Candida species, especially Candida albicans. Invasive candidiasis includes candidaemia, disseminated candidiasis with deep organ involvement and chronic disseminated candidiasis. During the last decades rare pathogenic fungi, such as Aspergillus species, Zygomycetes, Fusarium speciesand Scedosporium have also emerged. Timely diagnosis and proper treatment are of paramount importance for a favorable outcome. Besides blood cultures, several laboratory tests have been developed in the hope of facilitating an earlier detection of infection. The antifungal armamentarium has also been expanded allowing a treatment choice tailored to individual patients’ needs. The physician can choose among the old class of polyenes, the older and newer azoles and the echinocandins. Factors related to patient’s clinical situation and present co-morbidities, local epidemiology data and purpose of treatment  should be taken into account for the appropriate choice of antifungal agent.Invasive fungal infections are a growing problem in critically ill patients and are associated with increased morbidity and mortality. Most of them are due to  Candida species (spp) were predominantly isolated (17%) followed by Aspergillus species. Candidaemia is associated with a high mortality and increased length of hospital stay and cost [.High attributable mortality may be due to delayed diagnosis and treatment, development of resistance or severity of illness. The purpose of the present review is to provide a practical approach to diagnosis and treatment of invasive fungal infections in the critically ill.Great advances in contemporary medicine and especially in critical care achieved during the last decades have contributed not only to longer survival of patients, but also to the increasing incidence of opportunistic infections caused by fungi. Complex medical and surgical problems, disruption of natural barriers, multiple invasive procedures and prolonged antibiotic treatment are some of the factors contributing to the alarming increase of fungal infections in the Intensive Care Unit (ICU) setting ,2. The land cost ,5..High Candida species are ubiquitous and constitute part of the normal human flora. Only a small percentage of the identified species cause disease in humans. Candida spp. is responsible for an extremely large spectrum of diseases [Candida infections include candidaemia with or without endopthalmitis; disseminated haematogeneous infections; involvement of a single deep organ site  and chronic hepatosplenic candidiasis mostly in haematological patients [Candida infection can be endogenous  and exogenous  even leading to local outbreaks [diseases ,7. Invaspatients . Source utbreaks . Existinutbreaks .Candida bloodstream infections (BSIs) constitute the vast majority of nosocomial fungal infections. In a large nationwide surveillance study in United States (SCOPE) Candida spp were ranked fourth among the hospital-acquired BSIs (9%) [Candida infections (ICI) in the critically ill are available. In an Australian study of ICU-acquired BSIs, Candida species were ranked fourth (15.5%) while according to Meyer et al. the incidence of primary nosocomial candidaemia in 682 German ICUs remained stable in a 5-year period [Candida species [SIs (9%) . The majSIs (9%) . AlmiranSIs (9%) ,13. In aSIs (9%) . Limitedd fourth .5% whiler period . In a rer period . In a lar period . Finallyr period . In a to species . The dis species ,21.Candida BSIs are a well-recognized cause of morbidity and mortality among the critically ill. Though the crude mortality varies between studies, most authors report high percentages (39%–60%) and excess financial burden [et al. included seven studies with attributable mortality ranging between 5 and 71%. For six of them a considerable difference in mortality between cases and controls was observed. Moreover the length of stay and hospitalization cost was significantly higher than that of controls [l burden ,22. Attrcontrols . If candcontrols . Candida species which are pathogenic for humans, Candida albicans is the most frequently recognized followed by C. parapsilosis, C. glabrata and C. tropicalis [C. krusei, C. dublinensis, C. guillermondi, C. kefir, C. lusitaniae, and C. rugosa. Nevertheless the list of species formerly considered as “non-pathogenic” is expanding with the increase in the number of vulnerable population and the ability of the laboratory to isolate new species.During the last decades a shift to non-albicans species has been noted [Candida species in a 6-year period has changed with C. albicans decreasing by 10% though still remaining the most common isolated species [albicans species has been also observed in critically ill patients with considerable differences in the percentage of albicans vs non-albicans spp [C. albicans, followed by C.parapsilosis (23%), C. glabrata (15%), C. tropicalis (9%) and other species (13%) [C. albicans accounted for 57% of blood isolates followed by C. glabrata (16.7%), C. parapsilosis (7.5%), C. krusei (5.2%) and C. tropicalis (4.9%) [albicans species (NAS). In a case-comparator study Chow et al. showed that fluconazole exposure, presence of a central venous catheter and mean number of antibiotics per day were associated with an increased risk of BSIs due to NAS compared to C. albicans while in another study use of medical devices, steroids receipt and pre-existing candiduria were the risk factors associated with the presence of NAS [Candida species has its separate characteristics [Candida spp. is important because some of these species are resistant to fluconazole or other antifungal agents. Awareness of risk factors for the presence of NAS, rapid species identification and antifungal susceptibility testing are required for the proper and timely treatment of these infections.Among opicalis . Speciesen noted . Accordi species . This ch species . The inccans spp . In an Ies (13%) . In a prs (4.9%) . Severale of NAS ,32. Moreeristics ,34,35  is not an easy task because signs and symptoms vary from minimal to dramatic while existing diagnostic procedures present several drawbacks. Blood cultures (BCs) remain the cornerstone of diagnosis but they have suboptimal sensitivity (30%–50%) and need long incubation time . Moreovenfection .d-glucan tests whereas antibodies against the mannan antigen (anti-mannan) have also been developed [Candida; Biorad, Biorad laboratories GmBH); and an enzyme–linked immunosorbent assay (ELISA). Both methods have equal specificity (90%–100%) in diagnosing candidaemia [Candida species under consideration, being higher for C. albicans [d-glucan (BG) , which is a pan-fungal marker , demonstrates variable sensitivity depending on the cut-off diagnostic value and the Candida species under consideration [Candida infection and it must be interpreted with caution due to false positive results from albumin and/or immunoglobulin administration, haemodialysis or Gram-positive bacteraemia [et al. the BG test exhibited excellent positive  and negative  predictive values in critically ill patients [C. albicans  showing 77%–89% sensitivity and 91%–100% specificity [Candida species and it has not been validated in the critical care setting.In order to make timely diagnosis more feasible, several serological markers have been developed. These tests consist of the detection of various agents, components of the fungal cell wall such as the mannan and 1,3-β-eveloped ,39,40,41didaemia ,42 but Ealbicans ,44. In halbicans ,38. Combteraemia . A negatpatients . An indicificity ,47. ThouCandida infection [et al., both real–time PCR and BCs have comparable sensitivities in diagnosing candidaemia (60%) but PCR is invaluable in diagnosing deep–seated candidiasis with negative BCs (sensitivity 88% for PCR vs 17% for BCs) [et al. tested three real–time PCR assays for the detection of Candida spp in non-neutropenic critically ill patients with candidaemia, showing excellent sensitivity (90.9%) and specificity (100%) [Additionally nucleic acid-based detection methods  have been developed for the detection of nfection ,48,49. Afor BCs) . Moreovefor BCs) . Data coin situ hybridization (PNA FISH). This method is a rapid way to differentiate among the usually isolated Candida species in blood provided that blood cultures have developed Candida spp [Finally several recently introduced diagnostic tools include: (a) matrix- assisted laser desorption ionization—time of flight mass spectrometry (MALDI–TOF MS). The test seems a rapid and reliable tool for the identification of yeasts and yeast-like fungi ; (b) a cdida spp . In a redida spp . Candida infection leads to increased mortality in case of treatment delay. The lack of methods with a high sensitivity and specificity for the timely diagnosis of IC created the need for the identification of risk factors and evaluation methods in order to ensure timely treatment even in the absence of laboratory evidence of infection. These risk factors have been described in many studies [Invasive  studies ,54,55,56Candida colonisation patterns in combination with the established risk factors of Candida Prediction Rules have been established in many studies [Fungal colonisation has been associated with the development of IC, yet according to recent data, a small proportion (3%–25%) of colonised patients subsequently develop invasive disease ,58. Due  studies ,62,63,64Aspergillus spp are moulds which are able to cause life-threatening invasive disease in immunocompromised individuals and local disease in immunocompetent persons. The latter can present with a spectra ranging from localised infection of the lungs and sinuses to allergic reactions due to spore inhalation. The species present a worldwide distribution and are found in the environment, plants and decomposing organic matter. Among the hundreds of Aspergillus species few are able to cause disease to humans. The most commonly encountered include A. fumigatus followed by A. flavus and A. terreus. The epidemiology of aspergillosis in the ICU is difficult to establish due to the inhomogeneity of hospitalised patients, the diagnostic difficulties necessitating a biopsy and the difficulty in discriminating between colonisation and disease [Aspergillus in the ICU include improperly cleaned ventilation systems, water systems, or even computer consoles [ disease . Sometim disease  while a  disease . Possiblconsoles . ICU patconsoles . Anastomconsoles  while raconsoles ,71.The diagnostic approach includes evaluation of possible risk factors, clinical and radiological signs and the implementation of specific laboratory and “high technology” methods.etc. [Aspergillus to the brain can cause seizures, brain infarctions, intracranial haemorrhage and meningitis.Persons at greatest risk include patients with haematological malignancies, solid organ transplant recipients, haematopoietic stem cell transplant recipients (HSCT) but also prolonged steroid treatment before ICU admission, chronic obstructive pulmonary disease, severe burns, previous cardiac surgery etc. . Clinicaet al. recorded nonspecific infiltrates and consolidation as the commonest radiological findings in critically ill patients with Invasive Aspergillosis (IA) [Radiologic chest radiograph might be negative at the beginning of the disease, or might show nonspecific changes even during the late stages  and theysis (IA) .Aspergillus in invasive tissue sampling [Aspergillus from cultures of bronchial secretions, broncho alveolar lavage (BAL) or other body fluids or tissues, might represent colonisation rather than infection [Aspergillus in respiratory samples lies between 10%–80%, according to different studies [The gold standard of diagnosis of IA is the histopathological identification of sampling . Howevernfection . Howevernfection . Focusin studies ,79.d-glucan, two fungal cell wall components in blood or other body fluids. The first is released in body fluids during Aspergillus growth [d-glucan, false positive results might be associated to the use of immunoglobulin contaminated with fungal products, the presence of bacterial infections, the administration of several antibiotics and the use of cellulose filters of haemodialysis [Two non-culture-based diagnostic techniques are available: the detection of galactomannan (GM) and β-s growth . It can s growth . This mes growth . In non-s growth . Variabls growth . As for dialysis .et al., a whole blood quantitative real time PCR (qPCR) targeting to a special gene sequence of the fungus could prove as a clinically reliable technique for diagnosis of IA [An additional method for IA diagnosis is PCR . The senis of IA . Additiois of IA .proven, probable, and possible [proven diagnosis. Probable IPA is confirmed by a combination of clinical and host factors and positive mycological criteria, including cultures or detection of cell wall components. Possible IPA diagnosis is based on the presence of clinical and host features but without positive mycology. However, the aforementioned criteria have been validated in immunosuppressed patients, while in critically ill without classic risk factors this classification has been questioned [probable (“putative”) if there are compatible signs, abnormal medical images and either host risk factors or BAL culture positive for Aspergillus. This simple clinical algorithm has been validated in critically ill patients with histopathologically proven aspergillosis, showing specificity 61% and sensitivity 92%, and might prove to be useful in the IA diagnostics in critically ill patients.The European Organization for Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG) has incorporated the risk factors, clinical signs and laboratory tests in an attempt to stratify the diagnosis of invasive pulmonary aspergillosis (IPA) into possible . Histopaestioned . Therefoestioned . AccordiEntomophtorales and Mucorales. Entomophtorales are rarely implicated in a life-threatening angioinvasive human disease, whereas Mucorales are responsible for mucormycosis, the third commonest invasive fungal infection [Mucorales are traditionally divided in six families: Mucoraceae, Cunninghamellaceae, Saksenacea, Thamnidaceae, Syncephalastraceae and Mortierellaceae. Recently a seventh