills, chronic generalized fatigue and arthralgias [32]. At the cardiac level, arrhythmias have been described, with P-Q delay, atrial fibrillation being quite common, as well as myocarditis and pericarditis [23]. Bal A describes gastrointestinal involvement, characterized by diarrhea, jaundice, vomiting, pancreatitis, and cholecystitis [32]. The main organ affected is the kidney, as the Leptospira has a tropism for the kidney, leading to acute renal failure. Other affections that could be seen are acute liver failure, pneumonitis, haemolytic anaemia, haemorrhagic diathesis, adrenal insufficiency, multisystem failure and death .It may also cause encephalitis, meningoencephalitis, transverse myelitis and cranial nerve palsies [23].It could also have an involvement during pregnancy, from intrauterine growth retardation, foetal or placental ischaemia to much more severe consequences that can lead to foetal death and miscarriage, or congenital leptospirosis [4]. Systemic leptospirosis has a mortality of more than 10% in severe cases [10]. Cagliero J et al. have estimated that more than 60,000 deaths occur annually from leptospirosis [25].There are two terms that must be differentiated. The first one, is the most severe form of icteric leptospirosis that is called Weil’s syndrome. The second one, Weil’s disease, is an anicteric leptospirosis, renal failure and hepatic haemorrhage, multi-organ failure, vascular collapse and severe alterations in consciousness .The incidence of ocular signs varies from 2% to 90% during the acute phase. Ocular signs tend to occur in the immune phase . Rathinam et al. analyzed 394 eyes of 276 patients with seropositive leptospiral uveitis and found that 13.7% developed cataracts between 1-6 months after presentation and, of these, spontaneous absorption was observed in 18.5% with a median of 5 months after cataract onset [35]. Another article that included 6 young patients with leptospirosis, underlined an early onset and rapid progression to cataract formation within a period of three months [36].One of the early manifestations is chemosis, which may be as high as 85.7% and usually accompanies systemic manifestations. Sub-conjunctival haemorrhage may also occur in 19.5% of cases. Other clinical features of ophthalmological involvement are: cataract in 14% with rapid progression and spontaneous reabsorption, and glaucoma [6]. Rathinam et al. also reported a case of corneal melting secondary to ocular infection due to leptospirosis [37].The presence of icteric sclera and pericorneal congestion is considered pathognomonic of severe systemic leptospirosis. Interstitial keratitis may also be present in up to 18%  and it occurs in 40% of patients with systemic disease [30]. It usually occurs in two weeks after the infection up to 1 year later, with an average duration of 6 months. Uveitis can be anterior uveitis or panuveitis. The most common is anterior uveitis. Its course is usually insidious and mild . It may present with hypopyon in 2.7% to 12% of cases, and may be non-granulomatous or much more rarely granulomatous . Among the late findings, the most important is the uveitis. This is the most frequent ocular complication of the systemic disease. It can be unilateral or bilateral [38]. The other form, as it usually manifests itself, is as acute and severe panuveitis. In a study by Rathinam et al., which included 73 patients and 111 eyes with leptospirosis uveitis, panuveitis was found in 95.5% of the eyes. The most frequent findings were non-granulomatous reactions in 91.9%, keratic precipitates - 85.6%, vitritis in 76.6% of eyes, periphlebitis - 51.4%, posterior synechiae - 23.4%, hypopyon - 12.6%, choroiditis - 4.5%, papillitis - 3.6%, macular oedema - 2.7%, epiretinal membrane - 1.8%, pars planitis - 1.8% and arteritis - 0.9% [6].In a newer article from 2005, Rathinam SR described optic nerve hyperaemia in leptospirosis uveitis as ranging from 3% to 64%. He also reported neuroretinitis, optic neuritis and retrobulbar neuritis [42]. Moreover, Costa et al. in Brazil described a patient with sickle cell anaemia who presented with massive bilateral vitreous haemorrhage with retinal detachment and phthisis bulbis in the context of leptospiral infection [43].Martins et al. in Brazil described the ocular manifestations of leptospiral uveitis in 21 patients with conjunctivitis - 85.7%, increased retinal venous caliber - 57.1%, optic disc hyperaemia - 57.1%, and sub-conjunctival haemorrhage - 19.0%. Also, optic disc oedema, retinal vasculitis, retinal haemorrhage, hard exudates and papillitis were reported in 4.8% each . Similarly, rare cases of bilateral sixth cranial nerve palsies and acute bilateral keratouveitis have been reported [7].In a much more recent study undergone in 2016, Sivakumar R et al. reported that non-granulomatous panuveitis, hypopyon, and vitreous infiltration in the absence of retinochoroiditis are predictive parameters for leptospirosis uveitis with 86% sensitivity and 90.7% specificity [Diagnosis6].Diagnosis for systemic diseases can be made by laboratory tests that identify the spirochete directly, or indirectly by tests that show the presence of infection. It can be performed on blood, urine or cerebrospinal fluid. There are four groups of main tests. Being the first one, direct tests, which isolate the bacteria most frequently during the septicaemic phase of the disease, include darkfield microscopy, silver staining, immunohistochemical staining, and immunoperoxidase staining. A second group of tests is animal inoculation [6]. According to the World Health Organization, MAT is the gold standard for the diagnosis of leptospirosis. It has the disadvantage that it requires a specialized laboratory and trained personnel .The third group includes serological tests such as the macroscopic agglutination test, MAT test, haemagglutination test and the ELISA test. The latter test is rapid and commercially available and can have good sensitivity and specificity. Immunoglobulin G ELISA has been used. In addition, Immunoglobulin M ELISA, which can be detected 4 days after symptoms and can persist for up to 5 months, has a sensitivity of 88% and specificity of 96%. MAT, in which titers or concentrations greater than 1:400 are considered positive, and in chronic disease, values greater than 1:100 signify an infection, is also used . Haake et al. described the finding of 80% Leptospira DNA in aqueous humour by DNA enzymatic PCR, pulsed-field electrophoresis and restriction fragment length polymorphism techniques [31]. The last group is represented by molecular biology techniques like PCR, which is recommended when there is uncertainty with a MAT test or an ELISA test [6]. MAT detects uveitis due to leptospirosis in 50% of cases [30]. MAT and IgM ELISA together are positive for leptospirosis uveitis in 77% cases [29]. The use of lipopolysaccharides for the diagnosis of leptospiral uveitis is also being studied [48]. An article reported a sensitivity of 48% and a specificity of 90% with lipopolysaccharides [29]. Kits such as Lepto IgM MICROLISA and Leptocheck (Leptospira IgM Antibodies) have been tested for the diagnosis of leptospiral uveitis, but the sensitivity and specificity in the first was 60% and 55%, and for the second 80% and 59% respectively, having these results limitations for their use [34]. Due to leptospirosis, a protein produced by the bacterium, called HbpA, has been detected in uveitis. Anti-HbpA Ig are produced against this protein, which have been detected in 92% of patients with uveitis due to leptospirosis, being more sensitive than MAT and without cross-reaction like other types of uveitis [49].However, there in not a specific method for ocular diagnosis [Differential diagnosis50].Systemic differential diagnoses should be suggested when the patient is living in an endemic area and what should also be considered are influenza, rhizocystis disease, malaria, or typhoid [6], tuberculosis [44] or syphilis, brucellosis, or Eales disease [4].Leptospirosis uveitis is a pathology that is underdiagnosed and can simulate another type of uveitis. Among the uveitis with which a differential diagnosis must be made are HLA B27 uveitis, Adamantides-Behcet disease, both of which can present with hypopyon. Also, the other types of uveitis are sarcoidosis and endogenous endophthalmitis [Treatment51]. Cephalosporins such as ceftriaxone and cefotaxime have also been used for severe forms [52]. If the infection is mild to moderate, doxycycline can be used every 12 hours for one week [51].The treatment of severe systemic leptospirosis is performed with penicillin G, 1.5 units every 6 hours for one week [4].Regarding the Jarisch-Herxheimer reaction, which occurs in infections caused by spirochetes, there is controversy, but it is thought to be less prevalent in leptospirosis than in other spirochetes [4]. However, there are no clinical trials supporting antibiotic treatment in leptospirosis uveitis. In cases of leptospirosis panuveitis, topical, periocular or systemic corticosteroids should be added in addition to antibiotic treatment [51]. Anterior uveitis is usually self-limiting and resolves with the help of corticosteroids and mydriatics [53].Work is ongoing on developing a possible vaccine. LipL45 (lipopolysaccharide DNA) vaccine was used in mice and it induced a significant Th1 type immune response [Prognosis54].The mortality of systemic leptospirosis varies from 1% to 20% depending on the severity of the disease, the latter figure being a consequence of multi-organ failure [4].In leptospirosis uveitis, the prognosis is usually good despite the severe visual compromise that patients may have; patients recover their vision once the uveitis resolves [To sum up, it can be stated that leptospirosis and leptospiral uveitis are globally distributed, neglected and re-emerging pathologies and the aim of the present article was to make them visible and to make doctors and especially ophthalmologists aware of these pathologies.Conflict of Interest statementThe authors state no conflict of interest.AcknowledgementsNone.Sources of FundingNone.DisclosuresNone.


