text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 1, 2010
|Filing date
|Dec 15, 2005
|Priority date
|Dec 15, 2005
|Fee status
|Also published as
|Publication number
|John Gavin MacDonald, Stephanie M. Martin, Jason Lye
|Original Assignee
|Kimberly-Clark Worldwide, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (53), Non-Patent Citations (46), Referenced by (3), Classifications (12), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
Conjunctivitis is the most common eye disorder and may vary in severity from a mild inflammation with tearing to a severe inflammation that causes tissue injury. Infectious conjunctivitis is generally caused by the presence of bacteria or viruses. A variety of bacteria may cause bacterial conjunctivitis, such as S. pneumoniae, H. influenzae, P. aeruginosa, or S. pyogenes. Likewise, a variety of viruses may be responsible for viral conjunctivitis, such as adenovirus, herpes simplex virus (HSV), varicella-zoster virus (VZV), picornavirus (enterovirus 70, Coxsackie A24), poxvirus (molluscum contagiosum, vaccinia), and human immunodeficiency virus (HIV). Adenoviral conjunctivitis is the most common cause of viral conjunctivitis. Particular subtypes of adenoviral conjunctivitis include epidemic keratoconjunctivitis (pink eye) and pharyngoconjunctival fever. Primary ocular herpes simplex infection is also common, particularly in children, and is normally associated with a follicular conjunctivitis.
The treatment protocol for infectious conjunctivitis largely depends on whether the infection is viral or bacterial. Viral conjunctivitis is self-limiting and may require no specific treatment, other than drops to alleviate any symptoms associated with the infection. On the other hand, patients having bacterial conjunctivitis are typically treated with topical antibiotics (e.g., sulfacetamide sodium 10% drops or trimethoprim/polymyxin B qid). Conventional techniques for diagnosing the type of ocular infection rely primarily on clinical examination. For example, because the discharge in bacterial conjunctivitis is usually more purulent than the watery discharge of viral conjunctivitis, physical examination may be used in the diagnosis. Unfortunately, such diagnostic techniques are often unreliable and lead to the prescription of antibiotic drops in cases in which they are not needed.
As such, a need currently exists for a technique of rapidly screening for bacterial conjunctivitis in a patient.
In accordance with one embodiment of the present invention, a method for screening for bacterial conjunctivitis is disclosed. The method comprises contacting an ocular test sample with a chromogen. The chromogen undergoes a detectable color change in less than about 30 minutes in the presence of bacteria at a pathogenic concentration.
Other features and aspects of the present invention are discussed in greater detail below.
A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, which makes reference to the appended figures in which:
Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.
As used herein, the term "ocular test sample" generally refers to a biological material obtained directly or indirectly from the eye of a host, such as from ocular lens fluid (e.g., tears), discharge, tissue, etc. The test sample may be obtained in by any method desired, such as using a swab. The test sample may also be used as obtained or pretreated in some manner. For example, such pretreatment may include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc.
As used herein, the term "host" refers to any animal, preferably a human.
Reference now will be made in detail to various embodiments of the invention, one or more examples of which are set forth below. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, may be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present invention covers such modifications and variations as come within the scope of the appended claims and their equivalents.
Generally speaking, the present invention is directed to a method for rapidly screening for bacterial conjunctivitis in a host. The method includes contacting an ocular test sample with a chromogen (e.g., Reichardt's dye) that exhibits a color change in the presence of a microbe. The present inventors have discovered that the extent of the color change may vary depending on whether the microbe is a bacteria or virus. Without intending to be limited by theory, the present inventors believe that the chromogen interacts with the peptidoglycan-based cell wall structure of bacteria to induce a color change that is even more apparent at infectious levels. It is believed that this interaction occurs to a much greater extent in bacteria than in viruses. Accordingly, although the chromogen may still undergo a color change in the presence of the viruses, it is typically to a much lesser extent. In this manner, the degree of color change of the chromogen may be used in the present invention as a mechanism for differentiating between viral and bacterial conjunctivitis.