family, called Absidiaceae, was added. Rhizopus, Mucor, Rhizomucor, Absidia, Apophysomyces, members of the genera of the two families of Mucoraceae-Absidiaceae, are pathogens most commonly implicated in human disease [The class of Zygomycetes is divided in two orders, nfection . Mucoral disease .These organisms are ubiquitous saprophytes in nature rarely infecting organisms with intact immune system. Based on anatomic localization, mucormycosis can be classified in six forms: rhino- cerebral, pulmonary, cutaneous, gastrointestinal, disseminated and uncommon presentations . ClinicaRhizopus species [Sporadic mucormycosis is a life - threatening condition, always associated with certain risk factors, mainly neutropenia and prolonged acidosis of either diabetic or renal origin. Mucormycosis in the ICU setting is related more commonly to massive injuries e.g. from motor vehicle accidents, natural disasters ,96 or co species . Mucorales species has been recently described, but it cannot be routinely used [Aspergillus and Mucorales spp.The diagnosis is based on the examination of tissue samples. Stains of fixed tissues with hematoxylin-eosin, Grocott-methanamine-silver (GMS) or periodic acid-Schiff (PAS) are pathognomonic, showing broad nonseptated hyphae, irregularly branched at angles varying from 45–90° .Vascularely used ,101. TabRecent decades have seen an impressive progress in the development of our therapeutic antifungal armamentarium. Four classes of antifungal drugs are currently available for the treatment of invasive fungal diseases in critically ill patients. They include: (1) polyenes, (2) azoles, (3) echinocandins and (4) pyrimidine analogues.Candida species- with the exception of C lusitaniae and C guillermondii but also Cryptococcus neoformans and the Mucorales.The antifungal spectrum of the drug also includes filamentous fungi especially Aspergillus spp (with the exception of A. terreus) dimorphic fungi such as Histoplasma, Blastomyces, Coccidioidomyces, Paracoccidiodomyces, and emerging yeasts such as Trichosporon spp and Geotrichum spp although Trichosporon strains with high MICs to amphotericin B were detected in some studies [C. glabrata and C. krusei isolates presenting higher minimum inhibitory concentrations (MICs) [A. fumigatus, the species exhibiting usually susceptibility to amphotericin B, has shown an increase in resistance [alternative agents for the treatment of candidaemia [For several decades, amphotericin B deoxycholate (AmB) has been the mainstay of treatment for invasive fungal infections. It possesses a broad spectrum of activity against not only most of  studies . Its eff studies ,104. Dess (MICs) . On the s (MICs) . A. fumisistance . The usesistance  The risksistance ,110 and sistance  but havesistance . Moreovesistance . Infusiosistance . In contdidaemia  due to tdidaemia . This redidaemia . Dreyfusdidaemia .Four azole compounds are available for the treatment of invasive fungal diseases: itraconazole, fluconazole, voriconazole and posaconazole.Aspergillus spp. Once available as oral agent, it is now available in parenteral form as well. The role of itraconazole in critically ill patients with invasive fungal infections (IFI) has not been determined.The older agent of azoles, with a good activity against Candida with the exception of C. glabrata and C. krusei but not against Aspergillus or Zygomycetes. Fluconazole acts by inhibiting an enzyme necessary for the biosynthesis of cell membrane sterol ergosterol [et al have shown that levels of fentanyl and midazolam are increased with the co-administration of fluconazole or voriconazole [Fluconazole is active against most species of gosterol  and althgosterol ,120 it hgosterol . Especiagosterol  Skrobik conazole . ElevatiCandida species including the ones resistant to fluconazole, Cryptococous neoformans, Aspergillus and Fusarium species. Zygomycetes are not susceptible to voriconazole. It is the drug of choice for invasive forms of aspergillosis [A. fumigatus have also been ascribed to voriconazole [Voriconazole is a second-generation member of azole family. It possesses a broad spectrum of activity covering gillosis . It actsgillosis ,127,128.conazole . Adverseconazole ,130 but conazole  though tconazole . A wide conazole ,134. Facconazole ,123. Vorconazole .Candida spp, Aspergillus spp, Cryptococcus and Mucorales. It is available only in oral suspension form  therefore its use in the critically ill is very limited. Posaconazole is given to prevent the fungal infections in neutropenic patients with leukaemia [The newest compound of azoles is active against eukaemia . The comeukaemia .Candida and Aspergillus species. Their mode of action is the inhibition of the synthesis of 1,3-β-glucan, a polysaccharide which maintains the integrity of the cell wall and they are fungicidal in vitro against Candida but fungistatic for Aspergillus [.This is the newest class of antifungal agents developed and they include caspofungin, micafungin and anidulafungin. They possess a limited spectrum of activity covering only ergillus .C. parapsilosis is associated with higher MICs and therefore echinocandins are not recommended for candidaemia caused by these species. Their major advantages are the few or negligible interactions with other drugs, especially for micafungin and anidulafungin and their minor adverse effects  [They are given only intravenously in a slow infusion rate in order to avoid the rare infusion - related reactions. Since the active drug is not excreted into the urine their use in candiduria is not suggested. Moreover, the level of echinocandins in special compartments such as cerebrospinal fluid and intraocular compartment remains low. actions) . Differeactions) ,141,142 A significant advantage of echinocandins is that being fungicidal they offer much more rapid resolution of symptoms with fewer complications when compared to fluconazole . Due to  or other severe mycosis such as cryptococcosis, aspergillosis and chromoblastomycosis [Flucytosine is the main representative of the class. It is available only as oral formulation in USA but also in intravenous form in other countries and it is mostly used in combination with AmB for special forms of invasive candidiasis omycosis . Newer drugs under investigation include isavuconazole, ravuconazole and albaconazole while a human recombinant monoclonal antibody  has been used in combination with AmB showing favorable results ,145.Candida species and susceptibility tests [albicans species; prior treatment with fluconazole; site of infection; spectrum of activity; known adverse effects; pharmacodynamics /pharmacokinetics; cost of treatment; but the most important factor is the presence of haemodynamic instability [C. glabrata, echinocandins are preferred to fluconazole. Amphotericin B is characterized as an alternative choice in case of intolerance to the other two, refractory infection, resistant organism or suspicion of infection due to pathogens other than Candida (e.g. cryptococcus). This is a basic difference from the guidelines of 2004 where AmB was a first choice. A step-down from echinocandin to fluconazole is suggested provided there is a clinical improvement and sterilisation of blood cultures. The recent guidelines of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID 2011) [Candida sepsis are suggested also by Kullberg et al. [C. parapsilosis) an echinocandin is preferred. Finally the German speaking Mycological Society and the Paul – Ehrlich – Society for Chemotherapy suggest the use of echinocandins or liposomal amphotericin B in critically ill septic patients instead of fluconazole [The time of treatment initiation is a key factor for the favorable outcome of invasive candidiasis. Several studies have shown that delay of initiation of appropriate antimicrobial therapy over 24 or 48 h has a negative impact on mortality ,147,148.ty tests . Importatability ,150. Acctability . NeverthID 2011)  endorse g et al.  while thg et al. . Moreoveconazole .Candida is common or at least not negligible, empirical treatment with azoles cannot be recommended. Local and general resistance patterns are often published [An important parameter is that every institution should be aware of its resistance rates since in case that resistant ublished ,156 helpCandida spp supports this view. In case of catheter-related bloodstream infection, an antifungal drug with biofilm action is preferred (AmB or echinocandin). Finally, a fundoscopic examination is of pivotal importance in order to exclude disseminated endocular infection. If a form of localised candidiasis has to be treated, a longer treatment is suggested and the recommended agents are shown in Further important considerations for the practitioner include: (a) the length of therapy (b) the removal of central catheters (c) the fundoscopic examination. Serial blood cultures should be drawn after the start of treatment in order to ascertain blood sterilization. Duration of treatment is defined to 14 days after the last positive blood culture ,150,154.Combination of amphotericin B with flucytosine is recommended in cases of localised infection such as meningitis, osteomyelitis and intra-abdominal infections .Because the mortality associated with invasive candidiasis is high while the sensitivity of blood cultures is low other management options exist, including prophylactic, pre–emptive and empirical therapy.albicans species. A more judicious approach should include the careful validation of patients at risk taking into consideration local data on rates of different Candida species.Prophylaxis is defined as the administration of antifungal agents to high-risk patients without signs or symptoms of infection in order to prevent the development of invasive fungal infection. The agent given systematically for that purpose is mainly fluconazole while recently the use of echinocandins has been successfully tested . AlthougCandida colonisation, presence of gastrointestinal surgery or use of total parenteral nutrition are among the risk factors suggested for use in such cases [d-glucan was used for identifying ICU patients at highest risk to develop an IFI and for monitoring treatment response [Initiation of antifungal agents in the presence of multiple risk factors constitutes pre-emptive treatment. Prolonged ICU stay, use of broad-spectrum antimicrobials, multi-focal ch cases ,172. Thech cases . In a reresponse . NeverthCandida infection is due to NAS and increasing costs [If antifungal treatment is given to a patient with clinical signs of infection  and several risk factors for candidaemia without proof of invasive candidiasis, the term “empirical” may be used. Moreover this term can be applied if antifungal treatment is given to a patient with candidiasis proven by blood culture pending the results of susceptibility tests. In the first case the criteria for therapy initiation are vague in contrast to neutropenic patients and the benefits are not well established. The largest conducted study is the one by Shuster conducted in a SICU. No clear benefit of fluconazole use was proved in that study . IDSA gung costs . Canadiang costs . Fluconang costs . Figure Aspergillus spp and the drug’s side effects have limited its use for the treatment of IPA [Even a clinical suspicion of IPA should lead to the consideration of initiation of antifungal treatment, since the mortality of the disease remains high. AmB was the antifungal of choice for many years. However, the reported development of resistance among t of IPA . Lipid-bt of IPA .The treatment of choice for IA is voriconazole . The recAspergillus spp [. Combination of azoles and echinocandins might be used in refractory cases of IA [Other alternative drugs for the refractory cases of IPA are the echinocandins. Caspofungin has shown favorable results in patients with IA refractory to first line antifungals . Micafunllus spp . Combinaes of IA . The comes of IA ,181. Addes of IA .A.terreus shows primary resistance to AmB while similar susceptibility results have been demonstrated for several species of the Fumigati group [A. fumigatus possibly due to genotypically determined resistance mechanism. Cross-resistance to triazole has also been reported. According to IDSA guidelines, itraconazole should not be used for treatment of IPA refractory to voriconazole because of the possibility of cross-resistance and the subsequent toxicity [The duration of IPA treatment might last from several months to more than one year and should be tailored to patients’ response ,182. Cliti group ,180. Multoxicity . A reduction of the degree of immunosuppression by immunomodulatory therapy is a possible adjunctive factor to successful therapy of IPA. According to IDSA guidelines neutropenic patients with IPA might benefit from the administration of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) . There aAdditional measures for prevention of IPA are the use of high – efficiency particulate air filtration (HEPA) and the avoidance of hospitalisation in areas with construction procedures . RegardiMucorales spp. requires several simultaneous approaches: aggressive (sometimes disfiguring) surgical intervention, antifungal drug therapy and management of all underlying medical conditions that might predispose the patient to the disease. On diagnosis LipAmB should be started at a dose 5–10 mg/kg once daily. The maximal dose is indicated for mucormycosis with intracerebral extension. Posaconazole, a new triazole antifungal agent, is currently considered as a second-line drug for the treatment of mucormycosis at a dose of 400 mg twice daily. Its use is recommended in combination with LipAmB or as sequential long-term therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperbaric oxygen have been used adjunctively in neutropenic patients offering doubtful results [The treatment of  results .Invasive fungal infections in the critical care setting often constitute a challenging diagnostic and therapeutic problem. Clinical awareness, knowledge of local epidemiology and pharmaceutical considerations are factors of paramount importance for an early diagnosis and treatment of these potentially lethal infectious diseases.