In recent years, vegetarian and vegan diets became increasingly important as they are associated with beneficial health outcomes. Therefore, the NuEva study compares the impact of flexitarian, vegetarian, or vegan diets with omnivorous nutritional habits on nutrient intake and risk factors for non-communicable diseases.n = 65 (Median/Interquartile range: 33/17 yrs.), flexitarians, n = 70 (30/17 yrs.), ovo-lacto vegetarians, n = 65 (28/14 yrs.), vegans, n = 58 (25/10 yrs.)] on nutrient intake, nutrient concentrations in plasma, serum or 24h urine, body composition, and blood lipids.A dietary protocol was kept over five days and blood and 24h urine samples were collected to examine the impact of dietary habits [omnivores, p ≤ 0.05) and flexitarians . In omnivores vitamin A, vitamin E, ferritin, and the urinary excretion of selenium, iodine, and zinc were higher than in vegans (p ≤ 0.05). In contrast, vegans had the highest concentrations of biotin, folate, and vitamin C. Flexitarians, vegetarians, and vegans had a lower body weight, BMI, and body fat percentage in comparison to omnivores (p ≤ 0.05). In omnivores the concentrations on total cholesterol, total cholesterol/HDL cholesterol ratio, LDL cholesterol, LDL cholesterol/HDL cholesterol ratio, apolipoprotein B, and apolipoprotein B/ apolipoprotein A1 ratio were higher than in vegetarians and vegans (p ≤ 0.05).The increased exclusion of animal based foods in the diet (omnivores < flexitarians < vegetarians < vegans) is associated with a decreased intake of energy, saturated fat, cholesterol, disaccharides, and total sugar as well an increased intake of dietary fibers, beta carotene, vitamin E and K. The combined index of the B12 status (4cB12 score) in vegetarians (0.02/0.75) was lower compared to omnivores (0.34/0.58; The NuEva study confirms the position of the Academy of Nutrition and Dietetics that adequately planned vegetarian diets are healthy, nutritionally adequate, and may provide health benefits in the prevention and treatment of non-communicable diseases. Nevertheless, critical nutrients were identified for all groups studied. This highlights the need to develop individual nutritional concepts to ensure an adequate nutrient intake. Cardiovascular diseases (CVD) are one of the leading health problems worldwide and the main cause of death in German and the European region . Up to 4The predominant dietary pattern in Germany is characterized by high intake of foods of animal origin whereas the consumption of foods of plant origin is comparable low . This diThe hype surrounding the vegetarian and vegan diet and the high prevalence of the omnivorous dietary pattern in combination with the likelihood for over- and undersupply of nutrients following the adoption of these eating habits highlights the need of extensive data collection to develop evidence-based recommendations.In this context, a central objective of the NuEva study is to assess nutrient intake in the studied diets and compare of nutrient concentrations in plasma, serum and 24 h urine as well as the impact on cardiovascular risk factors.In summer/autumn 2018, healthy women and men between 18 to <70 years were recruited by press release and flier. The flier was distributed on the universities in Jena, Halle and Leipzig and in refectories, cafeterias, fitness studios, youth clubs, restaurants and coffee shops. Interested individuals initially complete a telephone pre-screening. As precondition, one of the four diets studied had been implemented since at least 1 year before enrollment. The adherence to one of the four diets  was assessed by a self-created questionnaire and a dietary protocol over 5d. The study protocol listed the following exclusion criteria :Patients with diseases of the parathyroid, diseases necessitating regular phlebotomies, or patients with acute or chronic disease, which could affect the results of the present study. In addition, the following treatments precluding participation (at least 3 months prior to study start) resulted in an exclusion from the NuEva studyWeight loss or weight gain (> 3 kg).Fundamental changes in dietary habits.Hormone replacement therapy.Elite athletes .Pregnancy or lactation.Following the informed consent and confirmation of the in- and exclusion criteria, participants are scheduled for the baseline assessment. In total, 65 omnivores , 70 flexitarians ; consumption of fish), 65 ovo-lacto vegetarians , and 58 vegans  participate on the NuEva screening . The die® version 6.4  and includes common foods and usual portion sizes. The template was adapted on the NuEva study by adding foods which are favored in vegetarian and vegan diets such as tofu, vegan yogurt alternatives and plant drinks, soy products, seitan, tempeh, maple and agave syrup . Foods which were basically not contained in PRODI® were created and the nutritional information was taken from the packaging . The daily energy and nutrient intake was calculated by the software package PRODI®. The nutrient intake from supplements was not considered in the calculation of nutrient intake by the dietary protocols .To record and document the variety in dietary practices within and between the groups, the run-in phase of the NuEva study included full self-reporting of individual dietary intake over 5 days. The dietary record based on the template “Freiburger Ernährungsprotokoll” which was provided by PRODIAvailable questionnaires from the German National Consumption Survey II (NVS II) and the German health interview and examination survey for adults (DEGS1) are used to consider the socio-economic status as a confounding factor . In deta® tubes á 9 ml were filled by the participants according to a standardized operating procedure. The aliquots were stored on a cool place and had to be hand at the study center until 10:00 am. Here the aliquots were frozen immediately (−20°C). Body weight, height, and waist circumferences were measured, respectively, by the same trained study nurse to the nearest half-kilogram or half-centimeter, with patients wearing light clothing with bare feet (one measurement). Waist circumference was measured midway between the lower rib margin and the iliac crest (a thumb's breadth above the navel). For measurement calibrated instruments were used .Blood was taken by venipuncture between 7:30 AM and 10:30 AM after at least 12 h overnight fast. The urine was collected over 24h directly before phlebotomy. The collection began after the morning urine on the day before. Afterward the urine was completely collected in a special container for the next 24 h. After the morning urine on day 2 (day of phlebotomy) the collection was finished. The total volume was documented, and eight SarstedtBody composition was assessed by Body Impedance Analyzer . The study protocol was reviewed and approved by the Ethical Committee of the Friedrich-Schiller-University of Jena (number: 5504-03/18). The NuEva study was registered before launching .®). Selenium and zinc were quantified by atomic absorption spectroscopy  for separation of plasma and serum. The 24h urine was aliquoted. Study parameters were analyzed immediately after blood sampling or urine collection or by using aliquots from serum, plasma, and 24h urine collections which were stored at −20°C (24h urine) or −80°C  until analysis. The samples were prepared according to standard operation procedures. The analyses of biotin, methylmalonic acid, vitamin B2, vitamin C, and iodine were performed by Dianovis GmbH . Furthertroscopy .t-test (using Benjamini-Hochberg correction). Otherwise, Kruskal-Wallis test with pairwise comparisons using Wilcoxon signed-rank tests (using Benjamini-Hochberg correction) were used.Statistical analyses were performed using the statistical software 'R‘ (version R i386 3.5.2). The same procedures were used for all studied diet groups. If the data of the four groups follow a normal distribution (tested with Shapiro-Wilk), one-way ANOVA was applied and the differences between specific groups were investigated using pairwise comparison with a two-sample The same analysis was performed for men and women subgroups and adjusted data sets as well. A standard ANCOVA was applied to the data to detect if a covariate  influenced a specific variable. This influence meant that the correlation of the variable and the covariate was similar in all four groups, large enough (correlation coefficient larger than 0.3 or lower than −0.3) and significant. If the conditions were met, the values of that variable were adjusted for age  or BMI . If sex had a significant influence, the statistical analysis was performed for men and women separately. All tests in this section were evaluated with α = 0.05.