Structure and function of mammalian cilia In the past half century , beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia , novel insights have been obtained regarding the structure and function of mammalian cilia . All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways , sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation . This view has had unanticipated consequences for our understanding of developmental processes and human disease . Introduction Although in 1835 , Purkinje and Valentin -LRB- 1835 -RRB- published the first treatise to include studies of mammalian cilia , the history of what may be considered modern research on mammalian cilia does not extend back more than a few years longer than the history of this journal . Motile mammalian cilia attracted the attention of microscopists before the advent of electron microscopy , because they moved and were obviously similar in this motion to protozoan cilia . Nevertheless , several light microscope structures whose later fine structure appearance -LRB- and molecular biology -RRB- had little in common with cilia were nevertheless labeled cilia , including the stereocilia of the hair cells , which are in fact modified microvilli . In 1954 , the electron microscope observations of Fawcett and Porter -LRB- 1954 -RRB- definitively characterized the 9 + 2 pattern of the cytoskeleton , the axoneme , of motile mammalian and other cilia . They showed that this structure was enclosed by an extension of the cell membrane , the ciliary membrane . The axoneme of true cilia was always an extension of a basal body and related to the nine-fold construction of the centriole . By 1956 , Porter -LRB- 1957 -RRB- and De Robertis -LRB- 1956 -RRB- realized that cilia were found as sensory structures in , for example mammalian photoreceptors , where the central pair of microtubules in the axoneme was absent , and the cilia were nonmotile , hence 9 + 0 . Finally , in the early 1960s , a number of electron microscopy studies showed that solitary 9 + 0 cilia , now termed primary cilia , are present on many differentiated cells of tissues of the mammalian and vertebrate body , including kidney cells , fibroblasts , neurons and even Schwann cells -LRB- Barnes 1961 ; Sorokin 1962 ; Grillo and Palay 1963 -RRB- . Although specific antibodies against detyrosinated and acetylated tubulin became available to detect primary cilia in immunofluorescence microscopy -LRB- Piperno et al. 1987 ; Cambray-Deakin and Burgoyne 1987 -RRB- , primary cilia were generally neglected as interesting cell organelles . The biggest problem was that primary cilia had no demonstrable function , even though considerable circumstantial evidence suggested that they were some form of chemical or mechanical sensor . On this basis , Poole et al. -LRB- 1985 -RRB- proposed that primary cilia of connective tissue cells functioned as a cellular cybernetic probe , or in current terminology , as a cellular global positioning system . Roth et al. -LRB- 1988 -RRB- then demonstrated that primary cilia on cultured kidney epithelial cells could be bent by flow , and Wheatley -LRB- 1995 -RRB- suggested that if primary cilia in renal epithelia did not function properly in sensing flow , there would be pathophysiological consequences . Nevertheless , many cell biologists dismissed the primary cilium as vestigial . While early studies of mammalian cilia focused on showing that the mechanism and biochemistry of motility was virtually identical to that discovered in other cilia , more recent studies have examined the way that cilia are built . These studies have led to novel insights for both motile and nonmotile , including primary , cilia . All cilia can now be viewed as sensory cellular antennae that coordinate a plethora of cellular signaling pathways , and this view has had unanticipated consequences for our understanding of developmental processes and disease . A recent review of mammalian cilia structure and function covering many of the topics presented here , in further detail , is found in Satir and Christensen -LRB- 2007 -RRB- . The mechanism of ciliary motility and primary ciliary dyskinesia The basic mechanism of ciliary motility is now reasonably well understood , although many details of how waveform is generated and propagated remain obscure . While much of the information about this mechanism was derived from protistan and invertebrate organisms -LRB- Satir 1985 -RRB- , the basic principles clearly apply to mammalian cilia of the respiratory and reproductive tract , and to brain ependymal cilia . An electron micrograph of cilia of the mouse oviduct is shown in Fig. 1 . Fig. 1Classic transmission electron micrograph of mouse oviduct cilia . Cross-sections show the 9 + 2 axoneme of motile cilia -LRB- asterisk -RRB- . The axoneme grows from a basal body , with a basal foot -LRB- arrowhead -RRB- pointing in the direction of the effective stroke . The transition zone between basal body and axoneme contains the ciliary necklace -LRB- arrow -RRB- . -LRB- From Dirksen and Satir 1972 , unpublished , with permission -RRB- Ciliary motility is caused by the relative sliding of the nine outer axonemal doublets , operating as two opposing sets , one -LRB- doublets 1 -- 4 -RRB- to produce the effective stroke -LRB- or principal bend in flagella -RRB- , one -LRB- doublets 6 -- 9 -RRB- to produce the recovery stroke -LRB- or reverse bend -RRB- , powered by ATP and a set of molecular motors , the axonemal dyneins . The axonemal dyneins are arranged in two sets of arms : the outer dynein arms -LRB- ODAs -RRB- consisting of two heavy-chain dynein isoforms in mammalian cilia , packaged with intermediate and light chains , with four identical ODAs , each aligned along 96-nm long doublet microtubule -LRB- Nicastro et al. 2006 -RRB- , and the more complex inner dynein arms -LRB- IDAs -RRB- . The IDAs , more centrally located , consist of at least seven heterodimeric and monomeric heavy-chain dynein isoforms per 96 nm . Generally , the ODAs and IDAs function together , but the ODAs principally regulate beat frequency , in part by cAMP-dependent phosphorylation of an ODA regulatory light chain , while the IDAs control beat form . Both frequency and form change by changing the rate of sliding and the switching of activity between the doublet sets . Sliding is converted into propagated bending , most efficiently by control of IDA activity via phosphorylation and mechanical interaction involving the radial spoke , central pair complex . In effect , the 9 + 2 axoneme is a nanomachine composed of perhaps over 600 proteins in molecular complexes , many of which also function independently as nanomachines . The enzymes that control axonemal response , for example protein kinase A , are structurally part of the complexes . Strong support for this mechanism of ciliary motility comes from studies of paralyzed and other mutants of protistan cilia , where a structural defect and a corresponding genetic mutation can directly be correlated with a change in ciliary beat . Similarly , strong support for the applicability of the mechanism to human cilia comes from studies , mainly of ciliary structural defects and genetic mutations associated with impaired mucociliary function in the respiratory system , which produces sinusitis and bronchiectasis . The genetic diseases are now called primary ciliary dyskinesia -LRB- PCD -RRB- . Clinical