The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.In immunocompromised patients, reactivation of latent BK polyomavirus (BKPyV) can cause disease with lytic infections of the kidneys and the lower urinary tract. Emerging evidence also links BKPyV to oncogenesis and high‐grade intrarenal and transitional cell carcinomas. These neoplasms strongly express polyomavirus large‐T antigen as a defining feature; that is, they are ‘large‐T‐positive carcinomas’. Such neoplasms arise in immunocompromised patients, typically in renal allograft recipients, and preferentially in tissues harbouring latent BKPyV. In recent articles in this journal, it was shown that tumour cells harbour replication‐incompetent clonal BKPyV. The virus can be truncated and randomly integrated into the genome, and/or it can be mutated in an episomal state. Truncation and/or deletions in the BKPyV non‐coding control region can hamper late viral gene expression, replication, and cell lysis, while facilitating overexpression of early genes, including that encoding large‐T. Biologically active fusion proteins or alterations in human tumour suppressor or promoter function have not been described so far, making uncontrolled large‐T gene expression in non‐lytically infected cells a prime suspect for neoplastic transformation. Current concepts of BKPyV‐induced disease, including recent reports from this journal, are discussed, and evolving paradigms of BKPyV‐associated oncogenesis are highlighted. © 2018 The Authors.  Lancet suggested that BKPyV was in search of a disease Polyomaviruses, including the BK polyomavirus (BKPyV) strain, are ubiquitous, and 40 years ago an editorial in the Well studied are BKPyV infections that start as clinically insignificant primary events in young individuals. After primary infection, the virus is usually not completely cleared from the body, but rather establishes latency, mainly in the urogenital tract , but also in other tissues and cells, such as tonsils, lymphocytes, blood mononuclear cells, and the brain. Latent BKPyV is usually in an episomal state and escapes standard morphological detection, necessitating the use of molecular techniques. Beyond infancy, latent infections can be found at any age, possibly driven by low‐level subclinical sporadic replication. By using polymerase chain reaction testing, we found latent BKPyV in the renal parenchyma (34%) and urothelium (42%) of asymptomatic adult patients .Under immune modulation, latent BKPyV can be reactivated and enter into a replicative cycle in permissive tissue. During the replicative cycle, early viral genes, including the large‐T gene, are expressed initially  in two neoplasms (each strain restricted to one tumour). Chapel Hill BKPyV 1 and 2 were linearised, truncated at viral capsid protein‐encoding sites, and randomly integrated into the tumour genome at a single locus. No BKPyV was found in adjacent non‐neoplastic parenchyma. Viral truncation with deletions in late gene sequences not only rendered Chapel Hill BKPyV 1 and 2 replication incompetent, but presumably also disrupted negative large‐T‐controlling feedback loops, resulting in unregulated and persistent expression of early viral genes. Overexpression of large‐T was presumably further promoted by deletions in the NCCR domain of Chapel Hill BKPyV 1 and resulting alterations in NCCR regulatory functions . No role in oncogenesis could be attributed to host gene integration or novel bioactive fusion proteins. Müller et al also described a linearised and truncated BKPyV randomly integrated at a single locus into the tumour genome of a micropapillary metastatic large‐T antigen expressing urothelial carcinoma of donor origin. Similarly to the observations of Kenan et al, truncation was noted in late viral gene sequences, rendering the integrated virus replication incompetent. What is especially intriguing is that Müller et al additionally detected episomal full‐length BKPyV that was not replicating because of a short, specific 17‐bp deletion in the NCCR P‐block impairing late but not early viral gene expression. As the same NCCR deletion was noted in the integrated viral gene sequence, the authors speculated that, during neoplastic transformation, mutation of the episomal BKPyV had occurred first, followed by truncation and chromosomal integration. Sole episomal mutated BKPyV without integration into the tumour genome was seen in one of our recently diagnosed high‐grade large‐T‐positive renal cell carcinomas. This tumour‐associated full‐length BKPyV showed deletions in the R‐block and S‐block and duplications of the O‐block, P‐block and Q‐block of the NCCR, promoting early and hampering late viral gene expression. In contrast to the report of Müller et al, however, no deletions were found in the NCCR P‐block . These modern‐era data are in line with those from older studies using electrophoresis and Southern blot techniques Three seminal publications by Kenan All studies combined show a common scheme in BKPyV‐associated cancer Figure ; Table 1et alCurrent observations, based on experience with simian virus 40 (SV40) and Merkel cell polyomaviruses, have culminated in the following hypothetical model(s) of BKPyV‐associated carcinogenesis Figure . In permet alet alRecently collected evidence on the role of BKPyV as a neoplastic driver is compelling. Seminal observations made by Müller All authors contributed to writing the manuscript.


Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population. Streptococcus suis is a major veterinary pathogen, causing a range of chronic and severe diseases in pigs such as arthritis and septicaemia   within a gene or promoter, or by the shuffling of sequences by homologous recombination between expressed and silent loci via inverted repeats (IRs) present in these loci . Phase vregulon) . These sfluenzae , Moraxelarrhalis , pathogearrhalis  and Streeumoniae . All thensferase . In contnsferase . All phansferase . This haStreptococcus suis isolates were grown on Brain-Heart Infusion (BHI) agar or Sheep-blood agar (Oxoid), with liquid cultures grown in Todd-Hewitt broth containing 0.2% yeast extract (Oxoid) at 37°C. Escherichia coli DH5α and BL21 were grown in Luria–Bertani media supplemented with 100 μg/ml ampicillin where appropriate, at 37°C with 200 rpm shaking. Prototype S. suis strains used in this study are detailed in Table Standard molecular biology techniques were used throughout . GenomichsdS in S. suis strain S735 were quantified utilising FAM labelled PCR coupled to a restriction digest in a method similar to that used previously for S. pneumoniae (hsdS locus was PCR amplified (3.02 kb) from extracted genomic DNA prepared from an entire plate of S. suis strain S735 grown overnight on BHI agar. PCR was carried out using GoTaq DNA polymerase (Promega) according to the manufacturer’s instructions, and utilising primers that bind outside the area of recombination (Ssu-T1-For-FAM and Ssu-T1-Rev). The PCR products were then digested in a double-digest with DraI and NciI (New England Biolabs). This digestion was predicted to yield different sized FAM-labelled fragments for each of the variant forms. The pool of restriction fragments was run on an ABI prism Gene Analyser (Life Technologies). The area of the peak given by each labelled fragment, each corresponding to the prevalence of one of the variant forms, was quantified using Peak Scanner v1.0.The variant alleles of eumoniae . The whoS. suis strain grown over-night on BHI agar.Fragment length analysis of the phase-variable Type III methyltransferase repeat tract was conducted using the fluorescently labelled forward primer SsuT3-F-FAM and the reverse primer SsuT3-R, and fragments were analysed by the Australian Genome Research Facility . PCR was carried out using cell suspensions using the relevant S. suis strain S735 using cells grown overnight on BHI plates, so as to replicate the growth conditions used for the Type I allele quantification assay , SS1028 (modS2) and YS77 (modS3). A vector containing an non-methyltransferase, siaB, used previously  to a final concentration of 0.5 mM overnight at 37°C with shaking at 200 rpm.PCR products for cloning into the NcoI/BamHI site of pET15b cloning vector (Merck) were prepared using KOD Hot-start DNA polymerase (Merck) according to manufacturer's instructions. Primers specific for the Type III system were designed to clone the gene containing only one GAGCA repeat so as to lock-on the methyltransferase (SsuT3-oE-F and SsuT3-oE-R). We cloned the three unique eviously  served aE. coli cells expressing each methyltransferase and a negative control expressing an the non-methyltransferase siaB, described previously (S. suis strain LSS66 was prepared from an overnight culture in Todd Hewitt broth (Oxoid) +0.2% Yeast Extract (Fisher Scientific) and cells lysed according to the Genomic DNA Handbook (Qiagen) using 100 mg/ml Lysozyme (Sigma) supplemented with 250 units/ml Mutanolysin (Sigma). High-molecular-weight genomic DNA was isolated using Genomic-tip 100/G columns (Qiagen) according to the manufacturer's instructions and DNA resuspended in 10 mM Tris–Cl, pH 8.5 (Qiagen) overnight at 4°C. A phenol:chloroform:isoamylalcohol (Sigma) clean-up was then performed before finally resuspending DNA in 10 mM Tris–Cl, pH 8.5 (Qiagen) overnight at 4°C. SMRT sequencing and methylome analysis was carried out as previously  containing 5% (v/v) sheep blood  as described previously (hsd region from S. suis P1/7 (Genbank locus tags SSU1271-SSU1274) and the mod locus from S. suis T15 (T15_1305) and used blastn against a custom blast database containing the collection and published complete genomes (S. suis strain P1/7 and using these as a tblastn query against a custom blast database containing the collection and published complete genomes (S. suis P1/7 (Genbank ID: AM946016) and which were present in every isolate were included in the core genome. A neighbour-joining tree of S. suis was made from this core genome using a pairwise distance matrix created with the ‘K80’ model  . This analysis examined the presence of both phase-variable Type I and Type III methyltransferases within S. suis genomes. Of these 393 S. suis strains, 262 strains contained a phase-variable Type I system, characterized by duplicate, variable hsdS loci containing IRs  . We alsoS. pneumoniae  of Type I alleles present in the expressed hsdS locus of the Type I system consist of 5′-TRD-1 and 3′-TRD-2, which we have designated allele B. Only two strains contain allele A, consisting of 5′-TRD-1 and 3′-TRD-1. No strains contained allele C (5′-TRD-2 and 3′-TRD-2) or allele D (5′-TRD-2 and 3′-TRD-1) in the expressed hsdS locus immediately downstream of the hsdM gene. However, this apparent lack of either allele C or allele D in the expressed hsdS locus of S. suis genomes could be a result of the genome annotation process – minority alleles do exist in a population but this locus will only be annotated as the majority allele in the final sequence annotation. In order to investigate if these duplicate, ‘tail-to-tail’ hsdS genes could undergo homologous recombination to produce variable hsdS sequences in the expressed locus immediately downstream of the hsdM gene, even in the absence of a locus-associated recombinase, we designed a fluorescent PCR reaction coupled to a restriction digest in order to type and quantify the alleles present within a population of S. suis  in the expressed hsdS locus , 5.14% of the population hsdS allele C (5′-TRD-2 and 3′-TRD-2)  , methyltransferase (HsdM) and specificity (HsdS) components Figure , with theumoniae , we demoi Figure , and tha′ Figure . Analysis Figure –E. Usings Figure  and E, c) Figure  and E, a) Figure  and E. Tallele B , and thaallele B . This deS. suis also demonstrated the presence of two additional Type I R-M loci (YYK_03085-YYK_03100 and YYK_07615-YYK_07625 in strain S735). Previous in depth analysis of Type I R-M systems in S. suis has shown that homologues of these loci are distributed across the majority of S. suis strains , with high diversity observed in the central TRD (<25% nucleotide identity), which dictates the DNA sequence to be methylated (mod gene with 5′-GAGCA(n) repeats, 29 contain an identical gene to the one we identified in a single strain of S. suis, strain T15, in our systematic study of all SSR-containing Type III mod genes in REBASE  repeat tract occurs in these genes, leading to variable expression of the mod gene, we performed FAM-labelled PCR coupled to fragment length analysis, over the repeat tract of the alleles identified in strains LSS89 and SS1028, as we have used previously to characterize and quantify expression of phase-variable genes  repeat tract length between each of the 41 Type III mod genes in our S. suis strain collection; this is also an excellent indication that phase-variable expression of this gene is occurring  sequencing and methylome analysis using plasmid DNA isolated from E. coli BL21 over-expressing each allelic variant demonstrated that these three alleles all methylate different DNA target sequences ATD-3′  methylates the sequence 5′-VTC(m6)ATC-3′ ANNNB-3′ ATC-3′) occurs in all three vectors, as would be expected in DNA isolated from E. coli , modS2 (SS1028) and modS3 (YS77) . This low number of repeats is highly stable, with low rates of phase-variation predicted based on previous analyses of SSR length and phase-variation rate ATC-3′  , with sy3 (YS77)  of S.SsuodS1 LSS8, modS2  immediately upstream of a putative Type III mod gene. However, closer examination of this region showed that a functional Type III mod gene containing SSR’s only occurred if a unique 119 bp sequence was present between the SSRs and the putative mod gene  repeats were present, sometimes in very high numbers  based on a Bayesian clustering method . Only strains T15 (Netherlands) and R61 (China) were not isolated in the UK. Interestingly, all strains from BAPS group 2 contained neither phase-variable methyltransferase, with a large proportion of BAPS group 4  also containing neither system , but that the phase-variable Type III methyltransferase has either been independently acquired at least three times in different populations, or alternatively been lost multiple times from isolates of S. suis.Our previous studies with g method  as well g et al. ). Upon eg et al.  and the g et al. . The phag et al. , and serm humans . All memm Figure . In summin silico analysis due to the genetic features associated with them , and thus exclude them as stably expressed antigens, the identification of members of a phasevarion can only be accomplished by studying the strains that contain phase-variable methyltransferases , the three minor variants  could all be detected using our bespoke FAM-labelled PCR assay  and modS2 (strain SS1028)  Figure . Phase-v) Figure , althoug) Figure ,15,46–48modS allelic variants present in S. suis  repeats in the genomes of our S. suis strain collection, but these are not always linked to the downstream mod gene due to the apparent requirement of the observed 119 bp ‘linker’ sequence we observed in the 41 strains containing a phase-variable Type III mod gene, it may be that acquisition of a phase-variable Type I locus leads to subsequent inactivation of the Type III mod gene by loss of the 119 bp ‘linker’ required for phase-variation of the Type III locus, but this would require subsequent investigation to confirm.Our demonstration of a distinct lineage-specific segregation of phase-variable methyltransferases in vergence . In the and modD , and strod genes ,39. The t et al.  but agaiS. suis leads to the possibility of multiple phasevarions in this species. The presence of phasevarions complicates the development of vaccines and therapeutics, as the stably expressed protein profile of S. suis can only be defined by dissecting which genes are controlled by phasevarions in this species.Our demonstration of the presence of several unique phase-variable methyltransferases in Supplementary DataClick here for additional data file.