*Power 3.1.9.2 as described in . In the NuEva study population, the higher age in omnivores and flexitarians differed significantly from the lower age in the vegan group .The NuEva participants' age ranged between 18 and 69 years and the collective consisted of 70% women and 30% men . In men,Generally, the omnivorous diet is practiced since birth or childhood whereas the flexitarian, vegetarian, and vegan diets were practiced on average for 13, 10, and 4 years .p ≤ 0.05). Most participants in all groups had a university entrance qualification, whereas the proportion with a secondary school leaving certificate was higher in omnivores. In omnivores and flexitarians, the number of subjects with a completed vocational training was higher than in the vegetarian/vegan groups . The intake of nutritional supplements was comparably high in vegans and low in omnivores .Marital and educational status differed significantly between the groups . The parp ≤ 0.01). The intake of carbohydrates, dietary fibers, protein, and fat also varied with substantial differences between the omnivores and the vegetarian/vegan diets (p ≤ 0.05). The intake of carbohydrates and dietary fibers increased in the following order: omnivores < flexitarians < vegetarians < vegans, the intake of protein and fat decreased in parallel (p ≤ 0.05). The intake of monosaccharides was similar in all the four diets. However, the intake of disaccharides was markedly lower in vegans than in flexitarians and vegetarians .The energy intake varied between the four studied groups with the highest intakes in both the omnivores and flexitarians and the lowest intake in vegans . The lowest amounts of SFA were consumed in the vegan group. The omnivores consumed the highest amounts of monounsaturated fatty acids (MUFA) and vegan participants had the lowest intake (p ≤ 0.001). In contrast to SFA and MUFA, the intake of PUFA was low in omnivores, higher in vegetarians and highest in the vegan group  was high in omnivores and notably lower in both flexitarians and vegetarians (p ≤ 0.05). The intake of stearic acid, arachidonic acid (n-6), eicosapentaenoic acid , and docosahexaenoic acid  differed also between the four groups, with decreasing of the intake in the following order: omnivores > flexitarians > vegetarians > vegans  and linoleic acid (LA) were similar in all four groups and the consumption of palmitic acid, oleic acid, and docosapentaenoic acid (n-3) were higher in omnivores than in the vegetarian/vegan diets (p ≤ 0.001). The average cholesterol intake in flexitarians, vegetarians and vegans matches the recommendations of the German Society of Nutrition (p ≤ 0.001). The average intake of pantothenic acid and vitamin A matches the recommendations only in omnivores. The lowest intake of vitamin A in the vegan group was partly compensated by a higher intake of beta carotene (p ≤ 0.001). Except for vitamin B2 and B12, the average intake of the B-vitamins complied with the recommendations for daily intake. The intake of vitamin B2 and B12 decrease as follows omnivores > flexitarians > vegetarians > vegans (p ≤ 0.05). In the vegan group, the average intake of vitamin B2 was below recommended levels. The recommended daily intake of Vitamin B12 (4 μg) was also not reached in vegetarians, flexitarians, and vegans . T. Tp ≤ 0.d vegans .p ≤ 0.05). In all groups, vitamin K intake was 3 to 4 times higher than recommended, with the highest intake in vegans . In women, the intake of chloride varied in the following order: omnivores > flexitarians/vegetarians > vegans (p ≤ 0.05). The comparably high intake of phosphor in omnivores, but also in flexitarians and in vegetarians differed from the lower intake in the vegan group (p ≤ 0.01).In group 1 to 3, the average calcium intake was almost at recommended level but was lower in vegans . The lowest zinc intake in the vegan subjects varied from the higher intake in omnivores, flexitarians, and vegetarians, respectively (p ≤ 0.01). In total, the average intakes of the vitamins B1, B2, B12, chloride, iron, copper, and zinc were higher in men than in women . Comparable differences were detected for waist circumference . The ECM and the ECM/BCM ratio did not differ between the four diet groups, except the slightly lower ratios in omnivorous and flexitarian men in comparison to the higher ratios in vegetarian men . The body fat mass was higher in omnivorous men in comparison to flexitarian and vegan men. In women and the entire study population, the higher values in omnivores differed from the values measured in the groups 2 to 4 (p ≤ 0.05).The body composition differed significantly between men and women . Extracep ≤ 0.05; p ≤ 0.05; In men of the studied diets, the body water fraction was similar. The highest amounts of approx. 35 L in omnivorous women differed from the values measured in the other three groups which varied between 28–42 L (p ≤ 0.05). Similar differences were found for the cell amount which describes the amount of BCM in the LBM  normally ranges between 5 to 7°. Values below the reference limit were found in groups 2 to 4 . The low the LBM . The higp ≤ 0.05). Apolipoprotein A1 concentration in groups 1 to 3 were higher than in vegans (p ≤ 0.05). HDL cholesterol and triacylglycerols did not differ between the four groups . Biotin values in omnivores were lower compared to flexitarians and vegans, respectively .Albumin and creatinine concentrations in 24h urine ranged from 5–82 mg/l and 1.4–21.9 mmol/l, respectively. The lowest values observed in vegetarians and vegans, diffed from omnivorous group's concentrations, respectively , potassium, and zinc fell below the DGE recommendations .In vegetarians, the intake of total fat, SFA, disaccharides, and total sugar was also above the recommendations while the intake of total protein, PUFA particularly n-3 PUFA, pantothenic acid, vitamin B12, vitamin D, calcium, iron (women), potassium, and zinc were below the recommendations for adequate nutrient intake .In vegans, the mean intake of total sugar was also higher than recommended. In this diet form, the sugar intake mainly arose from fruits while consumption of sugar such as chocolate or gummy bears was comparably lower, as these foods often contain animal-based ingredients , 32. StiThe average intake of iron was below the recommendations in flexitarians and vegetarians but almost reached the optimal levels in both omnivores and vegans. Considering the lower bioavailability of non-heme iron (iron from plant origin), the iron intake in vegetarian/vegan diets should be 1.8 times higher than in omnivores diets . In accoIn the diets under consideration, the average concentrations of all vitamins, minerals and trace elements analyzed, except for vitamin D and iodine, were within the reference range. Previous data indicate undersupply for vitamin D as a general problem independently from the diets studied , 36.n = 100), whereby the difference to the lower values in vegetarians  and vegans  was not significant.Evident from the dietary records, the intake of vitamin B2 and B12 decreased significantly in the following order omnivores > Flexitarians > vegetarians > vegans, because dairy products, fish and meat are the main food sources of these vitamins. Schüpbach et al.  found alp = 0.62), the low 4cB12 indices in vegetarians and vegans differed significantly from the index calculated in omnivores. Thus, our data indicate for the risk of undersupply with vitamin B12 in the vegetarian/vegan groups which can manifests in macrocytic anemia or neurological impairments and can lead to irreversible neurological damage if undetected , except for two participants showing hemoglobin values below 7.6 mmol/l, one omnivorous participant and one vegan with an additional transferrin saturation below 16%. Moreover, in one vegetarian and one vegan only transferrin saturation was below 16%, indicating latent iron deficiency.Depletion of iron stores, defined by ferritin concentrations below 20/30 μg/l for women and men, were detected in 20 and 0% of the omnivorous group, 43/14% flexitarians, 28/17% vegetarians, and 42/6% vegans, respectively. Since, ferritin is also an acute-phase reactant, its synthesis is upregulated by infection or inflammation . In the In accordance with the literature, men were marked by two to threefold higher ferritin concentrations than women . In addiAn iron deficiency anemia defined by decreased hemoglobin concentrations were found in 13% of women and 28% of men in the omnivorous group, 20/14% flexitarians, 21/17% vegetarians, and 29/18% vegans. Of the participants with anemia, reduced MCV values (<80 fl) were observed in 9% flexitarian women and one vegan (2%) woman. In men of this subgroup, MCV below the reference range was only detected in one vegetarian (6%).p ≤ 0.05). This indicates for a higher intake of vitamin C and thereof an improvement of the bioavailability of non-heme iron  vs. heme iron (approx. 