Any of a variety of bacteria and/or viruses may be detected in accordance with the present invention. For example, gram-positive cocci (e.g., Staphylococcus epidermidis, Streptococcus pyogenes, and Streptococcus pneumoniae); gram-negative cocci (e.g., P. aeruginosa, Neisseria meningitides, and Moraxella lacunata); and gram-negative rods (e.g., Haemophilus influenzae) are often associated with bacterial conjunctivitis and may be detected in the present invention. Gram-negative bacteria have a cell wall coated with lipopolysaccharide (LPS). Gram-positive bacteria are coated with thick peptidoglycan (or murein) sheet-like layers. The most prevalent causes of bacterial conjunctivitis are Streptococcus pneumoniae, Haemophilus influenzae, or Streptococcus pyogenes. Streptococcus pyogenes is a Gram-positive, nonmotile, nonsporeforming cocci that occurs in chains or in pairs of cells. Streptococcus pyogenes is a catalase-negative aerotolerant anaerobe (facultative anaerobe) and requires enriched medium containing blood in order to grow. Haemophilus influenzae is a small, nonmotile Gram-negative bacterium in the family Pasteurellaceae. Non-encapsulated strains of Haemophilus influenzae are believed to primarily cause conjunctivitis.
Viruses contain a nucleic acid enclosed within a protein organized in capsomer subunits ("capsid"). Some viruses also contain a glycoprotein envelope surrounding the capsid that is composed of two lipid layers interspersed with protein molecules (lipoprotein bilayer) and may contain material from the membrane of a host cell or viral origin. Viruses may also develop spikes made of glycoprotein on their envelopes that help them to attach to specific cell surfaces. The most common form of viral conjunctivitis that may be detected in accordance with the present invention is caused by adenoviruses of the family Adenoviridae. Epidemic keratoconjunctivitis, for example, is associated with adenovirus serotypes 8, 19, and 37. Adenoviruses are double-stranded DNA viruses that contain icosahedral capsids with twelve vertices and seven surface proteins. The virion is non-enveloped and spherical. Herpes viruses are also commonly associated with viral conjunctivitis. Herpes viruses contain icosahedral capsids surrounded by a thick layer of additional proteins (Tegument) and an outer envelope with spike-like glycoproteins. The capsid contains 162 capsomers enclosing a core structure containing double-stranded linear viral DNA. Herpes viruses are classified into three groups based on tissue tropism, pathogenicity, and behavior under culture conditions in the laboratory, i.e., α-, β- and γ-herpes viruses. Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) are in the α-subfamily of herpes viruses. HSV-1 is most commonly associated with viral conjunctivitis.
The chromogen is selected in accordance with the present invention to interact in some manner with the cell membrane of the microbe and/or the environment in which the microbe is present. As a result of this interaction, the chromogen undergoes a change in color that is readily detectable (e.g., with an unaided eye). The term "color" relates to the presence or absence of certain wavelengths of light reflected or emitted from objects in a visual field. Light entering the eye, for example, is subjected to a spectral analysis by three types of retinal cone cells that are sensitive to specific regions of the visible spectrum. Stimuli from these cells are in turn processed by retinal neurons, optic nerve neurons and the visual cortex so that a sensation of color is experienced. The chromogen employed in the present invention typically owes its color to the absorption of certain wavelengths of light. As such, the perceived color is usually the complement of the color associated with the wavelength of light being absorbed by the object. An object that appears to be red in color when viewed in white light, for example, is in fact selectively absorbing bluish light in the wavelength range of 490 to 500 nanometers. Similarly, an object that appears yellow in white light is in fact absorbing blue light in the wavelength range of 435 to 480 nanometers.
The absorption of visible light is associated with electronic transitions within a molecule and results in the generation of an excited state. The energy difference between the ground state of the molecule and the relevant excited state determines the wavelength of the light absorbed according to the Planck relationship:
E is energy,
h is Planck's constant, and
ν is the frequency of the photon of light absorbed, and is related to wavelength λ and the speed of light c by ν=c/λ.
A state diagram, such as shown below, may be used to graphically depict electronic transitions:
The energy of an absorbed photon is thus inversely proportional to the wavelength of the photon. Thus, photons of blue light (435-480 nanometers) have a higher energy than yellow light (580-595 nanometers). As such, the color of the chromogen is determined by the transition energy between the ground state of the chromogen and the first allowed excited state.