Cation [3]4+ features four pairs of olefins from the two tetrakis‐NHC ligands perfectly arranged for a subsequent [2+2] cycloaddition. Irradiation of [3](BF4)4 with a high pressure Hg lamp connects the two tetra‐NHC ligands through four cyclobutane linkers to give compound [4](BF4)4. Removal of the template metals yields the novel oktakisimidazolium salt H8‐5(BF4)8. The tetrakisimidazolium salt H4‐2(BF4)4 and the oktakisimidazolium salt H8‐5(BF4)8 have been used as multivalent anion receptors and their anion binding properties towards six different anions have been compared.The tetrakisimidazolium salt H Polyimidazolium cations: The octakisimidazolium salt H8‐5(BF4)8 has been obtained from two equivalents of tetrakisimidazolium salt H4‐2(BF4)4 in a template synthesis via the tetranuclear octakis‐NHC complex [Ag4(2)2] followed by postsynthetic irradiation and formation of four cyclobutane linkers between the two tetra‐NHC ligands and demetallation. The binding of selected anions by H8‐5(BF4)4 has been studied. N‐heterocyclic carbene (NHC) donors have appeared as promising ligands for the design of metallosupramolecular assemblies that can be utilized for PAM processes. Due to the stability of the M−CNHC bond, poly‐NHC‐derived metallosupramolecular architectures can be as stable or even more stable than supramolecular architectures derived from Werner‐type ligands.N‐olefin‐substituted poly‐NHCs have recently been developed as an effective PAM strategy for the connection of poly‐NHC ligands.C3‐symmetric polyimidazolium precursors, respectively. The preparation of these cage‐type polyimidazolium salts is of particular interest, given that imidazolium salts are considered privileged anion receptors due to their strong affinity to anions through (C−H)+⋅⋅⋅X− interactions, which combine hydrogen bonding with favourable electrostatic interactions.Coordination‐driven self‐assembly is currently one of the most effective strategies for the rational construction of discrete supramolecular coordination complexes (SSCs).Multivalent binding interactions between a host/receptor and a guest requires that the receptor possesses more than one binding site connected through suitable spacers.4‐2(BF4)4, 1,2,4,5‐tetrakis(diethoxyphosphinylmethyl)benzene1 featuring four internal olefin groups. In a subsequent reaction, compound 1 was alkylated at the imidazole nitrogen atoms using 1‐bromobutane to give H4‐2(Br)4. Anion exchange yielded finally the tetrakisimidazolium salt H4‐2(BF4)4 (Scheme 4‐2(X)4  were fully characterized by NMR spectroscopy, mass spectrometry and (for H4‐2(Br)4) by elemental analysis (see the Supporting Information).For the preparation of tetrakisimidazolium salt H4‐2(Br)4 with a slight excess of Ag2O followed by addition of AgBF4 yielded the sandwich‐type assembly [3](BF4)4 in good yield of 86 % (Scheme 3](BF4)4 was confirmed by 1H and 13C{1H} NMR spectroscopy. The resonances for the olefin protons were detected at δ=7.54 and 7.31 ppm  in the 1H NMR spectrum, only slightly shifted upfield compared to the equivalent resonances in H4‐2(BF4)4 . A characteristic doublet resonance for the CNHC carbon atoms at δ=178.1 ppm (1JAgC=149 Hz) was found in the 13C{1H} NMR spectrum. The strongest peak in the electrospray ionization mass spectrum  also confirmed the formation of the cationic assembly [3]4+ (see the Supporting Information). Transmetallation of the tetrakis‐NHC ligand from [3](BF4)4 to gold(I) in [6](BF4)4 was achieved by reaction of [3](BF4)4 with [AuCl(tht)] 4 suitable for an X‐ray diffraction study could not be obtained in spite of multiple attempts. However, stirring of an acetonitrile solution of [3](BF4)4 with four equiv of NaBPh4 in acetonitrile led to the isolation of the mixed anion salt [3](BF4)2(BPh4)2. Single crystals of [3](BF4)2(BPh4)2⋅4CH3CN were grown by slow diffusion of diethyl ether into a saturated solution of [3](BF4)2(BPh4)2 in acetonitrile. An X‐ray diffraction analysis with crystals [3](BF4)2(BPh4)2⋅4 CH3CN confirmed the formation of the sandwich‐type complex cation [3]4+ , Ag1⋅⋅⋅Ag1* 15.025(1) and Ag2⋅⋅⋅Ag2* 16.626(1) Å. The average Ag−CNHC bond length of 2.048 Å and the average N‐CNHC‐N angle of 102.0° fall in the typical range for Ag‐NHC complexes.centroid⋅⋅⋅Ccentroid measures 3.519 Å. The olefin units of the two tetrakis‐NHC ligands are arranged in pairs with the double bonds aligned in an almost parallel fashion. The average distance of the midpoints of the C=C bonds in each pair measures 3.6 Å which is a perfect separation for a subsequent [2+2] cycloaddition reaction.Crystals of [+ Figure . The tet3]4+, a post‐synthetic modification on the cation via a photochemically induced [2+2] cycloaddition reaction was investigated next. To this end, a sample of compound [3](BF4)4 was dissolved in acetonitrile. This solution was irradiated with a high pressure mercury lamp leading to conversion of [3](BF4)4 into complex [4](BF4)4 featuring four cyclobutane units as linkers between the two tetrakis‐NHC ligands (Scheme 4](BF4)4 was quantitative and stereospecific as confirmed by 1H and 13C{1H} NMR spectroscopy 4 features no resonances for the olefin protons anymore, but two new resonances at δ=5.32 and 5.18 ppm for the protons of the newly formed cyclobutane rings.Given the favourable orientation of the olefin groups in cation [ induced +2 cycloa3](BF4)4, crystals for an X‐ray diffraction study of [4](BF4)4 could not be obtained. However, stirring of an acetonitrile solution of [4](BF4)4 with 4 equiv of NaBPh4 lead, after addition of THF and cooling, to the precipitation of crystals of composition [4](BPh4)4 which were suitable for an X‐ray diffraction analysis.As was observed with salt [4](BPh4)4  and 15.119(2) Å. The average Ag−CNHC bond lengths and the average N‐CNHC‐N angle do not differ significantly from the equivalent parameters in [3]4+. The formation of the four cyclobutane rings in [4]4+ leads to a shortening of the distance between the midpoints of the two central benzene rings from 3.519 Å in [3]4+ to 2.837 Å in [4]4+. In addition, the central benzene rings are bent in a convex manner relative to the cyclobutane linkers 4 generated a precipitate of AgCl and the octakisimidazolium salt H8‐5(Cl)8. Anion exchange with NH4BF4 yielded H8‐5(BF4)8 in an overall yield of 76 % (Scheme 8‐5(BF4)8 was characterized by 1H and 13C{1H} NMR spectroscopy and by mass spectrometry (see the Supporting Information). The resonances at δ=9.70 and at δ=134.7 ppm in the 1H and 13C{1H} NMR spectra, respectively, confirmed the demetallation and formation of the octakisimidazolium salt. The resonances for the imidazolium H2 and C2 atoms fall, as expected, in the range previously recorded for the related resonances in the tetrakisimidazolium salt H4‐2(BF4)4. The resonances for the cyclobutane protons in cation H8‐58+ were detected as two singlets at δ=5.10 and 5.19 ppm, only slightly shifted upfield from the equivalent resonances in [4](BF4)4 (δ=5.32 and 5.18 ppm). The ESI mass spectrum shows strong peaks for various ions [H8‐5+nBF4](8−n)+ with the strongest peak (100 %) recorded at m/z=355.3715 .Addition of NH% Scheme . Salt H83](BF4)4 undergoes a transmetallation reaction with [AuCl(tht)] to give the tetranuclear gold(I) complex [6](BF4)4 in 70 % yield (Scheme 6](BF4)4 was confirmed by 1H and 13C{1H} NMR spectroscopy (δ(CNHC)=180.3 ppm, see the Supporting Information). The ESI mass spectrum shows the strongest peak (100 %) at m/z=684.5022 . Gold complex [6](BF4)4 also reacts quantitatively upon irradiation (high pressure mercury lamp) in a [2+2] cycloaddition reaction to give compound [7](BF4)4 featuring four cyclobutane linkers between the two tetra‐NHC ligands. Alternatively, compound [7](BF4)4 can be generated via a transmetallation reaction from silver complex [4](BF4)4 and [AuCl(tht)] in 59 % yield. Gold compound [7](BF4)4 was fully characterized by 1H and 13C{1H} NMR spectroscopy (δ(CNHC)=184.1 ppm) and by ESI mass spectrometry (see the Supporting Information). While gold complex [7](BF4)4 is easily accessible, it is a less suitable precursor for the liberation of the tetrakisimidazolium salt H8‐5(BF4)8 since the Au−CNHC bonds are less labile compared to the Ag−CNHC bonds. This causes problems in the liberation of the H8‐5(BF4)8 from [7](BF4)4.The tetranuclear silver(I) complex [d Scheme  similar 4‐2(BF4)4 and H8‐5(BF4)8 offers a good opportunity to compare their capabilities as multivalent anion receptors. The recognition and binding properties of the anions in the two salts were studied by 1H NMR titration experiments monitoring selected proton signals of the receptors upon addition of the tetrabutylammonium salts of the investigated anions. All titrations studies were carried out in [D6]DMSO. For the study we selected six anions of different geometry and charge density, namely chloride, bromide, nitrate, benzoate, adenosine triphosphate (ATP−) and 2‐(4‐isobutylphenyl)propionate (IBF−). The last two anions were selected in an effort to demonstrate the relevance of the two polyazolium salts as receptors of anions with biological and medical interest (2‐(4‐isobutylphenyl)propionic acid is ibuprofen, H‐IBF). Generally, addition of solutions containing the anions induced significant perturbations in the 1H NMR spectra of the polyimidazolium hosts.The preparation of the polyimidazolium salts H1H NMR spectra of H8‐5(BF4)8 upon titration with [NBu4+](IBF−). The spectra illustrate how the resonance for the eight equivalent acidic imidazolium protons (H2) is shifted progressively downfield upon addition of increasing amounts of IBF−. Together with this, one of the resonances due to the remaining protons of the imidazolium ring (H5) is slightly downfield shifted, while the resonance for the other one (H4) moves slightly upfield 4 as the host, the stoichiometry of the host:guest complexes formed was best fitted to a 1:2 stoichiometry (two anions bound to the tetrakisimidazolium receptor). This assumption was based on the analysis of the binding isotherms resulting from the titrations of host H4‐2(BF4)4 with all six anions. In all cases, the 1:2 stoichiometry gave the best distribution of residuals, compared to a 1:1 stoichiometry.The determination of the binding constants between the anions and H8‐5(BF4)8, the Job plot analyses suggested a stoichiometry that could vary between 1:3 or 1:4 . We are perfectly aware that the Job plot analysis has serious limitations, particularly when referred to models of high stoichiometry,4‐2(BF4)4, the 1:4 stoichiometry in which two arms of the receptor are bound to each anion seems to be the most plausible one.For the experiments performed with H8‐5(BF4)8 due to the limitations of the regression analysis used to process the data obtained from 1H NMR titrations. For the determination of the constants we also considered two different variants of the 1:2 binding model, depending on whether the two stepwise binding constants are linked , or not .For the determination of the binding constants, a 1:2 stoichiometry model was used for both the tetrakisimidazolium and the octakisimidazolium salts. The 1:4 (or 1:3) models were not used for evaluating the titrations with H4‐2(BF4)4, did not differ much regardless of whether we used a cooperative or non‐cooperative binding model (entries 1–6). This allows us to assume that the binding of the anions followed a non‐cooperative binding model, and this was the model that we used for the determination of the association constants when the octakisimidazolium salt H8‐5(BF4)8 was used (entries 7–12). The data reflect that the affinities shown for chloride are higher than that shown for bromide, in agreement with the larger basicity of the former one. Both receptors show larger affinities for carboxylates and ATP−. The two carboxylate anions (benzoate and IBF−) exhibited a rather similar affinity and the binding constant shown for ATP− was the largest found for both receptors, therefore showing large selectivity for this anion. The larger affinities observed for phosphate anions when polyazolium receptors are used have been observed before.8‐5(BF4)8 were between 3–8 times larger than those obtained when H4‐2(BF4)4 was used.As can be seen from the data in Table 8‐5(BF4)8 from two tetrakisimidazolium building blocks H4‐2(BF4)4. The intermediate octakis‐NHC complex [4](BF4)4 with four cyclobutane linkers features two non‐planar central benzene groups. The tetrakis‐ and the octakisimidazolium salts were tested as multivalent receptors of six different anions, including two with relevant biological and medical significance (ATP− and IBF−). From our study, we concluded that the octakisimidazolium salt exhibited a larger binding affinity for all six anions tested. Given that we did not find reasons to conclude that the stepwise binding of the anions followed a cooperative model, we believe that the enhancement of the binding affinity should be ascribed to the larger electrostatic attraction produced between the anionic guests and the octacationic octakisimidazolium receptor compared to the tetracationic tetrakisimidazolium one. With our work we proved that the template‐controlled preparation of polyimidazolium salts offers a unique opportunity to generate multivalent receptors with enhanced recognition abilities.In summary, we have demonstrated the template synthesis of the novel octakisimidazolium salt HFull details of synthesis and characterisation can be found in the Supporting Information.3](BF4)2(BPh4)2⋅4 MeCN), and 1972487 ([4](BPh4)4) Deposition Numbers 1972486  should be addressed to the authors.SupplementaryClick here for additional data file.


Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are common intestinal protozoans that infect humans and animals worldwide. A survey that assessed the prevalence, molecular characteristics, and zoonotic potential of these pathogens was conducted on a variety of dogs in Guangzhou, southern China. A total of 651 canine stool samples from household (n = 199), shelter (n = 149), breeding (n = 237), and pet market dogs (n = 66) were collected from eight districts in Guangzhou. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. were detected by PCR amplification of the SSU rRNA gene. Giardia duodenalis-positive specimens were further assigned into assemblages using the glutamate dehydrogenase gene. Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were found in 21 (3.2%), 20 (3.1%), and 35 (5.4%) samples, respectively. The overall prevalence of shelter dogs  was significantly higher than that of household , breeding , and pet market dogs  . Deworming in the past 12 months had a strong protective effect on the risk of contracting parasite infections (P < 0.001). No significant differences were detected between age or sex groups (P > 0.05). Dog-specific C. canis (n = 19) and zoonotic C. parvum (n = 2) were the only two Cryptosporidium species. Sequence analysis revealed the presence of three G. duodenalis assemblages: dog-specific assemblages D (n = 14) and C (n = 5), and cat-specific F (n = 1). Zoonotic Blastocystis ST3 (n = 28) was the dominant subtype, followed by ST1 (n = 6) and ST10 (n = 1). To our knowledge, this is the first large-scale investigation on the occurrence and molecular characteristics of Blastocystis sp. in dogs in China. Our results indicated that the dogs seemed to play a negligible role as reservoirs for Cryptosporidium spp. and G. duodenalis transmission to humans, but they are potential novel suitable hosts of Blastocystis sp. A strict sentinel surveillance system of dogs should be established to minimise the zoonotic risk of spreading blastocystosis among humans and dogs. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are cosmopolitan enteric protists with a wide range of hosts, including humans, non-human primates, companion animals, ruminants, birds, and wild mammals3. Although the pathogenicity of Blastocystis sp. is under strong debate, it may also be associated with gastrointestinal disease-causing agents like Cryptosporidium spp. and G. duodenalis, which cause issues such as self-limiting diarrhoea, abdominal pain, irritable bowel syndrome, and flatulence5. In particular, people with compromised immune systems (e.g. AIDS patients and organ transplant recipients) are susceptible to these infections7. Infections occur mainly by faecal–oral transmission after ingestion of infective forms (oocysts or cysts), usually via water, food, or direct contact8. Cryptosporidium parvum, C. hominis, C. meleagridis, C. canis, and C. muris are the five most common human pathogenic species of Cryptosporidium, of which C. canis is the most prevalent species in dogs11. G. duodenalis consists of eight distinct assemblages or genotypes (A–H). Assemblages A and B have a wide host range and are responsible for the majority of known human disease cases, whereas assemblages C–H seem to be host-specific for non-human species1. Dogs are predominantly infected by assemblages C and D14. Additionally, at least 17 distinct Blastocystis subtypes were identified. ST1–9 and ST12 are the common zoonotic subtypes, and ST10–11 and ST13–17 only infect non-human species15.Cryptosporidium spp., G. duodenalis, and Blastocystis sp. in dogs have been conducted worldwide, and numerous species/assemblages/subtypes have been detected, such as C. canis, C. parvum, C. muris, C. meleagridis, C. hominis, G. duodenalis assemblages A–F, and Blastocystis ST1–ST6 and ST1014  and companion animals (i.e. > 10.62 million pets in the city in 2015) . To date, one study of Cryptosporidium spp. and three molecular epidemiological studies of G. duodenalis have been published on dogs in the region36; however, nothing is known about the occurrence and molecular characterisation of Blastocystis sp.. The purpose of our study was to estimate the overall occurrence of Cryptosporidium spp., G. duodenalis, and Blastocystis sp. in dogs living in areas of Guangzhou and assess the zoonotic potential between humans and dogs.As intimate companions, dogs have close contact with humans. However, dogs often harbour intestinal protozoa, which can cause mild to severe disease in dogs and lead to zoonotic infections in humans. Among these protozoa, 2 and has a population of about 140 million. The annual average temperature is 20–22 °C and the average relative humidity is 77% . A total of 651 fresh faecal samples were randomly collected from 199 household dogs , 149 shelter dogs (from two shelters located in suburban Luogang and Huangpu Districts), 237 breeding dogs (from two breeding centres located in suburban Conghua and Nansha Districts), and 66 pet market dogs (from one pet market in urban Tianhe District), with or without a history of illness, on a single occasion between January and December 2018 ; it covers an area of 7434 mg for 5 min and precipitates were used for DNA extraction. Genomic DNA was extracted from 200 mg of each precipitate using an E.Z.N.A. Stool DNA Kit  according to the manufacturer’s instructions. To improve the quality of recovered DNA, the mixtures of stool samples and SLX-Mlus buffers were vibrated at maximum speed for 15 min until the stool samples were thoroughly homogenised. Extracted DNA was stored at − 20 °C.A 10-g aliquot of each sample was individually mixed with 30 ml of distilled water and passed through a ~ 250-μm-wide wire mesh sieve. Suspensions were centrifuged at 3000 × Cryptosporidium spp. was detected by nested PCR amplification of an approximately 830-bp fragment of the 18S rRNA gene as previously described37. G. duodenalis was detected by nested PCR amplifications of a 290-bp product of the 18S rRNA gene and a 520-bp polymorphic fragment of the glutamate dehydrogenase (GDH) gene as described39. To detect Blastocystis sp., an approximately 600-bp fragment of the 18S rRNA gene was amplified by single primer PCR as previously described , 2 mM MgCl2, 0.2 mM dNTP mixture, 0.625 U of TaKaRa Taq , and 1 μl of genomic DNA. Each specimen was analysed in duplicate using positive (cattle-derived DNA) and negative (sterile water) controls.https://www.ncbi.nlm.nih.gov/) database using Clustal X 2.1 . Phylogenetic analysis was performed by a neighbour-joining (NJ) analysis in MEGA 7.01 (https://www.megasoftware.net/) based on the Kimura 2-parameter model using 1000 bootstrap replicates. The p-distance model was selected as the most suitable model.The positive secondary PCR products were directly sequenced by GENEWIZ . Sequence accuracy was confirmed with two-directional sequencing. All raw sequencing data were viewed and aligned by eye in Chromas Pro 1.33 . The identity of species/assemblages/subtypes was established by comparing the obtained sequences with reference sequences from the National Center for Biotechnology Information  with 95% confidence intervals. Differences at P < 0.01 were considered significant.Differences between prevalence and the dog’s origin , age (≤ 6 months vs. > 6 months), sex, and deworming conditions (dewormed vs. non-dewormed in the past 12 months) were compared using a χPrior to fecal specimen collection, we showed an informed consent to the dogs owner. This informed consent provides some information, including the purpose of the study, research approval number, the benefits and risks that may bring to their animals participating in the study. The informed consent was obtained from the dog owners. Appropriate permission in written form was obtained from the animal owners. During specimen collection, all animal workstrictly followed the guidelines relating to the recommendations from the Guide for the Care and Use of LaboratoryAnimals of the Ministry of Health, China. Our protocol in written form was authorized by the Animal Ethics Procedures and Guidelines of the People's Republic of China and the approval of China Guangdong Province Science and Technology Department (Permit Number: SYXK (Yue) 2011–2018).Cryptosporidium spp., G. duodenalis, and Blastocystis sp. The prevalence and 95% confidence intervals are summarised in Table Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were detected in 21 (3.2%), 20 (3.1%), and 35 (5.4%) of the examined samples, respectively. Co-infection rates significantly differed based on the dogs’ origins. The overall prevalence of shelter dogs  was significantly higher than that of household , breeding , and pet market dogs  . Moreover, deworming in the past 12 months had a strong protective effect on the risk of contracting pathogens, with non-dewormed dogs having 15-times higher risk of overall positive infection rates than dewormed animals . No significant difference was observed between age categories  in the prevalence of Cryptosporidium spp. , G. duodenalis , or Blastocystis sp. . There were also no significant differences in overall  and pathogen-specific prevalence rates between the two sex groups of dogs .Faecal samples from 651 dogs were tested by PCR for the presence of SSU rRNA Cryptosporidium-positive canine samples revealed the presence of C. canis (n = 19) and C. parvum (n = 2)  Table . NucleotG. duodenalis assemblages: D (n = 14), C (n = 5), and F (n = 1). Three SSU rRNA nucleotide sequences of G. duodenalis assemblages D, C, and F were 100% identical to the GenBank reference sequences DQ385549, DQ385548, and JX275387, respectively. The genotypes identified by the GDH gene were fully consistent with those identified by the SSU rRNA gene and there were no overlapping nucleotides at any position, as shown by the chromatograms. No mixed assemblage infections were identified , and the remaining three sequences had minor differences from EF507636, including two single nucleotide polymorphism (SNPs) in four specimens (C to T substitution at position 162 and T to G substitution at position 324), three SNPs in two specimens , and three SNPs in three specimens . All GDH sub-assemblage C sequences were identical to each other and showed 99% sequence similarity to the EF507621 reference sequence, with one SNP difference at position 12 (T → C). The assemblage F nucleotide sequence was identical to the corresponding KF993737 sequence of from a cat in China , ST1 (n = 6), ST10 (n = 1), and unknown ST (n = 1). Twenty-seven nucleotide sequences identified as ST3 were identical to each other and had 100% similarity to the MK782518 reference sequence from urticaria patients in Brazil. Six ST1 nucleotide sequences included two different nucleotide sequences with 100% similarity to the GenBank reference sequences MK782501 in four specimens and MK782521 in two specimens. The ST10 nucleotide sequence had 100% similarity to a cattle-derived sequence from Malaysia in GenBank (MK240480). The remaining nucleotide sequence was not assigned a subtype by the sequence typing database, and was 100% homologous with the published sequence MK511788 from Malaysia.DNA sequencing of the Blastocystis subtypes obtained in the present study into three subtypes , and the unknown subtype was grouped into subtype ST1  in the surveyed canine populations. This finding is comparable to the prevalences in Poland 41, India 42, the United States (2.0% and 3.8% for Cryptosporidium)44, China (3.8% and 4.9% for Cryptosporidium)14, Italy (3.3% for Cryptosporidium)9, Australia (2.5% for Blastocystis)29, Brazil (2.6% for Blastocystis)31, and France (3.4% for Blastocystis)20. However, higher prevalences of 21% of Cryptosporidium from Japan, 36.5% of G. duodenalis from Spain, and 37.5% of Blastocystis from Colombia were also previously found in dogs33. Aside from geographical considerations, many factors can contribute to this difference in the prevalence, including a dog’s age, origin, health status, and examination methods used. Importantly, the true prevalences may be underestimated because of the intermittent shedding of oocysts/cysts, low parasitic burdens, and invalid amplification23. Therefore, it is important to perform a well-designed longitudinal study that includes appropriate sampling methods and a combination of diagnostic tests  to estimate the real prevalence.In this study, 23. Additionally, these shelter