25%) . The abseme iron .Besides iron, iodine, selenium, and zinc are further critical trace elements because of their lower content in vegetarian/vegan diets and particularly their lower bioavailability from plants, due to the presence of e.g., phytic acid . Since iMost of the Europeans practice one of the before mentioned dietary patterns, each of which varies in their amount of animal-based products. The Academy of Nutrition and Dietetics postulates that adequately planned vegetarian diets are healthy, nutritionally adequate, and may provide health benefits in the prevention and treatment of non-communicable diseases . OverallHowever, the NuEva screening reveals an insufficient dietary intake of selenium, zinc, potassium, iron (women), calcium, vitamin B12, n-3 LC-PUFA, and vitamin D particularly in vegetarian and vegan diets.A regular consumption of the following foods can counteract the weak points detected in the NuEva screening :i) nuts, seeds, wheat bran and barley flakes as sources for zinc and selenium,ii) paprika, pistachio, pumpkin seeds, cocoa etc. for optimal potassium intake,iii) sesame seeds or tahini, cocoa, amaranth, cashews, pine nuts, and oats in combination with vitamin C or other organic acids to ensure an adequate iron intake,iv) dairy products or plant-based foods enriched with vitamin B12, B12 supplements,v) seaweeds (Nori) or microalgae oils, e.g., from schizochytrium sp. or ulkenia sp. for an optimal n-3 LC-PUFA status,vi) adequate sun exposure is recommended to improve vitamin D status,vii) supplementation of calcium and vitamin D and ensuring an adequate intake of high-quality protein to avoid an elevated fracture risk or the development of osteoporosis.Future R&D activities should focus on the improvement of nutrient profiles of traditional plant food, e.g., by optimal variety selection of seeds, improvement of the soil quality, and reduction in processing steps of plant-based foods to avoid loss of their valuable nutrients.In summary, the NuEva study highlights the need for development and distribution of practical nutritional concepts adapted to individual dietary preferences to ensure an adequate nutrient intake and to avoid deficiency symptoms and risk of associated disorders for all diets under consideration.The NuEva study is designed to identify critical nutrients relating to the implementation of one of the studied diets  by evaluation of a self-reported dietary protocol and measured parameters reflecting nutrient status in serum, plasma, and 24h urine. Due to a comprehensive assessment of biomarkers and data on body composition, the NuEva study allows evaluating physiological benefits or possible physiological consequences resulting from the implementation of the studied nutritional habits with focus on cardiovascular risk.® version 6.4  for professional dietary counseling and therapy. The calculations on nutrients composition based on the “Bundeslebensmittelschlüssel” and further nutrition tables. In this context, differences between nutrient profiles calculated with the software and the nutrient composition of the consumed foods, adding to the limitations encountered in this study. Variations of nutrient profiles can depend on types, preparation conditions and feeding conditions.A one-time self-reported dietary protocol over a defined period is one of the limitations of the NuEva screening. Self-reports on dietary intake bear the risk of over-reporting of healthy foods and under-reporting of high-energy, low nutrient foods. The dietary protocols were calculated with the software PRODIA further limitation is the generalizability of the data which represent a regional sample as the participants were recruited from central East Germany.The calculated nutrient intake form self-reports was related to the recommendations for nutrient intake from the German Society of Nutrition (DGE e.V.). Currently, these guidelines do not consider the implementation of diets differing in their proportion of animal-based foods.The datasets presented in this article are not readily available because of an ongoing evaluation of the datasets by the study team. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethical Committee of the Friedrich Schiller University of Jena. The patients/participants provided their written informed consent to participate in this study.CD was responsible for acquisition of funding, conceptualization and trial design, conduction of the NuEva study, data acquisition, and she wrote the original draft. As study physician KD was responsible for medical care of the NuEva participants during the study. TW performed the statistical analysis of the data sets based on a constituted statistical plan. PS and CD supervised the statistical analysis. CR supported the data acquisition and sample management. MK was responsible for data acquisition and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by the German Federal Ministry of Research and Education (Grant Number: 01EA1708). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the type 2 diabetes group, plasma glucose and fructosamine concentrations were significantly higher than in the healthy group. In in vitro studies, collagen-induced thrombi formation in the blood of diabetic patients was 33% higher than in the healthy group. Resveratrol reduced thrombi by over 50% in the blood of healthy and diabetic patients. TXA2 production was 47% higher in diabetic platelets than in the healthy group. Resveratrol reduced TXA2 release by 38% in healthy platelets and by 79% in diabetic platelets. Resveratrol also reduced the activities of enzymes responsible for glycolysis and oxidative metabolism in the platelets of both groups. These data indicate that the resveratrol-induced inhibition of platelet metabolism and TXA2 release may lead to a reduction of platelet function and thrombus formation in patients with type 2 diabetes. Therefore, resveratrol may be beneficial to prevent vascular complications as a future complementary treatment in aspirin-resistant diabetic patients.Chronic hyperglycemia contributes to vascular complications in diabetes. Resveratrol exerts anti-diabetic and anti-platelet action. This study aimed to evaluate the effects of resveratrol on metabolism and the function of blood platelets under static and in in vitro flow conditions in patients with type 2 diabetes. Blood obtained from 8 healthy volunteers and 10 patients with type 2 diabetes was incubated with resveratrol and perfused over collagen-coated capillaries. Isolated blood platelets were incubated with resveratrol and activated by collagen to assess platelet function, metabolism, ATP release, TXA Th. Th2 proc events C 21]. F. F2 prodc events C 36,37],372 prodc events . This mac events C.2 production was more pronounced in patients with diabetes suggests that resveratrol may reduce thrombotic complications by the reduction of TXA2 production in hyperglycemia , directly incubated for 30 min with whole blood and blood platelets. In such conditions, resveratrol statistically reduced thrombus formation, platelet function, and metabolism. At the same time, it did not induce any toxicity , Table 2There are also results from in vivo studies on the effect of resveratrol in patients with diabetes. The clinical data showed that resveratrol at a wide range of concentrations (10 mg to 14 g daily) reduced fasting glucose, HBA1c, or increased insulin sensitivity, but the effect was observed after several weeks or months of the supplementation and was without evident adverse effects . TherefoStandard oral resveratrol supplementation, recommended to buy over the counter, is usually 100–2000 mg/d. There are, however, some issues of resveratrol bioavailability. Some studies revealed that a single oral dose of resveratrol (25–5000 mg) correlated with its mean peak plasma levels in healthy people, and resveratrol at 5000 mg gave plasma levels about 500 ng/mL ,19,20,212 production both in healthy and diabetic people. Therefore, our findings are only a tiny contribution to a wide discussion on the effectiveness and safety of resveratrol supplementation in healthy people and patients with diabetes. More research is required in the future to investigate the effect of the oral/intravenous supplementation of resveratrol on reducing thrombotic complications in patients with diabetes, but also to investigate any possible adverse effects, such as bleeding problems.However, in our studies, we focused on finding that an exposition of blood to resveratrol may have an anti-thrombotic effect, regulate glucose metabolism, and affect TXA2 production, and TBARS accumulation in patients with type 2 diabetes may be due to elevated activities of key enzymes responsible for glucose utilization in chronic hyperglycemia.Increased platelet aggregation, TXA2 production, which may suggest that platelet down-regulation by resveratrol is related to the reduction in the release and production of the secondary platelet mediators.Resveratrol reduced platelet adhesion, aggregation, and thrombus formation. The fact that this compound also inhibited the activities of platelet hexokinase, G-6-P-dehydrogenase, aconitase, and isocitrate dehydrogenase indicates that resveratrol reduced platelet function by the inhibition of glucose metabolism and energy production. Moreover, resveratrol reduced platelet ATP release and TXA2 production, or the inhibition of aconitase and isocitrate dehydrogenase activities, were more pronounced in the blood of patients exposed to hyperglycemia. Therefore, resveratrol may be useful in controlling platelet hyperactivation in type 2 diabetes.Resveratrol-related reduction of platelet function and TXA