features of PCD , which include chronic rhinitis/sinusitis , otitis media , male infertility and an increased incidence of hydrocephalus , point to physiological processes where ciliary motility is essential -LRB- Afzelius 2004 -RRB- . As might be anticipated , prominent causes of PCD are mutations that affect the assembly or function of the dynein arms -- most commonly a mutation in DNAH5 , a gene encoding a dynein heavy chain of the ODAs -LRB- Olbrich et al. 2002 -RRB- . Any mutation affecting the ciliary beat mechanism will potentially produce PCD , depending on the severity of beat impairment . An instructive example is hydin , a component of the central pair projection complex that interacts with the radial spokes in the switching mechanism between doublet sets . Hydin-deficient flagella of Chlamydomonas become intermittently paralyzed , stopping for long times at the end of the effective or recovery strokes , where the direction of beat is reversed -LRB- Lechtreck and Witman 2007 -RRB- . Mice defective in Hydin develop hydrocephalus and die shortly after birth . Lechtreck et al. -LRB- 2008 -RRB- analyzed ciliary structure and motility in these mice . The central pair projection assigned to the hydin complex was missing . Mutant cilia were unable to bend properly and frequently stalled , implying that because central pair structure was defective , interactions between the central pair and the radial spokes that affected dynein arm switching were abnormal , and consequently cilia-generated flow was impaired . Motility of tracheal cilia was also impaired . One would predict that human mutations in HYDIN are also likely to result in hydrocephalus or a form of PCD . Many people diagnosed with PCD have situs inversus totalis that is reversal of left -- right asymmetry of body organs such as the heart . This is now known to be produced , because fluid flow produced by motile cilia at the embryonic node is defective -LRB- Hirokawa et al. 2006 -RRB- . The usual flow pattern produced by the cilia is unidirectional right-to-left , and this activates a left-side-only gene cascade , probably by impinging on primary cilia at the left side of the node -LRB- McGrath et al. 2003 -RRB- . The nodal cilia are mainly missing the central pair of axonemal microtubules , hence , although motile , are often 9 + 0 , but with some unique ODA dynein isoforms -LRB- Supp et al. 1999 -RRB- . It is not known whether the response of the primary cilia at the node is to mechanical displacement -LRB- flow sensing -RRB- or if it involves morphogens produced in membrane parcels in the node -LRB- Tanaka et al. 2005 -RRB- . Morphogen-containing multimembrane vesicles are a feature of nodal response that could also apply respiratory and other ciliated epithelia , where flow occurs . While mouse mutants or knockouts that are missing cilia develop situs inversus , hydin mutants do not show this phenotype , presumably because their motility is sufficient to generate appropriate nodal flow . In a similar way , by examining the PCD phenotype of mutations in various ciliary proteins , identified by proteomics , it should be possible to dissect details of the function of the protein in the mechanism of beat generation -LRB- Avidor-Reiss et al. 2004 ; Badano et al. 2006 ; Blacque et al. 2005 -RRB- . Principles of ciliogenesis A major breakthrough in the understanding of the importance of cilia in the body has come from the study of Chlamydomonas ciliogenesis . In that cell , the cilia are traditionally called ` flagella ' . Ciliogenesis involves transport of materials into , along and out of the cilium , directly visualized as ` intraflagellar transport ' or IFT -LRB- reviewed by Rosenbaum and Witman 2002 -RRB- . IFT requires molecular motors , several kinesins for anterograde transport and a specialized cytoplasmic dynein for retrograde transport . Coupling the transported cargo to the molecular motors that move along the outer doublets of the axoneme are two protein complexes -LRB- Cole 2003 -RRB- comprising about 19 IFT proteins . Remarkably , this machinery is conserved almost universally wherever cilia are built , and orthologs of the motors and IFT proteins are found in sensory cilia and their derivatives , such as the mammalian photoreceptor -LRB- Baker et al. 2003 -RRB- and in primary cilia , as well as in protistan motile cilia . However , motile cilia of mammalian tissues often assemble in mass -LRB- Gaillard et al. 1989 ; Dirksen 1991 -RRB- . Centriologenesis of hundreds of basal bodies occurs from a fibrogranular mass in the cytoplasm and cilia sprout from the cell surface . It is unclear whether classical IFT is the major mechanism of ciliogenesis in respiratory epithelium , or what modifications of the process occur . Wheatley -LRB- 1969 -RRB- , Fonte et al. -LRB- 1971 -RRB- and Archer and Wheatley -LRB- 1971 -RRB- studied ciliogenesis of primary cilia of established cell lines in vitro . Primary cilia grow when the cells become confluent and reach stationary phase . Cells resorb their cilia shortly before entering mitosis . Postmitotic cells reassemble primary cilia in G1 and maintain the cilium as the cells enter growth arrest -LRB- G0 -RRB- and undergo differentiation . In contrast to motile cilia , the primary cilium emanates uniquely from the distal end of the existing mother centriole of the centrosome , which migrates to the cell surface during growth arrest . Ciliogenesis by IFT is initiated while the centrosome is positioned at the Golgi apparatus near the nucleus , while extensive IFT and elongation of the cilium take place after docking of the nascent cilium at the cell surface , where microtubule pairs are quickly assembled to form the mature axoneme . Two recent and extensive reviews of the complex interactions between IFT particles , molecular motors , centrosomal proteins and other microtubule-associated proteins in ciliogenesis are presented in Pedersen et al. -LRB- 2008 -RRB- and Blacque et al. -LRB- 2008 -RRB- . Ciliopathies The first human illness that could be linked to primary cilia in epithelial cells was polycystic kidney disease -LRB- PKD -RRB- . One of the orthologs of IFT88 is the mammalian protein polaris mutated in a transgenic mouse Tg737orpk , which was a model for the study of PKD . Chlamydomonas IFT88 mutants are defective in ciliogenesis , and Pazour et al. -LRB- 2000 -RRB- demonstrated that , similarly , cilia of the mouse kidney were abnormally short or missing , which suggested that PKD might be a ciliary disease . This conclusion was strengthened by two succeeding discoveries . Mechanical deflection of kidney tubule epithelial cells induces an increase in intracellular calcium -LRB- Praetorius and Spring 2001 -RRB- , and the PKD proteins , polycystins 1 and 2 in Ca2 + - signaling , localize to the ciliary membrane -LRB- Pazour et al. 2002 ; Yoder et al. 2002 -RRB- . Many other proteins whose functions are disrupted in cystic diseases , e.g. nephrocystins , have now been localized to the cilium or at the ciliary basal body , supporting the conclusion that the primary cilium in kidney tubules partly functions as a mechanosensor that , upon bending , activates a series of signaling pathways in the cilium to control development and homeostasis of the tissue -LRB- for reviews , see Yoder et al. 2007 ; Hildebrandt and Zhou 2007 -RRB- , as originally envisaged by Roth et al. -LRB- 1988 -RRB- and Wheatley -LRB- 