The light-absorbing portion of the chromogen employed in the present invention is a chromophore that is generally responsible for the color of the chromogen and is connected to a conjugated system. For instance, azo groups (e.g., azo dyes), polyene groups (e.g., carotene dye), carbonyl groups (e.g., anthraquinone dyes) are common chromophores. Auxochromes may induce a shift of the absorption maxima of the chromophore towards either the red end of the spectrum ("bathochromic shift") or the blue end of the spectrum ("hypsochromic shift"). Auxochromes may or may not be conjugated with the chromogen. For instance, an amino group conjugated to an azo group (chromophore) via, for instance, a benzene ring, will form an aminoazo chromogen. The type of absorption shift depends on the nature of the chromophore and, for example, on whether the auxochrome functions as an electron acceptor, in which a hypsochromic shift results, or whether the amino group functions as an electron donor, in which a bathochromic shift results. For example, a conjugated amino auxochrome will shift the absorption band of the azo group to longer wavelengths and increases the intensity of the absorption band. The absorption shift provides a color difference that is detectable, either visually or through instrumentation.
One particularly suitable class of chromogens that may undergo a detectable color change in the presence of bacteria is solvatochromic dyes. Solvatochromic dyes are dyes having spectroscopic characteristics (e.g., absorption) in the ultraviolet/visible/near-infrared spectrum and are sometimes influenced by the surrounding medium. The solvatochromic dyes may be positive or negative, which corresponds to bathochromic and hypsochromic shifts, respectively, of the emission band with increasing solvent polarity. In one embodiment, for instance, the solvatochromic dye undergoes a color change in a certain molecular environment based on solvent polarity and/or hydrogen bonding propensity. For example, a solvatochromic dye may be blue in a polar environment (e.g., water), but yellow or red in a non-polar environment (e.g., lipid-rich solution). The color produced by the solvatochromic dye depends on the molecular polarity difference between the ground and excited state of the dye.
Merocyanine dyes (e.g., mono-, di-, and tri-merocyanines) are one example of a type of solvatochromic dye that may be employed in the present invention. Merocyanine dyes, such as merocyanine 540, fall within the donor-simple acceptor chromogen classification of Griffiths as discussed in "Colour and Constitution of Organic Molecules" Academic Press, London (1976). More specifically, merocyanine dyes have a basic nucleus and acidic nucleus separated by a conjugated chain having an even number of methine carbons. Such dyes possess a carbonyl group that acts as an electron acceptor moiety. The electron acceptor is conjugated to an electron donating group, such as a hydroxyl or amino group. The merocyanine dyes may be cyclic or acyclic (e.g., vinylalogous amides of cyclic merocyanine dyes). For example, cyclic merocyanine dyes generally have the following structure:
wherein, n is any integer, including 0. As indicated above by the general structures 1 and 1′, merocyanine dyes typically have a charge separated (i.e., "zwifterionic") resonance form. Zwitterionic dyes are those that contain both positive and negative charges and are net neutral, but highly charged. Without intending to be limited by theory, it is believed that the zwitterionic form contributes significantly to the ground state of the dye. The color produced by such dyes thus depends on the molecular polarity difference between the ground and excited state of the dye. One particular example of a merocyanine dye that has a ground state more polar than the excited state is set forth below as structure 2.
The charge-separated left hand canonical 2 is a major contributor to the ground state whereas the right hand canonical 2′ is a major contributor to the first excited state. Still other examples of suitable merocyanine dyes are set forth below in the following structures 3-13.
wherein, "R" is a group, such as methyl, alkyl, aryl, phenyl, etc.
Indigo is another example of a suitable solvatochromic dye for use in the present invention. Indigo has a ground state that is significantly less polar than the excited state. For example, indigo generally has the following structure 14:
The left hand canonical form 14 is a major contributor to the ground state of the dye, whereas the right hand canonical 14′ is a major contributor to the excited state.