The morbidity and mortality of lung cancer are increasing, seriously threatening human health and life. Non-small cell lung cancer (NSCLC) has an insidious onset and is not easy to be diagnosed in its early stage. Distant metastasis often occurs and the prognosis is poor. Radiotherapy (RT) combined with immunotherapy, especially with immune checkpoint inhibitors (ICIs), has become the focus of research in NSCLC. The efficacy of immunoradiotherapy (iRT) is promising, but further optimization is necessary. DNA methylation has been involved in immune escape and radioresistance, and becomes a game changer in iRT. In this review, we focused on the regulation of DNA methylation on ICIs treatment resistance and radioresistance in NSCLC and elucidated the potential synergistic effects of DNA methyltransferases inhibitors (DNMTis) with iRT. Taken together, we outlined evidence suggesting that a combination of DNMTis, RT, and immunotherapy could be a promising treatment strategy to improve NSCLC outcomes. There hGiven the respective limitations of ICIs and RT, promising results from the combination of these therapies in NSCLC have been reported in a growing number of studies, with synergistic effect by combined ICIs and RT significantly improving patients’ outcome. ICIs blocked inhibitory immune checkpoints such as PD-1, PD-L1, and CTLA-4 to reboot the cancer-immunity cycle (CIC) and prevent tumor immune evasion, thus restoring and maintaining anti-tumor immunity. RT plays a vital role in anti-tumor immunity together with ICIs during CIC. The mechanisms by which RT enhances the anti-tumor immune response are versatile. Firstly, RT can promote the release of neoantigens and the expression of MHC-I molecules, thereby activating antigen-presenting cells such as dendritic cells (DCs) , 7. SecoThe synergistic mechanism of RT and ICIs has laid a solid foundation for their combined application in clinical practice. Currently, numerous phase 3 clinical trials are being launched or have been completed for various stages of NSCLC, as summarized in DNA methylation is the most common and best studied epigenetic modification that has been involved in immune evasion and radioresistance in numerous studies. Emerging evidence suggested that targeting DNA methylation could be a promising strategy to substantially enhance the effect of iRT. In this review, we focus on the regulation of DNA methylation on ICIs resistance and radioresistance in NSCLC and elucidate the potential synergistic effects of DNA methylase inhibitors (DNMTis) in patients treated with iRT. The combination of DNMTis, RT, and immunotherapy is worthy of further study to improve NSCLC outcomes.2in vitro. For example, sodium bisulfite can convert unmethylated cytosine into uracil, but cannot change cytosine within methylated cytosine guanine (CpG) islands. the living state showed a decrease in gene expression activity. Through this modification, DNA conformation, DNA stability, DNA-protein interaction, and chromatin structure will change, thereby controlling gene expression . There are four main DNMTs: DNMT1, DNMT3A, DNMT3B, and DNMT3L. After DNA replication is completed, DNMT1 is the most important enzyme in the methylation reaction that catalyzes the transfer of methyl groups to newly synthesized DNA strands, a phenomenon known as maintenance methylation. DNMT3A and DNMT3B are responsible for catalyzing the reaction of new methylation sites on the nucleic acid chain, called de novo methylation. DNMT3L is a regulatory enzyme in the DNA methyltransferase family without methyltransferase activity, and its main role is to regulate the activity of other methyltransferases  of the promoter region and the first exon region of the gene, the CpG sequence density is high, more than 5 times the average levels. These regions are characterized by repeated guanine and cytosine-rich fragments called CpG islands and have typical lengths of 200 bp ~ 1 kb. As an important epigenetic event in the occurrence and development of cancer, methylation of CpG islands plays a key role in gene silencing . Because3.1telomerase reverse transcriptase (TERT) gene promoters has been shown to enhance TERT expression in most tumor types , and that FANCF methylation was related to the age of smoking and drinking  and expression of PD-L1, loss of neoantigen expression, deficiency of antigen presentation, activation of driver genes, and dysfunction of specific pathways in tumor cells. These changes in tumor cells can lead to the development of immune resistance.4.1.1.1TMB is a critical biomarker, which can serve as a predictor of the efficacy of ICIs. Yarchoan et al. demonstrated that a significant positive correlation could be observed between TMB and objective response rate (ORR) with ICIs in 27 tumor types . Based o4.1.1.2In addition to TMB, PD-L1 is another valid predictor, which is widely utilized in clinical practice. Although numerous studies have demonstrated that high expression of PD-L1 in tumor cells can mediate immune escape, current clinical trials have proved that high expression of PD-L1 can make tumor cells more sensitive to ICIs –75. A de4.1.1.3in vitro, indicating a lasting immune memory  is a vital member in the immune process, involving in antigen processing and presentation. The engagement of MHC I molecule on the surface of cancer cells and the T cell receptor (TCR) on the surface of CD8+T cells promotes the activation of CD8+T cells . Beta-2 sistance . NSCLC psistance . When thsistance . In addisistance –92.4.1.1.5The abnormality of the special signal pathway can also cause immunotherapy resistance. The absence of an IFNγ signal protects tumor cells from recognition and attack by immune cells. IFNγ is produced by tumor-specific T cells and performs an effective anti-tumor immune response by recognizing corresponding receptors on cancer cells or antigen-presenting cells. Moreover, IFNγ has the ability to boost tumor antigen presentation by increasing MHC I expression. At the same time, it can directly repress the proliferation of cancer cells, promote apoptosis, and recruit immune cells to cause anti-tumor effects , 93. Thu4.1.2The tumor-extrinsic mechanism is mainly caused by the change in the host immune microenvironment, which refers to the reduction of cellular components and cytokines related to immune activation in TME and the increase of cellular components and cytokines related to immune suppression in TME. TME is not only the internal environment for tumor cells to survive and develop but also the “main battlefield” for immune cells to kill tumor cells. As a result of low infiltration levels and exhaustion of immune effector cells, immune suppression occurs, thereby mediating immune escape . Accordi4.2The concept of immunotherapy has brought new progress in the treatment of cancer. However, cancer cells and host immune cells are prone to immune tolerance after the interaction. In the context of cancer, epigenetics can reshape the TME, promote tumor growth, and escape the immune system. For example, aberrant DNA methylation can provide survival benefits to tumor cells by silencing genes necessary for antitumor activity. DNMTis can reduce DNA hypermethylation in CG-rich regions (CpG islands) of tumor suppressor gene promoters and restore transcriptional activity at these sites, thereby altering the immune response , 110. Zhde novo DNA methylation acquired after CD8 + T cell exhaustion can lead to further exhaustion of T cells, while DNMTis can reduce CD8 + T cell exhaustion by inhibiting DNMT3a-mediated de novo DNA methylation  and endogenous retroviruses (ERVs) , 113. Afhylation . The acthylation . For exahylation . Furtherhylation , 126. Fihylation , 128, mahylation . These s4.3DNMTis have the ability to inhibit methylation formation in the CpG region . The use4.3.1The landmark drugs of clinically approved epigenetic therapy are azacitidine (AZA) and 5-2′-deoxycytidine (decitabine), two nucleoside classes of DNMTis that were discovered by researchers in the 1960s . After sIn the past few decades, some novel nucleoside DNMT inhibitors and non-nucleoside DNMT inhibitors have been identified and used as antitumor drugs. We summarize the commonly used DNMTis in Recently, a phase 1 clinical trial showed that CC-486  as monotherapy has a good therapeutic effect on advanced nasopharyngeal carcinoma. 