1995 -RRB- . Thus , even if primary cilia are present , unless these proteins are targeted appropriately to the cilia or the ciliary basal body , pathology develops . It is now clear that the ciliary membrane is a privileged and specialized compartment for receptor signaling and that the polycystins are only one type of many membrane proteins that must be targeted to the primary cilium to function properly . Other carefully documented examples include the receptor tyrosine kinase PDGFRα -LRB- Schneider et al. 2005 -RRB- and the patched receptors for hedgehog signaling -LRB- Rohatgi et al. 2007 -RRB- that control cell growth and differentiation processes . Further , essential downstream components in PDGFRα and hedgehog signaling as well as signaling in neurotransmission , Wnt pathways , extracellular matrix interaction and osmolyte transportation uniquely localize to the cilium and/or to the ciliary centrosome -LRB- Gerdes et al. 2007 ; Corbit et al. 2008 ; for reviews , see Singla and Reiter 2006 ; Michaud and Yoder 2006 ; Christensen et al. 2007 ; Christensen and Ott 2007 -RRB- . In many of these signaling systems , the cellular response is initiated by receptor activation in the primary cilium . Deficiencies in placement of receptors and their immediate downstream signaling components in the cilium or at the ciliary base results in ciliopathy , including cystic kidney , pancreatic and liver diseases , retinitis pigmentosa , cancer , defective neurogenesis , Bardet-Biedl syndrome , polydactyly , anosmia and other developmental defects . In the Tg737 mouse , these defects lead to early perinatal death . As shown in Fig. 2 , primary cilia with hedgehog receptor pathway signaling are found on human embryonic stem cells -LRB- Kiprilov et al. 2008 -RRB- , which suggests that ciliary signaling is involved in differentiation from the beginning of embryogenesis . In embryogenesis , signaling through the primary cilium is necessary for normal development , probably because such signaling regulates the balance between cell division , polarity , migration , differentiation and apoptosis for many tissues . Fig. 2Primary cilia of human embryonic stem cells . Immunofluoresence microscopy using acetylated α tubulin antibody -LRB- tb -RRB- reveals the presence of primary cilia -LRB- arrows -RRB- on human embryonic stem cells . In the absence of stimulation , the hedgehog receptor ` patched ' -LRB- Ptc -RRB- colocalizes with the acetylated α tubulin all along the ciliary membrane . Red and green channels are displaced in the images to define colocalization more clearly . Nuclei are stained with DAPI -LRB- blue -RRB- . Upon stimulation , as part of the signaling cascade , Ptc leaves the cilium and the smoothened receptor -LRB- Smo -RRB- enters to activate the hedgehog signaling cascade . Asterisk marks the ciliary base . -LRB- From Kiprilov et al. 2008 , with permission , courtesy of The Journal of Cell Biology -RRB- Primary cilia in adult tissues Primary cilia persist in many differentiated cells , including kidney tubule epithelial cells , fibroblasts and neurons , after organogenesis is complete and cell division rates fall . Presumably , as semipermanent structures , the cilia function as mechano - or chemosensors and as a cellular global positioning system to detect changes in the surrounding environment , to initiate cellular replacement after damage , for example . To test this hypothesis , techniques are being used to knock out primary cilia or ciliary proteins in specific tissues of adult organisms . Davenport et al. -LRB- 2007 -RRB- have completed one of the first of these studies , using an inducible system in adult mice to disrupt IFT in several different ways , causing loss of primary cilia . Respiratory motile cilia are probably more stable and relatively unaffected . Surprisingly , when primary cilia are lost from all adult tissues , the devastating abnormalities and lethality seen after embryonic loss of cilia are not observed . PKD eventually develops a year after induction . The same delay has been reported by Piontek et al. -LRB- 2007 -RRB- after adult-specific knockout of polycystin-1 . This delay correlates well with a greatly reduced rate of cell division and a different pattern of gene expression in the mature kidney and could explain the increase in human PKD with age . Such changes in cell proliferation rate may relegate the primary cilium of adult tissues to a less immediate role , involving long-term homeostasis of the tissue . Because signaling through primary cilia is coupled to cell cycle events , for example in PDGFRαα signaling in fibroblasts , long-term disruption of ciliary signaling could be a factor in oncogenesis . One adult tissue responds immediately to ciliary knockout however -- that is nervous tissue , most specifically neurons in the hypothalamus . Knockout of all adult primary cilia in the mouse , or specifically only of primary cilia on POMC neurons , leads to hyperphagia -- compulsive and excessive eating -- leading to obesity . Obesity then causes numerous secondary defects resembling type II diabetes . These defects do not occur if the knockout mice are kept on a restricted diet . Eating behavior is regulated by the hormone leptin . These fndings suggest that the leptin receptor might be located in the membrane of the primary cilia of the POMC neurons . The hypothalamic hormone somatostatin is a negative regulator of leptin . While leptin leads to reduced eating , somatostatin increases eating behavior . The somatostatin receptor sst 3 is localized to primary cilia in hypothalamic neurons -LRB- Stepanyan et al. 2007 -RRB- . The results of Davenport et al. -LRB- 2007 -RRB- suggest that , much like the patched and smoothened receptors work in hedgehog signaling , the leptin receptor and a somatostatin receptor could work in a Yin-Yang relationship within the POMC primary cilium -LRB- Satir 2007 -RRB- . Whatever the precise cell biological explanation of the relationship between ciliary knockout and hyperphagia , the effect of knockout is behavioral . We might expect that mutations in other proteins of neuronal primary cilia could lead to other behavioral responses . There are reports that the rages that fueled the famous Hatfield-McCoy feud of Appalachia probably were driven by a family suffering from Von Hippel-Landau -LRB- VHL -RRB- syndrome . VHL protein controls ciliogenesis and is localized to cilia -LRB- Schermer et al. 2006 -RRB- . Perhaps a more ciliocentric view of neuronal activity affecting recurrent or addictive actions is warranted . Conclusions Beginning with important electron microscopic studies and culminating in immunolocalization combined with molecular genetic technology , much has been learned about motile , sensory and primary cilia in mammals . Defects in building the primary cilium or mutations in ciliary membrane or axonemal proteins lead to ciliopathies , important human diseases . The cilium has moved to a prominent place in studies of embryogenesis and tissue differentiation and maintenance . There are hints that the fundamental cell biology of cilia will also be important in oncogenesis , aging diseases and human behavioral disorders . The strides of the past half century in understanding this organelle have been impressive , and the promise of discovery in the next half century is compelling .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