dogs originally roamed free in the nearby streets before they were found by local citizens and sent to the shelters, which increased their exposure to a variety of pathogens. No significant age- and sex-associated differences were detected in the prevalence of these three pathogens, which is consistent with observations of previous studies in China, Japan, Colorado, France44 , and Australia45. As expected, deworming had a significant negative effect on the risk of overall and single pathogen infections. This could be related to the fact that the anthelminthic ingredients used for dogs in China mainly include ivermectin, pyrantel, praziquantel, pyrantel pamoate, febantel, nitazoxanide, metronidazole, and fenbendazole, most of which are also effective against protozoan infections, such as Giardia spp., Cryptosporidium spp., and Blastocystis sp.48. Thus, pet hygiene management is suggested to be a major risk factor for contracting these pathogens in dogs.In risk factor analysis, the overall and pathogen-specific prevalences of shelter dogs were significantly higher than those of household, breeding, and pet market dogs. Poor care conditions may be a significant factor that contributed to the high prevalence in shelter dogsC. canis. Another interesting outcome was the identification of C. parvum in two of the canine isolates genotyped. Cryptosporidium parvum is the most frequent species known in cryptosporidium infections of humans and has resulted in several zoonotic outbreaks49. However, because of the omnivorous nature of dogs, C. parvum detected from dogs in this study may have been present because of accidental acquisition or mechanical carriage of C. parvum oocysts of anthroponotic origin via environmental contamination. Although the host-specific species C. canis also colonised individuals in hospitals, including children, HIV patients, and even immunocompetent individuals52, the infections in humans were likely transient53. Based on our data and that of other studies, we conclude that dogs do not seem to be suitable reservoirs for Cryptosporidium spp. transmission to humans, therefore, posed a limited risk to humans. This is consistent with the findings reported in dog populations in eastern Spain, where most of the genotypes identified seemed to be primarily transmitted within canine cycles24.The sequencing data revealed that dogs were predominantly infected by the expected host-specific species G. duodenalis, dogs were infected by assemblages D and C. Surprisingly, the supposedly cat-specific assemblage F was also found in one household dog, which was consistent with a previous report in Beijing, China14. Considering there was little possibility of the specimen being contaminated with cat faeces, as individual faecal samples were freshly collected by pet dog owners who kept only a single companion animal, it is more likely that the dog was transiently infected by ingesting parasite cysts of cat origin. Assemblages D and C have strong host specificities and have been mainly detected in canines. Both are considered of limited zoonotic relevance, although sporadic cases of human infections have been frequently detected in travellers, children, and diarrhoeal outpatients55. The dominant appearances of G. duodenalis assemblages D and C in dogs in our study suggested that zoonotic transmission of giardiasis rarely occurs between humans and dogs.Regarding Blastocystis subtypes has been identified in dogs worldwide and the subtype constitution was observed to differ among geographical regions, such as ST1 and ST4 in China; ST1, ST4, ST5, and ST6 in India; ST1 and ST10 in the USA; ST1, ST3, and ST4 in Australia; and ST1 and ST2 in Thailand57. This is the first large-scale survey on the prevalence and genetic characteristics of Blastocystis sp. in dogs of various origins in China, and we detected the presence of subtypes ST3, ST1, and ST10. Among the subtypes in this study, ST3 and ST1 are the two predominant subtypes. The subtype distribution of Blastocystis in dogs in our study consistent with most populations in humans around the world58. In China, in a cross-sectional survey in humans from four epidemiological settings, ST3 was the predominant type, accounting for 60.4% (n = 116) of 192 positive specimens, followed by subtype ST1, accounting for 24.5% (n = 47)58. A report from Argentina also showed that Blastocystis ST3 was the most prevalent subtype (48 cases) among 76 patients infected with Blastocystis, and other subtypes identified were ST1 (14.9%), ST6 (7.5%), and ST2 (5.9%)57. Moreover, ST3 and ST1 were found in dogs and their owners in Australia, the Philippines, and Turkey59. All of these studies suggested that ST3 and ST1 might be the important sources of human-to-human or animal-to-human transmission of Blastocystis, although more environmental factors and or other animal sources should be included. Blastocystis ST10 was found in one dog sample; this subtype is frequently identified in common livestock, including cattle, sheep, goats, and deer60, and occasionally found in wild animals, pigs, dogs, and cats27. Taken together, our results revealed that canines are a novel reservoir for Blastocystis.A high diversity of 62. However, our epidemiological study still generated baseline information and determined the genetic diversity of these pathogens in the investigated region.We conducted partial assessment of the zoonotic potential of our canine isolates using only molecular epidemiological data, because assessing the risk for zoonotic transmission of these pathogens from dogs to/from humans is difficult. The only way to properly determine zoonotic transmission is by conducting case–control studies that assess the genotypes/subtypes of these pathogens by using appropriate molecular typing tools in human and canine populations that maintain permanent close contact in the same spatial and temporal settingCryptosporidium spp., G. duodenalis, and Blastocystis sp. were determined in dogs in Guangzhou. Our risk factor analysis showed that management of pet hygiene may be a major risk factor for contracting these pathogens in dogs. Our results suggest that dogs do not seem to be suitable reservoirs of human giardiasis or cryptosporidiosis in the investigated region, but may act as novel suitable hosts of human blastocystosis. Strict sentinel surveillance of dogs, especially stray dogs, should be established to minimise the risk of spreading blastocystosis among humans and dogs.The prevalence and molecular characteristics of Supplementary Table S1.Supplementary Table S2.


A previous study showed early statin administration in patients with acute ischemic stroke (AIS) was associated with a lower risk of early-onset seizure (ES), which is a high risk of epilepsy, but this retrospective study design may not have eliminated confounding factor effects. We aimed to verify the determinants and prognostic significance of ES and clarify the effects of statin administration. Consecutive AIS patients without a history of epilepsy were enrolled. The relationship between ES (within 7 days of index-stroke) and statin treatment was assessed using multivariate and propensity scores (PS). Of 2,969 patients with AIS, 1,623 (54.6%) were treated with statin, and 66 (2.2%) developed ES. In logistic regression models, cortical stroke lesion [odds ratio (OR), 2.82; 95% confidence interval (CI), 1.29–7.28) and pre-morbid modified Rankin Scale (per 1 point)  were higher risks for ES, while statin significantly reduced the risk of ES . In accordance with PS-matching, statin treatment produced consistent results for ES after adjusting by inverse probability of treatment-weighting PS . In conclusion, as previously, statin treatment was independently associated with a lower risk of ES in AIS. Stroke is the most common comorbidity in epilepsy in elderly people2. Recent reviews have reported acute symptomatic seizure in acute ischemic stroke (AIS), so called “early-onset seizure” (ES), was a risk of post-stroke epilepsy (PSE)5. Estimates of the rate of ES in patients with an AIS in the last decade range from 2% to 6.5%6. Recently, the predicting score of PSE with AIS was published, with ES the most significant risk factor7.Over the last decade, there have been notable improvements in the treatment of acute stroke. While the number of patients surviving stroke is expected to increase, optimal management of post-stroke patients remains problematic9. Statin is an inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase and a key drug in the management of acute phase or for the prevention of atherosclerotic diseases, including stroke and coronary artery disease, and is thought to possess neuroprotective properties10. Statin administration can lead to the modification of epileptogenic processes, presumably from its AED effects16. Early use of statins reduced the risk of ES in the management of AIS, and was associated with a lower risk of progression of ES into PSE11; however, the retrospective design and small number of participants in the study meant confounding factors may not have been eliminated. For example, cardioembolic strokes occur in cortical areas more frequently and are treated with statin less frequently in the acute phase. Stroke subtypes should therefore be acknowledged in any retrospective analysis.The lack of robust evidence in previous studies has meant prophylactic use of anti-epileptic drugs (AED) for ES or PSE remains controversial; indeed, current stroke guidelines do not recommend use of AED for such purposesThere is a paucity of clinical evidence on the association between statin and seizure. We therefore aimed to verify the predisposing factors in ES in patients with AIS from our observational data, as well as clarify the association between statin administration and ES using propensity score (PS) methods.All patients admitted to the Department of Stroke and Cerebrovascular Diseases of the National Cerebral and Cardiovascular Center were registered in a database . We identified consecutive patients with AIS admitted within 3 days of onset between August 2012 and July 2016. AIS was defined as the acute onset of focal neurological symptoms lasting 24 h or longer and confirmed by MRI. The following patients were excluded: (i) those diagnosed with epilepsy before index-stroke; (ii) those with stroke due to a trauma, intracerebral hemorrhage, or subarachnoid hemorrhage; (iii) those diagnosed with transient ischemic attack after admission; and (iv) those who did not undergo MRI.The present study was approved by the Institutional Ethical Committee of the National Cerebral and Cardiovascular Center and conducted in accordance with relevant institutional guidelines. The ethics committee granted a waiver to conduct this study without written informed consent.The collected data included information regarding patient clinical history and presentation, laboratory, imaging, electroencephalographic, treatment, and outcomes [modified Rankin Scale (mRS) score and mortality] at discharge. The data collectors were unaware of the current study.17. We classified stroke subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification18. We estimated vascular territory according to a published atlas19, and the AIS lesions according to the Alberata Stroke Program Early CT Stroke score for MRI (DWI-ASPECT)20.Stroke severity was measured using the National Institutes of Health Stroke Scale (NIHSS) score on hospital admission, and trichotomized into three levels 21. We assessed seizure semiology before treatment, the deterioration of consciousness, and any subtle neurological manifestations in the stroke care unit or stroke ward. An imaging study was performed to exclude recurrence of stroke. Electroencephalography (EEG) was also performed if patients previously had, or were deemed likely to have, seizure. The EEG findings were obtained by two trained neurologists in neurophysiology .In the current study, patients were recruited if any occurrence of seizure had occurred, after careful evaluation by a trained neurologist(s). ES was defined as a seizure within 7 days of stroke8. Statins were used in patients whose stroke was presumed to be of atherosclerotic origin, including small-vessel occlusion and large-artery atherosclerosis, with or without consideration of the low-density lipoprotein cholesterol level, and not for the prevention of seizure8. Statin use was