37.5% of patients had partial response (PR), and 50% of patients achieved stable disease (SD) . CC-486 4.3.2https://clinicaltrials.gov/ct2/show/NCT00978250). The PFS of 25 NSCLC patients was 2.3 months. Severe AEs occurred in 38 of the 93 patients, the most common of which was gastrointestinal reactions. Schiffmann et al. included 10 patients with refractory advanced NSCLC in a phase 1/2 trial to evaluate the tolerance of azacitidine combined with atenolol. The treatment was well tolerated and an objective response was observed. The median survival time was 6.4 months  and decitabine (5-aza-2 ‘ -deoxycytidine), which are cytidine analogs. DNMTis have a wide range of cellular effects, through phosphorylation and DNA binding, inducing DNA hypomethylation, apoptosis, or activation of specific genes, such as tumor suppressor genes, to exert anti-tumor effects . Momparl4 months .4.3.3.In previous studies, when NSCLC patients only received single-agent methylation inhibitors, such as DNMTi azacytidine, only 4% of patients showed an objective response, and the effect was not ideal . Wranglehttps://clinicaltrials.gov/ct2/show/NCT01928576). In 2016, 13 NSCLC evaluation studies were recruited to evaluate whether the drug tetrahydrouridine-decitabine (THU-Dec) combined with nivolumab is more effective than nivolumab alone in the treatment of NSCLC patients . Among them, 8 were in an experimental group and 5 were in the control group. The latest results in August 2022 showed that 4 of the 8 patients in the experimental group developed progression, 2 were SD, and 2 were PR; among the 5 patients in the control group, 1 progressed, 3 were stable, and 1 was partially relieved. The PFS was 69 and 227 days, respectively. As of the latest results, the OS for the experimental and control groups was 389.5 days and 844 days, respectively. In the same year, a 1/2 phase study  was conducted in USA, UK, and Spain to evaluate the safety and tolerance of azacitidine combined with pembrolizumab and epalrestat. The study included 70 subjects with advanced solid tumors and previous stage IIIB or IV NSCLC and stage IV microsatellite-stabilized colorectal cancer. The study was permanently discontinued in February 2019 and there were no patients in treatment groups B and C. Treatment group A was divided into two groups according to different doses, 62 patients with 100 mg INC B24360 and 8 patients with 300 mg INC B24360. These results demonstrated that the all-cause mortality rate of the 100 mg INCB24360 group was 66.13%, and the risk of serious adverse events was 45.16%. The all-cause mortality of patients with 300 mg INCB24360 was 50%. The risk of serious AEs (adverse events) was 37.5%. In 2017, 34 castration-resistant prostatic cancer and NSCLC were included to evaluate the safety and toxicity of decitabine (SGI-110) combined with pembrolizumab (MK3475) in patients with refractory solid tumors . In the same year, 28 NSCLC patients were included to assess the safety and dose selection of a 1/1b phase study  in the treatment of patients with advanced NSCLC with bortezomib combined with guadicitabine and moxitinib. The clinical trial was first published in 2017 and is expected to be completed in July 2023. The trial results have not yet been released. Overall, it is safe to use DNMTis. Epigenetic therapy does not directly affect cell cycle progression or apoptosis, but rather regulate some genes to cause global changes in cellular transcriptional programs, and therefore do not have an immediate cytotoxic effect. But there is much more to learn in how to make better use of DNMTis in clinical settings, such as a more detailed understanding of drug resistance mechanisms and more clinical trials.In a follow-up study of combined epigenetic therapy for advanced treatment-refractory NSCLC, six patients received ICIs, three of whom achieved PR and two achieved SD, all of which lasted more than 8 months . The goo5RT is a vital treatment for NSCLC, however, radioresistance is considered to be the main cause of the poor effect of RT for NSCLC. The underlying mechanism of radioresistance remains to be clarified. To improve the radiosensitization, investigators concentrate mainly on ameliorating the hypoxia state, increasing DNA damage, and affecting the cell cycle. At present, the commonly used radiosensitizers in clinical practice are 5-fluorouracil, platinum, and gemcitabine –153. Nev5.15.1.1.DNA methylation is combined with histone modification, resulting in RNA polymerase binding to this region, causing the silence of related genes, especially tumor-suppressive genes , 156. It5.1.2HIF-1 α  homologous recombination (HR) repair, augments cell cycle arrest, and increases cell death induced by ionizing radiation (IR). OTUD4 could be a potential target for radiosensitizing NSCLC. Furthermore, a study indicates that OTUD4 radiosensitizes NSCLC via ATM/CHK2/P53 signaling and suppresses homology-directed repair of DNA DSBs induced by IR  revealed higher expression of SERPINB5 in radiosensitive lung cancer cells than radioresistant cells (TM4SF4) protein is a cell surface glycoprotein that has the function of modulating cell proliferation  –180. A s pathway ..5.2Although numerous preclinical studies have demonstrated the significant radiosensitization effect of DNMTis, there are no result related to DNMTis combined with radiotherapy for NSCLC. The clinical trials such as NCT03445858, NCT01707004, and NCT04174196, that are currently being recruited or have been completed, focusing on lymphoma and hematological neoplasms, have shown the promise of DNMTis combined with RT. Therefore, increasing clinical trials are needed to undertake to explore the potential benefit of DNMTis combined with RT.6.RT has a good local control effect on the tumor, but its systemic anti-tumor effect is relatively limited, while immunotherapy is the contrary. RT can promote anti-tumor immunity by increasing neoantigen release and changing TME and other mechanisms. The combination of DNMTis and the former two can not only enhance the sensitivity of RT to increase the ability of tumor local control but also promote the further release of tumor antigens and improve immunogenicity. At the same time, DNMTis can also increase the production of neoantigens and the expression of MHC I molecule, and improve the antigen presentation ability of immune cells. In addition, DNMTis can activate cytotoxic T lymphocytes, inhibit the function of immunosuppressive cells, up-regulate the positive cytokines of immune regulation, create a TME conducive to immunotherapy, and improve the systemic effect of immunotherapy. The three complement each other and play a better and more powerful anti-tumor effect However, the toxicity of DNMTis is a problem that cannot be underestimated. Some DNMTis have been extensively used in clinical practice with controllable AEs, but the AEs of treatment in combination with ICIs have not been widely studied, especially in long-term outcome. With the advent of new DNMTis, the toxicity aspect of the drug is acceptable. In addition, clinical studies associated with low-dose DNMTis and RT as induction before ICIs are also worthy of future exploration. For different stages of NSCLC, in combination with different ICIs, more in-depth exploration of the dose, frequency, time, and sequence of RT, as well as better control of the side effects, can make patients more benefit from combination therapy.7DNA methylation is a very stable sign that can be detected in various types of biological samples, including tumor tissue and body fluids . At pres8The advent of iRT has been a boon for patients with NSCLC and has proven to be a fruitful research field for NSCLC. Future research will focus more on improving existing iRT. DNMTis are promising therapeutic agents for reversing radiation resistance as well as immune resistance due to their ability to sensitize tumors to radiation and significantly activate tumor immune responses. We eagerly look forward to the emergence of clinical studies of iRT combined with DNMTis, using epigenetic therapy to reverse tumor treatment resistance and sensitize to iRT, benefiting a broader group of patients than currently exists.CX provided fund support and designed the conception of the review. CJ, RL, and QW wrote the main manuscript text. CJ and RL were responsible for drawing the figure and designing tables. QW, ZW, and YC critically revised the article. All authors contributed to the article and approved the submitted version.