motile cilia
primary cilia
motility
signaling
ciliopathies


Refractive properties of the healthy human eye during acute hyperglycemia Purpose To measure the refractive properties of the healthy human eye during acute hyperglycemia by means of Scheimpflug imaging and Hartmann-Shack aberrometry . Transient refractive changes , due to a variation in blood glucose levels , are well-known complications of diabetes mellitus -LRB- DM -RRB- . Both myopic shifts -LSB- 2 , 6 , 10 , 23 , 31 , 36 -RSB- and hyperopic shifts -LSB- 9 , 13 , 18 , 26 , 32 , 37 -RSB- have been reported in patients with DM after several days or weeks of hyperglycemia . It has been suggested that the predominant cause of the refractive changes is a change in the thickness of the lens -LSB- 16 , 17 , 19 , 25 , 29 -RSB- or shape of the lens -LSB- 21 -RSB- , and/or a change in its refractive index -LSB- 24 , 27 , 29 , 33 -RSB- . Two studies have been conducted in which hyperglycemia was induced under controlled circumstances to investigate refractive changes during hyperglycemia . Firstly , Gwinup et al. -LSB- 15 -RSB- administered glucose intravenously to ten patients with DM . A myopic shift of maximal -0.75 D was measured with autorefractometry . Secondly , Furushima et al. -LSB- 11 -RSB- induced hyperglycemia in seven healthy , young subjects through somatostatin injections and an oral glucose load . By means of autorefractometry and ultrasound biometry a large change in ocular refraction -LRB- -2.0 diopters -LRB- D -RRB- -RRB- and thickness of the lens -LRB- 1.0 mm -RRB- was measured within 150 minutes after administration of the glucose load -LRB- maximal hyperglycemia : 13.9 mmol/l -RRB- . The results of these two studies indicated that induced hyperglycemia can cause changes in refraction and that these changes appeared to be larger in healthy subjects . In the study of Furushima et al. -LSB- 11 -RSB- , only the thickness of the lens was measured , and there is no information about the change in the shape and the refractive index of the lens due to acute hyperglycemia . Therefore , the aim of our study was to induce hyperglycemia in healthy subjects under controlled circumstances , and accurately measure ocular geometry and refraction by means of corrected Scheimpflug imaging and Hartmann-Shack aberrometry , in order to investigate the mechanism underlying refractive changes during hyperglycemia . From the ocular geometry and refraction , in combination with the measurement of the axial length of the eye , it is also possible to calculate the equivalent refractive index of the lens -LSB- 4 , 5 -RSB- . Subjects and methods Five healthy Caucasian subjects -LRB- two males and three females -RRB- were recruited for this study . Mean age was 24.8 years -LRB- SD ± 4.6 and range 21.2 -- 32.6 -RRB- and mean body mass index -LRB- BMI -RRB- was 24.2 kg/m2 -LRB- SD ± 3.2 and range 21.4 - 29.7 -RRB- . The Medical Ethics Committee of the VU University Medical Center in Amsterdam approved the study protocol , and written informed consent was obtained from all subjects after the nature of the study had been explained . Subjects with a history of diabetes mellitus -LRB- or a fasting plasma glucose > 5.5 mmol/l -RRB- , a BMI of > 30 kg/m2 , visual acuity of < 0.5 -LRB- Snellen -RRB- , or a history of ocular pathology were excluded from the study . Procedure to induce hyperglycemia After a 10-hour overnight fast , the subjects received a subcutaneous injection of 100 µg synthetic somatostatin -LRB- Sandostatin , Novartis , Basel , Switzerland -RRB- in order to suppress the endogenous insulin secretion during glucose loading . Each subject had an oral glucose tolerance test -LRB- OGTT -RRB- -LRB- glucose 75 g -RRB- 30 minutes after the somatostatin injection . Blood glucose levels were measured with a blood glucose analyzer -LRB- HemoCue Diagnostics BV , Oisterwijk , the Netherlands -RRB- . Endogenous insulin levels were determined with immunometric assays -LRB- Luminescence , Bayer Diagnostics , Mijdrecht , the Netherlands -RRB- in the Laboratory of Endocrinology of the Department of Clinical Chemistry in the VU University Medical Center . The subjects remained in a fasting state during the entire procedure . Measurement of ocular parameters Before and 120 minutes after the OGTT , 1.0 % cyclopentolate and 5.0 % phenylephrine eye-drops were administered to the right eye of the subjects . Hartmann-Shack aberrometry was performed with an IRX3 aberrometer -LRB- Imagine Eye Optics , Paris , France -RRB- and Scheimpflug imaging was performed with a Topcon SL-45 Scheimpflug camera , in which the film was replaced by a charge-coupled device -LRB- CCD -RRB- camera -LRB- St-9XE , SBIG astronomical instruments -RRB- with a range of 16 bits of grey values -LRB- 512 × 512 pixels , pixel size 20 × 20 µm , magnification : 1 × -RRB- . Before and every 30 minutes after the OGTT , three measurements were made with each apparatus . To obtain accurate measurements of the shape of the lens , ray-tracing was performed to correct the Scheimpflug images for the distortion that is inherent to this technique -LSB- 4 , 5 -RSB- . By combining the measurements of the corneal thickness -LRB- d1 -RRB- , the depth of the anterior chamber -LRB- ACD -RRB- , the anterior -LRB- R1 -RRB- and posterior -LRB- R2 -RRB- radius of the cornea , the lens thickness -LRB- d3 -RRB- and the anterior -LRB- R3 -RRB- and posterior -LRB- R4 -RRB- radius of the lens , the axial length of the eye , and ocular refraction , it was possible to calculate the equivalent refractive index of the lens -LRB- n lens -RRB- by means of an iterative process -LSB- 5 -RSB- . In these calculations it was assumed that the refractive indices of the cornea , aqueous humor , and vitreous do not change , because in vitro experiments have shown that large changes in glucose levels induce negligible changes in the refractive indices of the ocular media -LSB- 8 , 22 -RSB- . The axial length of the eye was measured with an IOL-master -LRB- Carl Zeiss Inc. , North America -RRB- . The equivalent refractive error -LRB- ERE -RRB- was calculated as : ERE = sphere + -LRB- cylinder/2 -RRB- . Furthermore , the higher order aberrations -LRB- HOA -RRB- of each eye were analyzed for a pupil size of 5.7 mm and they were summarized in root mean square errors -LRB- RMS -RRB- , including the third up to the eighth Zernike orders -LSB- 34 -RSB- . Furthermore , the HOA , including the spherical aberrations , were analyzed separately . Measurements at baseline and during hyperglycemia were compared in the whole group by means of Wilcoxon signed rank sum tests . A refractive change of more than 0.2 D and a change in HOA of more than 0.025 µm were considered to be meaningful , according to the precision -LRB- defined as 95 % confidence interval -RRB- for measuring the ERE and HOA of the aberrometer -LSB- 3 , 30 -RSB- . Error analysis indicated that a change in R1 , R2 , d1 , ACD , d3 , R3 , R4 , and n lens of more than 0.05 mm , 0.05 mm , 0.02 mm , 0.14 mm , 0.15 mm , 0.30 mm and 0.40 mm , and 0.007 respectively , could be considered as significant , according to the precision of corrected Scheimpflug imaging -LSB- 4 -RSB- . In each subject individually , the significance of a change was determined from the precision of the measurements and the difference in the ocular parameters at baseline and during hyperglycemia . P values ≤ 0.05 were considered statistically significant . Results Figure 1 shows the changes in blood glucose after the OGTT . In all subjects the mean blood glucose levels rose from 4.0 mmol/l -LRB- range 3.4 -- 4.5 mmol/l -RRB- to 18.4 mmol/l -LRB- range 16.1 -- 22.0 mmol/l -RRB- in 126 minutes -LRB- range 90 -- 210 minutes -RRB- after the OGTT . Subject 1 had a delayed elevation of blood glucose level , and therefore this subject received a second 75 g oral glucose load at time = 30 minutes . Furthermore , venous blood samples of subject 1 were taken from the antecubital vein , which was kept open with 0.9 % saline -LRB- 100 cl -RRB- . Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1 pmol/l -LRB- range 0.4 -- 4.5 pmol/l -RRB- , and remained below basal secretion level -LRB- < 110.0 pmol/l -RRB- for 147 minutes -LRB- range 75 -- 270 minutes -RRB- . Fig. 1Changes in blood glucose -LRB- BG -RRB- levels in the five subjects after the administration of somatostatin and glucose ; the oral glucose load -LRB- 75 g -RRB- was administered at time = 0 minutes No significant change in ERE was found in the whole group or in four of the subjects individually . In subject 1 , a hyperopic shift in ERE of 0.4 D and an increase in the spherical aberration -LRB- ± SE -RRB- from 0.03 ± 0.01 µm at T = 0 to 0.11 ± 0.01 µm during hyperglycemia was measured -LRB- p < 0.001 -RRB- . The other HOA did not change in subject 1 . Furthermore , there was no significant change in the HOA separately or the RMS values in the group or in any of the other subjects individually . No changes in corneal thickness -LRB- d1 -RRB- , corneal shape -LRB- R1 and R2 -RRB- , anterior chamber depth -LRB- ACD -RRB- , lens thickness -LRB- d3 -RRB- , or posterior lens shape -LRB- R4 -RRB- were found in the group or in any of the subjects individually . In the whole group and in four subjects , no changes were found in the anterior radius of the lens -LRB- R3 -RRB- or refractive index -LRB- n lens -RRB- . However , in subject 01 , the R3 and n lens changed significantly during hyperglycemia , compared to normal conditions ; R3 decreased from 11.65 to 9.69 mm -LRB- mean decrease R3 = 1.96 mm ; p < 0.001 -RRB- and n lens decreased from 1.436 to 1.422 -LRB- mean decrease n lens = 0.014 ; p = 0.003 -RRB- . Figure 2 presents graphs of the normalized ERE , ACD , d3 , R3 , R4 and n lens of the five subjects . Figure 3 shows two corrected Scheimpflug images and a schematic drawing of the lens of subject 1 , in order to illustrate the differences in lens geometry during hyperglycemia compared to baseline . In all subjects , both hematological and ocular parameters normalized within 6 hours after the OGTT . Fig. 2Graphs of the normalized equivalent refractive error -LRB- ERE -RRB- , lens thickness -LRB- d3 -RRB- , anterior chamber depth -LRB- ACD -RRB- , anterior -LRB- R3 -RRB- and posterior -LRB- R4 -RRB- radius of the lens , and the refractive index of the lens -LRB- n lens -RRB- of the five subjects . Data are normalized by subtracting the value at baseline from the measured value in each subject . In subject 01 a hyperopic shift of 0.4 D , an increase in R3 and decrease in n lens were found during hyperglycemia at T = 240 minutes -LRB- p < 0.01 -RRB- . The oral glucose load was administered at T = 0Fig . 3Corrected Scheimpflug images of the right eye of subject 1 . a At baseline time = 0 minutes , the shape of the cornea and lens are indicated with a solid line . b During hyperglycemia at time = 240 minutes after the first oral glucose load , the shape of the cornea and lens are indicated with a line of dashes . c A drawing of the changes in the shape of the lens in hyperglycemic condition -LRB- line of dashes -RRB- compared to the normal condition -LRB- solid line -RRB- Discussion Refractive changes occur frequently in patients with DM . The underlying mechanism is still unclear , and therefore the aim of our study was to measure ocular refraction and geometry during hyperglycemia , in an attempt to identify a possible explanation for these refractive changes . The effect of reproducible hyperglycemia was studied in healthy subjects without the systemic effects of DM . An OGTT in combination with a somatostatin injection was used to induce hyperglycemia . In an earlier study , this was shown to induce large changes in the refractive error and lens thickness -LSB- 11 -RSB- . Somatostatin inhibits insulin secretion -LSB- 7 -RSB- , and to our knowledge there are no reports of refractive errors or changes in the lens due to this agent . In general , and in four of the five subjects individually , no changes in ocular refraction or geometry were found during hyperglycemia . It could be that a more prolonged and severe hyperglycemia is needed to induce changes in refractive error or geometry of the eye . Glucose within the lens is metabolized via the sorbitol pathway , which consists of two enzymes that catalyze the conversion of glucose into its sugar alcohol sorbitol and the further conversion of sorbitol to fructose . These sugar alcohols tend to accumulate within the lens , because they penetrate cell membranes poorly . This accumulation of sugar alcohols causes the lens to swell -LSB- 12 -RSB- . This process might have taken longer than the observation period of the present study . Furthermore , the subjects measured in our study were young , and it could be that their lenses , which still have a fast metabolic reaction capacity , tolerate short hyperglycemic stress without swelling . The results of our study are not in accordance with the findings of Furushima et al. -LSB- 11 -RSB- , who observed a large myopic shift -LRB- -2 D -RRB- and a large increase in the thickness of the lens -LRB- 1 mm -RRB- during hyperglycemia in healthy subjects . It must be noted that in our study the mean increase in the blood glucose level was even higher than that of the study of Furushima et al. . One main difference between our study and the study by Furushima et al. is the methods that were used to measure ocular refraction -LRB- aberrometry versus autorefractometry -RRB- and ocular geometry -LRB- corrected Scheimpflug imaging versus ultrasound biometry -RRB- . However , the precision of aberrometry and autorefractometry are comparable for the measurement of defocus , astigmatism and , consequently , the equivalent refractive error -LSB- 3 , 30 , 35 -RSB- . Furthermore , the precision of corrected Scheimpflug imaging and ultrasound biometry are comparable as well . It could be that the difference in ethnicity -LRB- Caucasian subjects in the present study versus Asian subjects in the study by Furushima et al. -RRB- caused the inconsistency between the two studies , since Asian people generally have a more myopic ocular refraction than Caucasian subjects -LSB- 14 , 38 -RSB- . In our study , small but significant changes in ocular refraction and lens geometry were found in one subject . A hyperopic shift of 0.4 D was found , in combination with an increase in anterior convexity of the lens . A combination of hyperopia and an increase in lens thickness during hyperglycemia has been described by Kluxen et al. -LSB- 21 -RSB- , who found a 6 D hyperopic shift and a 0.4 mm increase in lens thickness in a diabetic patient with hyperglycemia . Saito et al. -LSB- 29 -RSB- reported hyperopic shifts -LRB- 1.1 to 4.9 D -RRB- and an increase of approximately 0.2 mm in the thickness of the lens in five diabetic subjects during hyperglycemia . Therefore , it has been suggested that hyperglycemia causes a change in the refractive index of the lens -LSB- 21 , 24 , 27 , 29 , 33 -RSB- . The results of our study support this hypothesis ; in subject 1 , a decrease in the equivalent refractive index of the lens was calculated during hyperglycemia . It could be suggested that if the change in the shape of the lens is small , hyperopia will predominate during hyperglycemia due to a decrease in the refractive index of the lens . Alternatively , if the change in the shape of the lens is large in comparison to the decrease in the refractive index of the lens , the overall refractive error will result in myopia . The controversy in the literature with regard to refractive changes during hyperglycemia could be explained by this underlying mechanism of a balance between changes in the shape or the refractive index of the lens , which eventually determine the overall refractive outcome -LSB- 20 , 23 , 28 , 37 -RSB- . It is surprising that a change in refraction and ocular parameters could be determined in only one subject . It must be noted that the procedures for inducing hyperglycemia and monitoring blood glucose were , to some extent , different for subject 1 compared to the other subjects . Because of a delayed elevation in blood glucose level , a second oral glucose load -LRB- 150 g instead of 75 g glucose -RRB- was administered . Nevertheless , the maximum blood glucose value of subject 1 did not exceed that of the other subjects , and the endogenous insulin level was adequately suppressed during the glucose loading . Furthermore , in order to obtain sufficient blood samples , 0.9 % saline had to be administered to keep the antecubital vein open . Therefore , it could not be excluded that the administration of saline contributed to the refractive change and alterations in the lens of subject 1 . However , no studies have yet reported any refractive change due to saline administration . The change in refractive error in patients with hyperglycemia could also be caused by a change in the shape of the cornea . However , previous research has shown that hyperglycemia has no influence on the shape of the anterior corneal surface -LSB- 10 , 24 , 29 , 32 -RSB- . The results of our study also agree with these findings . No change in the anterior or posterior corneal radius was found during hyperglycemia . Therefore , the cornea does not seem to play a role in the explanation of refractive changes during hyperglycemia . This also applies for the higher order aberrations . Applegate et al. -LSB- 1 -RSB- reported that an increase in the higher order aberrations could cause a decrease in visual acuity . However , no changes in the higher order aberrations were found in any of the subjects in our study . Therefore , it can be assumed that blurred vision in hyperglycemia can not be explained by changes in the higher order aberrations of the eye . In sum , the results of this study show that induced hyperglycemia generally does not cause changes in the refractive properties of the healthy human eye . However , there were interindividual variations , as illustrated by subject 1 , who had a hyperopic shift of refraction and a change in shape and equivalent refractive index of the lens during hyperglycemia . This could provide an explanation for the mechanism underlying the refractive changes often experienced by patients with DM and hyperglycemia .