classified into two phases: before stroke or in the acute phase. Statin treatment in acute phase was defined as starting within 3 days of index-stroke onset. Types of statin included pitavastatin, atorvastatin, simvastatin, rosuvastatin and pravastatin. AEDs were administered according to the current guidelines on seizures22 if patients needed treatment, regardless of EEG findings.All patients were undergoing treatment for strokes, including antiplatelet agents, anticoagulants, and edaravone as a neuroprotectant, with some having endovascular treatment or decompressive craniectomy, in line with current guidelinesWe examined the differences between the groups using Pearson’s chi-square test and Fisher’s exact test for categorical data, and Welch’s t test and Wilcoxon’s rank sum test for continuous variables. All P-values were 2-sided, with P-values of <0.05 considered statistically significant.Firstly, logistic regression was used for multivariate analysis of clinical characteristics pertaining to ES, and the associations between ES and clinical outcomes. Pre-specified variables determined to be clinically important were also included in the model.23. ES and outcome at discharge were analyzed as dependent variables. In PS-matching methods, rigorous adjustment was performed with PS matching using the following algorithm: 1:1 optimal match with caliper width 1/5 logit of the SD and no replacement24. In the IPTW method, patients were weighted by the inverse probability of receiving statin, the average treatment effect (ATE) and average treatment effect on the untreated (ATT) were estimated by computing the difference between the observed and potential outcomes resulting from the presence and absence of statin treatment for each subject.Secondly, PS were estimated using a logistic regression model. Possible confounders were chosen for their potential association with statin on clinical knowledge. The predicted probability of preprocedural statins was calculated by fitting a logistic regression model, using all clinically relevant variables. PS was used with PS-matching and inverse-probability-of-treatment weighted (IPTW) methodshttps://www.R-project.org/.).Two-hundred sixteen subjects with missing values were excluded from the multivariate and PS analysis. Statistical analysis was performed using R version 3.5.0 (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL We identified 4595 patients with acute stroke, including intracranial hemorrhage, transient ischemic attack, and 2969 patients with AIS. Of the patients with AIS , 1623 (54.6%) were treated with statin (93 only before index-stroke and 1530 in acute phase after admission), and 66 (2.2%) developed ES during the hospitalization. Comparison of the baseline characteristics between the patients with ES (ES group) and those without ES (non-ES group) are shown in Table Among the 66 patients with ES, 28 (42.4%) had a focal aware seizure, 21 (31.8%) a focal impaired awareness seizure, and 27 (40.9%) a secondary generalized convulsion. Of those with ES, 22 (33.3%) developed status epilepticus. The majority  of all ES occurred during the first 24 hours, 12 (18.2%) occurred during 24 to 48 hours, and 6 (9.1%) occurred 3–7 days after index-stroke. Of 66 patients with ES, 57 (86.3%) had EEG and 20 (30.3%) showed epileptiform discharges. Nine patients with ES did not undergo EEG examination due to the following reasons: the seizure episode taking place out-of-hours for EEG examination in four, too critical medical condition to be eligible for EEG examination in three, and unknown reasons in two.Comparison of treatment and outcomes at discharge between the ES and non-ES groups are shown in Table In logistic regression models adjusted by age, sex, body mass index (BMI), pre-morbid mRS, initial NIHSS, LDL-cholesterol level, subtypes of stroke (TOAST classification), cortical stroke lesion, and statin treatment, cortical stroke lesion [odds ratio (OR), 2.83; 95% confidence interval (CI), 1.29–7.28; P = 0.008), pre-morbid mRS (per 1 point) , and initial NIHSS (per 10 point)  were higher risks of ES, while statin treatment significantly reduced the risk of ES   or mortality  at discharge.Propensity score (PS) precision was evaluated using a receiver operating characteristic curve, with the area under the curve showing good precision equaling 0.730 . Of patients who received statin, 54.6% (n = 886) were matched to similar patients who did not. The covariate balance in the PS-matched patients was enormously improved Table . RegardiThe major findings of this study were as follows: (i) cortical stroke lesion, pre-morbid mRS, and initial NIHSS were higher risks for ES, while statin treatment was significantly associated with a lower risk for ES in patients with AIS, and (ii) statin treatment still had a consistent result after adjusting by PS methods.26. We found that cortical stroke lesion, higher NIHSS and higher degree of disability, represented by pre-morbid mRS, were independently associated with ES in the study subjects. Our study confirmed these factors also promote ES after AIS.Several previous studies have reported AIS patients with cortical stroke lesion, higher stroke severity or other comorbidities had acute symptomatic seizures more frequently than those without11 and PSE16 after stroke. Statin was also associated with a lower risk of epilepsy in the absence of stroke14. Our study demonstrates early use of statin reduced ES in two PS analyses, adjusted by baseline characteristics, stroke severity, stroke subtypes, and lipid profile. These findings suggest statin is independently associated with lower incidence of ES than other factors. Using these two methods, confounding factors could be reduced or eliminated by measured covariates23. The effect size of statin was almost equivalent to that found in previous studies16. The current study, however, differs from previous analyses by reducing selection biases through the addition of PS analyses, adding robustness regarding the inhibitory effect of statin for ES. Although the rate of ES was relatively low compared to the other ES study11, it is intriguing that the effect of statin, represented by ATT or ATE, was estimated at 1.9% and 1.7%, respectively.Recent studies have shown that statin was associated with reduced risk of ES5. ES was indicated to be a significant risk factor for PSE in a meta-analysis3. Experimental studies have provided convincing evidence that statins have a neuroprotective, and anti-seizure, effect. It has been reported that lipophilic statins, such as atorvastatin, pitavastatin and simvastatin, but also hydrophilic statins, including simvastatin, have a suppressive effect on seizure development in an experimental animal model30. The possible anti-seizure mechanism of statins may be related to the reduction in neuroinflammation mediated by a decrease in glutamate, pro-inflammatory cytokines  and action in the nitrergic system30. In accordance with our findings and a previous study11, early use of statin was independently associated with a lower incidence of ES, which may be related to one or more of the mechanisms described above.ES is considered a transient metabolic and biochemical phenomenon, incorporating hypoxia, metabolic dysfunction, global hypoperfusion, hyperperfusion, glutamate excitotoxicity, ion channel dysfunction, and blood-brain barrier disruption in acute phase32. A post-hoc analysis of randomized clinical trials showed patients with ischemic stroke of small-vessel subtypes and treated by high dose of atorvastatin had a higher incidence of hemorrhagic stroke, though hemorrhage risk was not increased in patients with stroke of a large-artery atherosclerosis or cardioembolic origin31. In addition, active statin therapy was not associated with significant increase in ICH, and had a significant reduction in all stroke and all-cause mortality in a meta-analysis of 31 randomized controlled trials of statin therapy32. Therefore, future prospective studies testing the effect of statins on early seizure after stroke should be considered with such factors in mind.Regardless of stroke subtype or cholesterol levels, our study suggested that statin imparted a protective effect against ES; however, the risk of statin-induced hemorrhage must also be consideredWe acknowledge some limitations in this study. Firstly, the study was performed in a single-center, with retrospective design, and its observation period was short . Although the 165 patients were initiated statin after acute phase, the reasons behind why they were not initiated within 3 days could not be found. We also excluded subjects with missing data from the analysis and could not include all potential factors not to start statin, i.e. dysphasia, drug hypersensitivity, progression or recurrence of stroke, infection, etc. Such factors may have caused selection bias and limited generalizability. Secondly, acute statin treatment for stroke is not well-established, and our management of AIS could not be sufficiently aligned to other stroke guidelines, further limiting generalizability. Thirdly, there was potential for misdiagnosis regarding limb-shaking TIA for patients with severe carotid stenosis or occlusion; however, this is a rare phenomenon in carotid disease, and with MRI and EEG assessment by a professional neurologist, the risk of misdiagnosis should be minimized. Fourthly, we could not assess doses or types of statins because of the difficulty in acquiring subjects in each group. The neuroprotective effects of statins likely changed according to different doses and type of drug, for example. The study, however, did demonstrate early use of statins was significantly beneficial, including in low doses of commonly-prescribed types. We hope future prospective studies will be performed to address some of the limitations of the current study.In conclusion, using PS analysis, this study showed robustly that statin treatment can be associated with a lower risk of ES in AIS.Online Supplemental Material.


Brechemia lineata is a well-known medicinal plant in the Rhamnaceae family and widely used in traditional Chinese medicine. Here, we sequenced the complete chloroplast genome using Illumina pair-end sequencing data. The chloroplast genome was 154,962 bp in length, consisting of a large single-copy (LSC) region of 82,928 bp, a small single-copy (SSC) region of 17,376 bp, and a pair of inverted repeat (IR) regions of 27,329 bp. The chloroplast genome consists of 112 unique genes, including 78 protein-coding genes, 30 transfer RNA, and 4 ribosomal RNA genes. The overall GC content of the chloroplast genome was 37.0%. The phylogenetic analysis suggests close relationship of B. lineata with other Berchemia species. These genomic resources will be valuable resource for systematic and phylogenetic studies of Berchemia genus. Berchemia is a genus of Rhamnaceae family, comprises of about 32 species and mainly distributed in temperate and tropical areas of East to Southeast Asia (Chen and Carsten Berchemia lineata (L.) DC. is a traditional Chinese medicinal species distributed in China , Japan and Vietnam. Berchemia lineata roots and leaves are used medicinally for relieving coughs and reducing sputum and for treating injuries, trauma, and snakebites  and voucher specimen (Zhou2019-18) was deposited in the Herbarium of Sun Yat-sen University (SYS). Genomic DNA was isolated using Tiangen plant genomic DNA kits  and sequenced on the Illumina Hi-Seq 2500 platform. GetOrganelle pipeline were used for de novo chloroplast genome assembly  region of 82,928 bp, a small single-copy (SSC) region of 17,376 bp, and a pair of inverted repeat (IR) regions of 27,329 bp. The chloroplast genome contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. The overall GC content of the chloroplast genome was 37.0% and the corresponding values of LSC, SSC, and IR regions were 34.7%, 31.2%, and 42.4%, respectively.B. lineata within the Rhamnaceae family was inferred using the previously published eight chloroplast genome from the Rhamnaceae family and Hippophae rhamnoides and Elaeagnus macrophylla (Elaeagnaceae) as out group. The chloroplast genome of these species were aligned using MAFFT v7.3  (Cheon et al. B. lineata will provide a fundamental resource for studying the systematic position and conservation of this important medicinal species.Phylogenetic relationship of te clade  might be