Clostridioides difficile infection (CDI), prolonged hospitalization, and 30-day reoperation after lumbar spine surgery for noninfectious indications, and to report adherence to current guidelines.To describe the association between duration of antimicrobial prophylaxis (AMP) and 30-day surgical site infection (SSI), 7-day acute kidney injury (AKI), 90-day Survey.The study cohort comprised 6,198 patients who underwent lumbar spine surgery for noninfectious indications across 137 Veterans’ Health Administration surgery centers between 2016 and 2020.Used univariate and multivariate logistic regression to determine the association between type and duration of AMP with 30-day SSI, 7-day AKI, 90-day CDI, prolonged hospitalization, and 30-day reoperation.P = .056).Only 1,160 participants (18.7%) received the recommended duration of AMP. On multivariate analysis, the use of multiple prophylactic antimicrobials was associated with increased odds of 90-day CDI  and 30-day reoperation . Courses of antimicrobials ≥3 days were associated with increased odds of prolonged hospitalization  and 30-day reoperation . In univariate analysis, increasing days of AMP was associated with a trend toward increasing odds of 90-day CDI (cOR, 1.4; 95% CI, 1.0–1.8 per additional day; Longer courses of AMP after lumbar spine surgery were associated with higher odds of CDI, prolonged hospitalization, and reoperation, but not with lower odds of SSI. However, adherence to the recommended duration of AMP is very low, hinting at a wide evidence-to-practice gap that needs to be addressed by spine surgeons and antimicrobial stewardship programs. Antimicrobial prophylaxis (AMP) has been shown to decrease SSIs after spine surgery, but the duration of AMP remains a frequent subject of debate between antimicrobial stewardship programs (ASPs) and spine surgeons.Surgical site infections (SSIs) complicate ∼3% of spine surgeries. Consequently, despite recommendations to limit prophylactic antimicrobials to a single dose or a single day, in practice, the duration of prophylactic therapy ranges from 1 to 10 days, and many patients receive antibiotic courses that may be unnecessarily long and lead to increased acute kidney injury (AKI), Clostridioides difficile infection (CDI), and healthcare costs.To date, most of the available evidence has not shown a difference in the frequency of SSIs after short and long courses of prophylactic antimicrobials, but some studies have yielded conflicting results.Our primary aim was to determine the association between the duration of AMP and 30-day SSI, 7-day AKI, 90-day CDI, prolonged hospital length of stay (LOS), and 30-day reoperation after lumbar spine surgery for noninfectious indications. Additional aims included determining the effect of antimicrobial selection on these outcomes and describing the degree of adherence to guidelines regarding selection and duration of prophylactic antimicrobials. For this study, we matched perioperative variables from the Veterans’ Affairs Surgical Quality Improvement Program (VASQIP) database to bar-code medication administration (BCMA) records from a database created by the VA Informatics and Computing Infrastructure (VINCI) team. The VASQIP uses a validated sampling algorithm to manually review 105 perioperative variables from ∼70% of all major surgical cases performed within the VHA. The database created by VINCI is a relational database that contains detailed clinical, pharmacy, and laboratory data abstracted directly from the VHA integrated electronic health record (EHR). We included all veterans from the VASQIP database who underwent lumbar spine surgery  between January 1, 2016, and September 30, 2020, and received at least 1 day of AMP with cefazolin, clindamycin, and/or vancomycin at any time starting on the date of surgery up to, and including, 7 days after the date of surgery. For patients who underwent multiple spine surgeries within the study period, we included only the first episode of care in this analysis. We excluded individuals who received other antibiotics and/or had postoperative International Classification of Disease, Tenth Revision (ICD-10) codes for epidural abscess , osteomyelitis, discitis, other infectious spondylopathy , or infection and inflammatory reaction due to prosthetic material .The Veterans’ Health Administration (VHA) is the largest integrated healthcare system in the United States, offering surgical care at 109 inpatient and 28 ambulatory surgery centers. If a patient received >1 of the specified prophylactic antimicrobials, their duration of therapy was the sum of the days of therapy of all administered agents.Individuals were categorized by type  and duration  of prophylactic antimicrobials. The duration of therapy was defined as the total sum of days for which any amount of cefazolin, clindamycin, and/or vancomycin was administered between the date of surgery and 7 days after the date of surgery. We defined 7-day AKI according to Acute Kidney Injury Network (AKIN) criteria  or as determined by the VASQIP reviewer. Only 1,155 patients had enough data for the 7-day AKI outcome. The 90-day CDI outcome was defined as the presence of a positive C. difficile laboratory result in the 90 days following the date of surgery. Prolonged hospital length of stay (LOS) was defined as an LOS greater than the mean LOS for the entire cohort (4 days). The 30-day reoperation outcome was defined as a return to the operating room for any reason in the 30 days after the date of surgery.The primary outcome of interest was 30-day SSI, which was defined as the development of superficial, deep, or organ-space SSI in the 30 days after the date of surgery. The VASQIP reviewer used the National Healthcare Safety Network (NHSN) definitions to categorize the different SSI categories.2, history of diabetes mellitus, history of hypertension, American Society of Anesthesiologists (ASA) classification), substance or medication use , surgical details  in the 3 days after the date of surgery), and postoperative infections. Postoperative sepsis was defined as documentation of sepsis in the EHR by the primary physician or the presence of infection plus ≥2 of the following in the immediate postoperative period: temperature >38°C or <36°C, heart rate >90 beats per minute, respiratory rate >20 breaths per minute, PaCO2 <32 mmHg, leukocyte count >12,000 or <4,000 cells per mm, or >10% immature (band) forms with or without hypotension. Postoperative pneumonia and urinary tract infection (UTI) were defined according to NHSN criteria.Covariates were selected prior to data collection based on published risk factors for our outcomes of interest. These included demographic details , comorbid conditions . Our multivariate logistic regression model adjusted for age, BMI, diabetes mellitus, hypertension, ASA classification, tobacco use, corticosteroid use, surgery type, duration of surgery, and antimicrobial type or duration. For the 7-day AKI, 90-day CDI, and prolonged hospital LOS outcomes, we also adjusted for other postoperative infections . Lastly, we stratified our analysis of the impact of duration on these outcomes by selected antimicrobial agent. Analyses were conducted in Stata version 15.1 software . All tests were 2-sided, and The study was approved by the institutional review board of the Veterans’ Affairs Puget Sound Healthcare System. The requirement for informed consent was waived.We included 6,198 veterans who underwent lumbar spine surgery: 2,041 (32.9%) underwent arthrodesis or fusion, 858 (13.8%) underwent discectomy, and 3,299 (53.2%) underwent laminectomy. Of all participants, 5,384 (86.9%) received the recommended agent (cefazolin), but only 1,160 (18.7%) received the recommended duration (≤1 day) of prophylactic antimicrobials.The average age of this patient cohort was 60.9 years (SD ±12.6), 5,748 (92.7%) were men, 4,543 (73.3%) were classified as ASA class 3, and 2,010 (32.4%) had used tobacco <1 year before surgery. The average duration of surgery was 3.1 hours (SD ±1.7).The rate of 30-day SSI was 1.3% (n = 83), including 44 (0.7%) superficial SSIs, 30 (0.5%) deep SSIs, and 9 (0.1%) organ-space SSIs. The rate of 7-day AKI was 7.1% ; the rate of 90-day CDI was 0.3% (n = 18), and the rate of 30-day reoperation was 3.6% (n = 223). The average hospital LOS was 3.9 days (SD ±9.4)  and 30-day reoperation . Additionally, patients who received >1 agent also had a higher rate of prolonged hospital LOS in univariate analysis only  (Table P = .056) .We did not find a significant association between the duration of prophylactic antibiotics and the odds of 30-day SSI, 7-day AKI, and 90-day CDI. However, we identified a trend toward increasing 90-day CDI with increasing antimicrobial days on univariate analysis  received the recommended duration of AMP (≤1 day). In a study of 1,579 patients undergoing lumbar spine surgery, no differences were detected in the rate of SSI after a single dose or 5–7 days of AMP. This finding has also been replicated in studies of patients requiring implantation of prosthetic material and temporary drains. More recently, however, in a study of 1,922 patients undergoing instrumented spine surgery, patients who received a single dose of cefazolin had a significantly higher frequency of SSI compared to patients who received 3 days of cefazolin . In another recent study of 336 individuals requiring temporary drain placement after cervical and lumbar spine surgery, continuing AMP for as long as the drain remained in place was associated with a trend toward decreased SSI. Our findings support the conclusion that continuing prophylactic therapy beyond 24 hours is unlikely to result in a meaningful decrease in SSI for most patients undergoing spine surgery. Admittedly, since the VASQIP database does not classify patients according to whether they required implantation of prosthetic material or a temporary drain, we could not evaluate the association between duration of AMP and SSI in these specific populations. We did, however, control for potential correlates of instrumentation in our multivariate analysis, including surgery type and duration of surgery.Current guidelines recommend a single dose of prophylactic antimicrobials for spine surgery because most of the available evidence shows that increasing the duration of therapy beyond 24 hours does not decrease the frequency of SSI. In contrast, in our study, additional days of antimicrobials only led to a trend toward increased 90-day CDI. This discrepancy may be due to our focus on procedures and antibiotics that do not independently increase the odds of AKI  and CDI . Notably, our study did not find an association between duration of vancomycin and 7-day AKI or clindamycin and 90-day CDI as in the Branch-Elliman et al study, but this may be due to the small number of these events in these subgroups.Evidence regarding the effect of duration of prophylactic antibiotics on the rates of AKI and CDI after spine surgery is limited. However, these relationships have been investigated in other perioperative settings. A study of 79,058 veterans undergoing cardiac, colorectal, orthopedic joint replacement, and vascular surgery, found that increased duration of AMP significantly increased the odds of 7-day AKI  and 90-day CDI .P < .05). The increased LOS may simply be a reflection of a longer duration of prophylactic therapy, or a result of complications stemming from the surgery itself or from inappropriate antimicrobial use .In our study, patients who received prophylactic therapy for ≥3 days had increased odds of prolonged hospitalization. In a study of 135 patients undergoing lumbar spine study, patients who received 9 days of AMP had significantly longer average hospital LOS than patients who received 2 days of AMP . Conversely, it is possible that patients with an SSI that eventually required revision received longer courses of antibiotics as part of their initial management. So, it is not surprising that the use of multiple prophylactic agents was associated with increased odds of CDI. However, because it was unclear which antibiotics were used and whether they were used concurrently or sequentially, we could not determine why this association occurred in our study. Similarly, the effect of multiple antimicrobial use on prolonged hospitalization and reoperation was difficult to interpret. These outcomes may have been due to using multiple redundant agents or switching from one agent to another due to an adverse reaction.The effect of antibiotic use on CDI risk is cumulative. In our study, only 1,160 patients (18.7%) received the recommended duration of prophylactic therapy. Notably, this figure may overestimate the frequency of appropriate postprocedural antimicrobial use since current guidelines recommend administering prophylactic antimicrobials immediately before surgery and every 4 hours during surgery, rather than for a full day. Studies regarding the drivers of inappropriate antimicrobial use in spine surgery are limited. Surveys of cardiothoracic and general surgeons cite contradictory guidelines and use of alternate sources of information . Both, the Surgical Infection Society (SIS) and the National Association of Spine Specialists (NASS) recommend limiting prophylactic antimicrobials to a single dose or a single day. However, the NASS suggests that a prolonged duration of therapy may be reasonable for certain patients, including obese and diabetic patients and patients who require prolonged or instrumented surgery. Recent studies suggesting that prolonged antimicrobial therapy may decrease SSI in patients requiring implantation of prosthetic material or temporary drains may also confound surgeon attitudes about the duration of prophylactic therapy. Additional drivers may be the paucity of surgeons in antimicrobial stewardship roles, and the perception that antimicrobials are a relatively risk-free intervention. Regardless, this is an area of opportunity for ASPs.Continuing prophylactic therapy beyond 24 hours is the most common cause of inappropriate postprocedural antimicrobial use.Our study has several strengths. To our knowledge, this is the largest study of the association between type and duration of AMP and postoperative outcomes after spine surgery and the first to describe adherence to published guidelines among spine surgeons in a large integrated healthcare system. Additionally, because the VASQIP database is manually validated by trained reviewers and has been utilized in similar analyses, we can be relatively confident about the accuracy of our variables. Second, the follow-up for our SSI outcome was limited to 30 days. In most studies, however, a diagnosis of SSI was typically made within 30 days of surgery. Third, because we focused exclusively on patients who underwent lumbar spine surgery and received 1 of only 3 potential prophylactic antimicrobials, we could not account for the effect of surgery at other spinal levels or different antibiotics on our outcomes. However, the incidence of SSI does not vary significantly according to spinal level  and cefazolin, clindamycin, and vancomycin are the recommended and most widely prescribed prophylactic agents. Additionally, because the individuals in our study were generally older and harbored more risk factors for SSI, our findings may not be generalizable to other populations. Lastly, we were unable to stratify patients according to whether they required implantation of prosthetic material or temporary drains.Our study also had several limitations. First, there is the inherent bias and confounding introduced by our retrospective design. However, since SSI, AKI, and CDI are rare after spine surgery, a randomized trial would need to enroll a prohibitively large number of patients to be sufficiently powered to detect differences in these outcomes.In conclusion, among individuals undergoing lumbar spine surgery, continuing prophylactic antimicrobials beyond 24 hours does not decrease the odds of SSI. However, a longer duration of prophylactic therapy may increase the odds of CDI, prolonged hospitalization, and reoperation. Furthermore, administering multiple agents also increases these outcomes. These findings add to the growing body of evidence that continuing prophylactic antibiotics beyond 24 hours offers minimal benefits and may, in fact, contribute to suboptimal clinical outcomes after spine surgery. Notably, our 18.7% rate of adherence to the recommended duration of prophylactic antibiotics hints at a large evidence-to-practice gap that needs to be addressed by spine surgeons and ASPs. To help bridge this gap, future studies should focus on patients undergoing instrumented spine surgery and implementation of stewardship interventions that address the concerns of our surgical colleagues.