O O O O O O O O O B O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O

hyperglycemia
diabetes mellitus
scheimpflug imaging
refractive errors
lens


Treatment

of

invasive

fungal

infections

in

clinical

practice

a

multi-centre

survey

on

customary

dosing

treatment

indications

efficacy

and

safety

of

voriconazole

Invasive

fungal

infections

are

frequent

and

often

deadly

complications

in

patients

with

malignant

hematological

diseases

Voriconazole

is

a

third

generation

triazole

antifungal

with

broad

activity

against

most

clinically

relevant

fungal

pathogens

Clinical

practice

often

deviates

from

insights

gained

from

controlled

randomized

trials

We

conducted

a

multi-centre

survey

to

evaluate

efficacy

safety

treatment

indications

and

dosing

of

voriconazole

outside

clinical

trials

Patients

receiving

voriconazole

were

documented

via

electronic

data

capturing

An

analysis

was

conducted

after

submission

of

episodes

from

September

to

November

Voriconazole

was

administered

for

suspected

or

proven

invasive

fungal

infection

-LRB-

IFI

-RRB-

-LRB-

-RRB-

as

empirical

treatment

in

patients

with

fever

of

unknown

origin

-LRB-

-RRB-

and

secondary

-LRB-

-RRB-

as

well

as

primary

-LRB-

-RRB-

prophylaxis

of

IFI

Investigators

assessment

of

fungal

infection

often

diverted

from

EORTC/MSG

criteria

A

favorable

response

was

reported

in

for

suspected

or

proven

IFI

and

for

empirical

treatment

Mortality

was

of

which

was

attributable

to

IFI

Breakthrough

fungal

infections

occurred

in

four

-LRB-

-RRB-

patients

with

voriconazole

as

secondary

prophylaxis

Toxicity

and

adverse

events

comprised

elevated

liver

enzymes

and

visual

disturbances

Although

indications

frequently

deviated

from

clinical

evidence

and

legal

approval

voriconazole

showed

efficacy

and

safety

comparable

to

major

controlled

clinical

trials

Data

from

this

survey

demonstrate

the

difficulty

of

putting

drugs

to

their

approved

use

in

IFI

Introduction

Patients

with

malignant

hematological

diseases

receiving

remission-induction

chemotherapy

are

at

a

high

risk

of

contracting

severe

and

sometimes

lethal

invasive

fungal

infections

especially

invasive

pulmonary

aspergillosis

-LRB-

IPA

-RRB-

-LSB-

-RSB-

Voriconazole

follows

itraconazole

as

the

second

triazole-antifungal

agent

with

an

enhanced

spectrum

of

activity

against

numerous

clinically

important

fungi

This

activity

is

achieved

by

inhibition

of

fungal

cytochrome

demethylase

a

key

enzyme

in

ergosterol

biosynthesis

Subsequent

loss

of

ergosterol

in

the

fungal

cell

wall

and

accumulation

of

sterols

are

the

primary

mode

of

action

of

voriconazole

Among

the

properties

of

voriconazole

are

in

vitro

fungistatic

activity

against

most

Candida

spp.

including

those

resistant

to

fluconazole

-LSB-

-RSB-

fungicidal

activity

against

several

Aspergillus

spp.

-LSB-

-RSB-

as

well

as

profound

activity

against

Fusarium

spp.

-LSB-

-RSB-

Scedosporium

spp.

-LSB-

-RSB-

Cryptococcus

spp.

-LSB-

-RSB-

and

other

rare

yeasts

molds

and

dermatophytes

such

as

Trichosporon

spp.

Blastomyces

spp.

Histoplasma

capsulatum

and

Coccidioides

immitis

-LSB-

-RSB-

In

a

controlled

clinical

trial

voriconazole

proved

superior

efficacy

and

improved

survival

when

compared

with

amphotericin

B

deoxycholate

in

the

treatment

of

invasive

aspergillosis

-LSB-

-RSB-

Voriconazole

has

been

approved

by

the

US

Food

and

Drug

Administration

-LRB-

FDA

-RRB-

and

European

Medicines

Agency

-LRB-

EMEA

-RRB-

for

treatment

of

invasive

aspergillosis

invasive

candidiasis

in

non-neutropenic

patients

esophageal

candidiasis

as

well

as

serious

infections

caused

by

Scedosporium

apiospermum

and

Fusarium

spp.

in

cases

refractory

to

or

intolerant

of

first

line

treatment

-LSB-

-RSB-

Availability

of

oral

-LRB-

tablets

suspension

-RRB-

and

intravenous

preparations

along

with

a

favorable

safety

profile

make

voriconazole

a

popular

drug

among

clinicians

in

cases

of

suspected

or

proven

invasive

fungal

infection

alike

Voriconazole

has

become

a

well-accepted

treatment

option

in

probable

and

proven

IFI

-LSB-

-RSB-

Controlled

clinical

trials

are

an

imperative

tool

for

testing

drug

efficacy

and

safety

in

an

idealized

study

population

To

allow

comparison

between

clinical

trials

the

European

Organisation

for

Research

and

Treatment

of

Cancer

-LRB-

EORTC

-RRB-

and

the

Mycoses

Study

Group

-LRB-

MSG

-RRB-

have

established

criteria

for

the

diagnosis

of

IFIs

-LSB-

-RSB-

These

criteria

classify

IFIs

as

possible

probable

or

proven

according

to

diagnostic

test

results

Basically

these

criteria

demand

major

clinical

signs

in

combination

with

host

factors

for

a

possible

diagnosis

For

a

probable

diagnosis

certain

microbiological

criteria

-LRB-

e.g.

serum

galactomannan

-RRB-

must

be

fulfilled

while

a

proven

diagnosis

demands

histopathological

findings

or

positive

culture

from

a

primary

sterile

site

However

trial

results

do

not

always

translate

into

everyday

clinical

practice

We

therefore

conducted

a

multi-centre

survey

to

evaluate

efficacy

and

safety

as

well

as

common

treatment

indications

and

prescribed

dosing

of

voriconazole

outside

clinical

trials

Methods

Participating

investigators

were

asked

to

document

all

patients

receiving

voriconazole

via

online

electronic

data

capture

Data

were

collected

retrospectively

after

treatment

completion

Collected

data

comprised

demographic

information

underlying

disease

indication

for

treatment

earlier

antifungal

treatment

risk

factors

for

invasive

fungal

infection

clinical

outcome

-LRB-

including

results

of

diagnostic

imaging

microbiology

lab

results

vital

signs

and

survival

-RRB-

occurrence

of

adverse

events

concurrent

medication

and

evaluation

of

response

Treatment

outcome

was

evaluated

by

the

investigator

Data

were

monitored

electronically

and

manually

for

plausibility

and

completeness

and

queries

were

raised

to

the

investigators

in

cases

that

were

unclear

or

incomplete

An

analysis

was

conducted

after

submission

of

episodes

The

objective

of

the

analysis

was

the

evaluation

of

drug

safety

and

efficacy

as

well

as

dosing

habits

and

indications

for

voriconazole

therapy

by

means

of

descriptive

analysis

All

episodes

were

registered

in

the

period

from

September

to

November

Drug

toxicities

were

evaluated

applying

common

toxicity

criteria

-LRB-

CTC

-RRB-

-LSB-

-RSB-

Results

One-hundred

patient

courses

from

the

hematological

units

of

five

different

centers

-LRB-

from

Frankfurt

am

Main

from

Oldenburg

from

Cologne

from

Frankfurt

an

der

Oder

and

from

Mainz

-RRB-

were

successfully

enrolled

in

the

survey

All

relevant

data

concerning

the

survey

objectives

were

present

The

most

frequent

underlying

disease

was

hematological

malignancy

-LRB-

-RRB-

A

total

of

patients

underwent

stem

cell

transplantation

of

these

were

allogeneic

and

showed

signs

of

acute

or

chronic

graft-versus-host

disease

-LRB-

GvHD

-RRB-

The

five

patients

with

other

hematological

diseases

were

two

patients

with

aplastic

anemia

two

with

myelodysplastic

syndrome

and

one

with

osteomyelofibrosis

Almost

half

of

the

patients

-LRB-

N

-RRB-

were

isolated

by

means

of

reverse

isolation

patients

had

air

conditioning

with

high-efficiency

particulate

air

filters

-LRB-

HEPA

-RRB-

Only

patients

were

accommodated

with

no

isolation

at

all

-LRB-

Table

-RRB-

Table

characteristics

at

baseline

and

subsequent

voriconazole

exposure

-LRB-

N

-RRB-

Age

-LRB-

years

-RRB-

a

-LRB-

-RRB-

-LRB-

-RRB-

Mean

weight

in

kg

-LRB-

SD

-RRB-

-LRB-

-RRB-

Underlying

diseaseb

Acute

myeloid

Acute

lymphoblastic

Low

grade

non-Hodgkin

Other

hematological

Other

non-malignant

hematological

Solid

cell

Unrelated

Sibling

Reverse

HEPAc

No

HEPAc

reverse

indication

Primary

Empirical

Suspected

or

proven

Secondary

on

voriconazole

-LRB-

SD

-RRB-

-LRB-

-RRB-

aMedian

bTwo

patients

with

multiple

cancers

cHigh

efficiency

particulate

air

filter

The

most

common

indication

for

voriconazole

treatment

was

suspected

or

proven

IFI

by

assessment

of

the

investigator

-LRB-

N

-RRB-

patients

received

voriconazole

as

empirical

treatment

for

an

episode

of

fever

of

unknown

origin

-LRB-

FUO

-RRB-

while

considered

at

risk

for

an

IFI

Other

treatment

indications

were

prophylaxis

of

IFI

-LRB-

N

-RRB-

of

which

were

considered

as

secondary

prophylaxis

after

an

earlier

episode

of

IFI

On

average

treatment

duration

was

days

-LRB-

Table

-RRB-

Fifteen

patients

died

during

the

observation

period

Causes

of

death

were

severe

sepsis

with

multi-organ

failure

-LRB-

N

-RRB-

progression

of

the

underlying

disease

-LRB-

N

-RRB-

progression

of

IFI

-LRB-

N

-RRB-

and

severe

arrhythmia

with

cardiac

arrest

-LRB-

N

-RRB-

The

investigator

attributed

the

lethal

arrhythmia

to

hypokalemia

several

days

after

switching

antifungal

therapy

from

voriconazole

to

liposomal

amphotericin

B.

For

the

patients

receiving

voriconazole

for

treatment

of

suspected

or

proven

IFI

the

lung

was

the

most

common

site

of

infection

-LRB-

N

-RRB-

Microbiological

evidence

was

obtained

in

two

cases

of

candidemia

one

invasive

pulmonary

aspergillosis

and

one

Geotrichum

capitatum

fungemia

All

other

patients

had

suspected

invasive

pulmonary

aspergillosis

by

the

investigator

's

assessment

Frequent

risk

factors

were

exposures

to

dust

due

to

nearby

construction

sites

-LRB-

N

-RRB-

leukopenia

-LRB-

N

-RRB-

indwelling

central

venous

catheters

-LRB-

N

-RRB-

immunosuppressive

therapy

-LRB-

N

-RRB-

and

mucositis

-LRB-

N

-RRB-

The

initial

daily

voriconazole

dose

averaged

mg/kg

Almost

half

of

the

patients

were

treated

orally

from

the

beginning

-LRB-

N

-RRB-

In

of

the

remaining

patients

therapy

was

later

switched

to

oral

administration

Treatment

success

was

rated

by

the

investigators

as

complete

or

partial

response

in

-LRB-

-RRB-

patients

Disease

remained

stable

in

another

-LRB-

-RRB-

patients

Progressive

disease

was

diagnosed

in

the

remaining

patients

-LRB-

-RRB-

Fifteen

-LRB-

-RRB-

patients

were

switched

to

a

different

antifungal

class

-LRB-

Table

-RRB-

Table

of

patients

with

suspected

or

proven

IFI

by

assessment

of

the

investigator

-LRB-

N

-RRB-

Site

of

infectiona

-LRB-

-RRB-

Blood

-LRB-

fungemia

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Risk

factors

Diabetes

-LRB-

-RRB-

-LRB-

-RRB-

Leukopeniab

-LRB-

-RRB-

-LRB-

-RRB-

Prior

-LRB-

-RRB-

Central

venous

-LRB-

-RRB-

Dust

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Purine

-LRB-

-RRB-

-LRB-

-RRB-

Other

-LRB-

-RRB-

Days

with

fever

-LRB-

SD

-RRB-

-LRB-

-RRB-

Total

days

on

antibiotic

treatment

-LRB-

SD

-RRB-

-LRB-

-RRB-

Days

on

voriconazole

-LRB-

SD

-RRB-

-LRB-

-RRB-

Average

initial

voriconazole

mg/kg

-LRB-

range

-RRB-

-LRB-

-RRB-

Route

of

administration

Oral

-LRB-

-RRB-

Switch

to

-LRB-

-RRB-

Switch

to

-LRB-

-RRB-

Outcomec

Complete

-LRB-

-RRB-

Partial

-LRB-

-RRB-

Stable

-LRB-

-RRB-

Progressive

-LRB-

-RRB-

Switch

to

other

-LRB-

-RRB-

Liposomal

amphotericin

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aSuper

additive

because

of

patients

with

multiple

infection

sites

bAs

defined

as

less

than

leukocytes/μl

cAs

assessed

by

investigator

An

evaluation

of

the

investigators

adherence

to

EORTC/MSG

criteria

showed

a

discrepancy

between

the

softer

criteria

used

in

recent

clinical

trials

-LSB-

-RSB-

and

the

official

criteria

-LSB-

-RSB-

While

recent

trials

allowed

the

investigator

to

rate

evidence

as

probable

without

microbiological

findings

the

official

criteria

demand

at

least

one

microbiological

criterion

Thus

of

the

cases

with

suspected

or

proven

IFI

had

probable

IFI

according

to

the

softer

criteria

but

only

four

patients

had

probable

IFI

when

applying

the

official

EORTC/MSG

criteria

Independent

of

the

applied

edition

of

the

EORTC/MSG

criteria

patients

did

not

meet

the

requirements

for

classification

of

evidence

even

as

possible

-LRB-

Table

-RRB-

Table

rating

for

patients

with

IFI

suspected

or

proven

by

the

investigator

-LRB-

N

-RRB-

Investigator

assessmentAccording

to

criteria

from

recent

clinical

trials

-LSB-

-RSB-

According

to

EORTC/MSG

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Not

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Values

in

parenthesis

are

in

percentage

In

the

patients

where

voriconazole

was

given

as

empirical

therapy

oral

administration

was

preferred

-LRB-

N

-RRB-

Average

duration

of

fever

was

higher

than

in

those

with

suspected

or

proven

IFI

averaging

days

Treatment

duration

averaged

days

markedly

shorter

than

the

other

subgroups

Eleven

patients

improved

under

empirical

treatment

six

were

stable

while

four

showed

signs

of

progressive

infection

There

was

a

frequent

switch

of

therapy

to

antifungals

of

different

classes

i.e.

liposomal

amphotericin

B

-LRB-

N

-RRB-

caspofungin

-LRB-

N

-RRB-

or

their

combination

-LRB-

N

-RRB-

-LRB-

Table

-RRB-

Table

dosing

and

treatment

outcomeEmpirical

treatment

-LRB-

N

-RRB-

Secondary

prophylaxis

-LRB-

N

-RRB-

Average

initial

voriconazole

dose

in

mg/kg

-LRB-

range

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Route

of

administration

Oral

-LRB-

-RRB-

-LRB-

-RRB-

Switch

to

-LRB-

-RRB-

Switch

to

intravenous

-LRB-

-RRB-

Days

with

fever

-LRB-

SD

-RRB-

-LRB-

-RRB-

n.

a.Days

on

antibiotic

treatment

-LRB-

SD

-RRB-

-LRB-

-RRB-

n.

a.Days

on

voriconazole

-LRB-

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Breakthrough

IFIn

a.

-LRB-

-RRB-

Outcomea

Progressive

-LRB-

-RRB-

n.

a.

Stable

-LRB-

-RRB-

n.

a.

Partial

-LRB-

-RRB-

n.

a.

Complete

-LRB-

-RRB-

n.

a.Switch

to

other

-LRB-

-RRB-

-LRB-

-RRB-

Liposomal

amphotericin

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Liposomal

amphotericin

B

-LRB-

-RRB-

Almost

all

patients

on

voriconazole

as

secondary

prophylaxis

-LRB-

N

-RRB-

received

their

treatment

orally

-LRB-

N

-RRB-

Mean

treatment

duration

was

days

Four

breakthrough

IFIs

were

reported

Therapy

was

switched

to

other

antifungals

in

seven

cases

most

often

to

caspofungin

-LRB-

N

-RRB-

-LRB-

Table

-RRB-

Table

adverse

events

-LRB-

N

-RRB-

Visual

events

at

least

possibly

related

to

voriconazole

by

judgment

of

the

investigator

The

most

frequent

adverse

events

at

least

possibly

related

to

voriconazole

treatment

by

the

investigators

were

visual

disturbances

-LRB-

N

-RRB-

nausea

-LRB-

N

-RRB-

and

rash

-LRB-

N

-RRB-

-LRB-

Table

-RRB-

At

least

one

liver

function

test

showed

grade

toxicity

applying

the

common

toxicity

criteria

in

cases

seven

of

which

were

attributed

to

voriconazole

by

the

investigators

In

this

survey

gamma

glutamyl

transpeptidase

was

the

most

sensitive

marker

for

voriconazole

toxicity

showing

a

marked

elevation

in

cases

of

investigator

attributed

toxicity

However

no

definite

preference

could

be

detected

for

any

of

the

liver

parameters

assessed

of

the

four

patients

with

grade

nephrotoxicity

were

attributed

to

voriconazole

-LRB-

Table

-RRB-

Table

grade

of

treatment-emergent

renal

and

hepatic

adverse

eventsCTC-toxicity

-LSB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

N

evaluableLiver

GOT

-LRB-

AST

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

GPT

-LRB-

ALT

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

Kidney

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

-LRB-

-RRB-

a

Values

given

in

parenthesis

are

in

percentage

aToxicity

judged

at

least

possibly

attributable

to

voriconazole

treatment

by

the

investigator

Discussion

In

our

survey

voriconazole

was

efficacious

in

of

patients

treated

for

suspected

or

proven

IFI

-LRB-

by

assessment

of

the

investigator

-RRB-

Favorable

response

was

documented

for

of

patients

receiving

voriconazole

as

empirical

therapy

This

observation

is

comparable

to

prior

results

For

the

treatment

of

suspected

or

proven

IFI

especially

IPA

investigator

assessed

favorable

results

defined

as

complete

or

partial

responses

were

obtained

in

as

compared

to

in

Herbrecht

's

trial

-LSB-

-RSB-

In

the

empirical

setting

favorable

response

was

reported

in

while

only

a

overall

response

was

reported

previously

-LSB-

-RSB-

However

the

outcome

evaluation

was

supposedly

far

more

rigid

in

the

clinical

trials

than

the

self-assessment

of

the

registry

contributors

When

compared

to

earlier

prospective

controlled

clinical

trials

-LSB-

-RSB-

adverse

events

were

less

common

Visual

disturbances

were

only

reported

in

and

hallucinations

in

as

compared

to

and

respectively

in

aspergillosis

-LSB-

-RSB-

and

to

and

respectively

in

febrile

neutropenia

-LSB-

-RSB-

However

adverse

event

reporting

is

naturally

less

stringent

in

a

survey

than

in

controlled

clinical

trials

Analysis

of

survey

data

did

not

reveal

yet

unknown

toxicities

of

voriconazole

Treatment-related

grade

or

hepatotoxicity

was

observed

in

patients

Further

frequent

adverse

events

were

visual

disturbances

-LRB-

-RRB-

rash

-LRB-

-RRB-

and

nausea

-LRB-

-RRB-

Significant

elevations

of

serum

alkaline-phosphatase

activities

-LRB-

baseline

value

-RRB-

occurred

in

compared

to

of

grade

CTC

toxicity

in

our

study

aspartate

aminotransferase

-LRB-

baseline

value

-RRB-

in

versus

alanine

aminotransferase

-LRB-

baseline

value

-RRB-

in

versus

and

bilirubin

-LRB-

baseline

value

-RRB-

in

versus

-LSB-

-RSB-

Our

survey

demonstrates

that

clinical

reality

regularly

strays

from

approved

indications

and

evidence

based

choice

of

treatment

A

total

of

of

the

reported

patients

received

voriconazole

prophylactically

or

empirically

One

third

of

the

patients

with

voriconazole

for

treatment

of

actual

IFI

lacked

sufficient

evidence

of

IFI

according

to

EORTC/MSG

criteria

-LSB-

-RSB-

Thus

a

rate

of

as

much

as

may

be

considered

as

off-label

use

Furthermore

all

randomized

controlled

clinical

trials

on

voriconazole

used

the

intravenous

formulation

as

initial

treatment

Despite

this

of

patients

with

at

least

possible

IFI

were

started

on

oral

voriconazole

Looking

at

these

figures

one

could

claim

that

only

of

the

patients

reported

were

treated

as

approved

by

the

German

health

administration

and

according

to

best

scientific

evidence

But

does

this

mean

patients

were

not

treated

in

their

best

interest

The

difficulty

of

proving

IFI

remains

a

dilemma

in

choosing

adequate

treatment

There

is

still

no

sensitive

and

specific

non-invasive

method

for

detection

of

invasive

pulmonary

aspergillosis

-LRB-

IPA

-RRB-

-LSB-

-RSB-

Controlled

clinical

trials

on

antifungal

agents

normally

use

the

current

EORTC/MSG

criteria

with

or

without

modifications

while

the

consensus

committee

clearly

discourages

from

clinical

decision-making

based

on

their

guideline

-LSB-

-RSB-

Additionally

especially

in

palliative

situations

long-term

inpatient

treatment

with

intravenous

antifungals

is

often

undesirable

Finally

no

antifungal

has

yet

been

approved

for

secondary

prophylaxis

of

IPA

In

the

above

clinical

situations

voriconazole

offers

a

flexible

and

tolerable

treatment

option

Still

the

many

difficulties

in

the

diagnosis

and

treatment

of

invasive

fungal

infections

should

not

be

used

as

a

carte

blanche

in

clinical

decision

making

Physicians

should

strive

towards

establishing

the

most

accurate

diagnosis

possible

and

then

treat

according

to

current

evidence

O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

antifungals
aspergillosis
candidiasis
fungal infection
voriconazole
prophylaxis