Other suitable solvatochromic dyes that may be used in the present invention include those that possess a permanent zwitterionic form. That is, these dyes have formal positive and negative charges contained within a contiguous π-electron system. Contrary to the merocyanine dyes referenced above, a neutral resonance structure cannot be drawn for such permanent zwitterionic chromogens. Exemplary dyes of this class include N-phenolate betaine dyes, such as those having the following general structure:
wherein R1-R5 are independently selected from the group consisting of hydrogen, a nitro group (e.g., nitrogen), a halogen, or a linear, branched, or cyclic C1 to C20 group (e.g., alkyl, phenyl, aryl, pyridinyl, etc.), which may be saturated or unsaturated and unsubstituted or optionally substituted at the same or at different carbon atoms with one, two or more halogen, nitro, cyano, hydroxy, alkoxy, amino, phenyl, aryl, pyridinyl, or alkylamino groups. For example, the N-phenolate betaine dye may be 4-(2,4,6-triphenylpyridinium-1-yl)-2,6-diphenylphenolate (Reichardt's dye) having the following general structure 15:
Reichardt's dye shows strong negative solvatochromism. That is, Reichardt's dye displays a shift in absorbance to a shorter wavelength and thus has visible color changes as solvent eluent strength (polarity) increases. Still other examples of suitable negatively solvatochromic pyridinium N-phenolate betaine dyes are set forth below in structures 16-23:
wherein, R is hydrogen, —C(CH3)3, —CF3, or C6F13.
Still additional examples of dyes having a permanent zwitterionic form include dyes having the following general structure 24:
wherein, n is 0 or greater, and X is oxygen, carbon, nitrogen, sulfur, etc. Particular examples of the permanent zwitterionic dye shown in structure 24 include the following structures 25-33.
Still other suitable solvatochromic dyes may include, but are not limited to 4-dicyanmethylene -2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM); 6-propionyl -2-(dimethylamino)naphthalene (PRODAN); 9-(diethylamino)-5H-benzo[a]phenox-azin-5-one (Nile Red); 4-(dicyanovinyl)julolidine (DCVJ); phenol blue; stilbazolium dyes; coumarin dyes; ketocyanine dyes; N,N-dimethyl-4-nitroaniline (NDMNA) and N-methyl-2-nitroaniline (NM2NA); Nile blue; 1-anilinonaphthalene -8-sulfonic acid (1,8-ANS), and dapoxylbutylsulfonamide (DBS) and other dapoxyl analogs. Besides the above-mentioned dyes, still other suitable dyes that may be used in the present invention include, but are not limited to, 4-[2-N-substituted -1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one, red pyrazolone dyes, azomethine dyes, indoaniline dyes, and mixtures thereof.
Although the above-referenced dyes are classified as solvatochromic, it should be understood that the present invention is not necessarily limited to any particular mechanism for the color change of the chromogen. Even when a solvatochromic dye is employed, other mechanisms may actually be wholly or partially responsible for the color change of the dye. For example, acid-base or proton donation reactions between the dye and microbe may result in the color change. As an example, highly organized acid moieties on bacteria cell walls may protonate certain dyes, resulting in a loss of color. Redox reactions between the dye and microbe may likewise contribute to the color change.
The chromogen may be used alone or as part of a detection composition. If desired, the detection composition may contain a carrier for the chromogen that functions as a mobile phase. The carrier may be a liquid, gas, gel, etc., and may be selected to provide the desired performance (time for change of color, contrast between different areas, and sensitivity) of the chromogen. In some embodiments, for instance, the carrier may be an aqueous solvent, such as water, as well as a non-aqueous solvent, such as glycols (e.g., propylene glycol, butylene glycol, triethylene glycol, hexylene glycol, polyethylene glycols, ethoxydiglycol, and dipropyleneglycol); alcohols (e.g., methanol, ethanol, n-propanol, and isopropanol); triglycerides; ethyl acetate; acetone; triacetin; acetonitrile, tetrahydrafuran; xylenes; formaldehydes (e.g., dimethylformamide); etc. The amount of the carrier and chromogen in the detection composition may generally vary based on the level of microbe sensitivity and-color pattern or design utilized. For instance, in some embodiments, the chromogen may be present in the detection composition at a concentration from about 0.1 to about 100 milligrams per milliliter of carrier, in some embodiments from about 0.5 to about 60 milligrams per milliliter of carrier, and in some embodiments, from about 1 to about 40 milligrams per milliliter of carrier.
Besides the chromogen and carrier, the detection composition may also contain a variety of other components. In one embodiment, for instance, additives are incorporated into the detection composition that enhance the performance of the chromogen. For example, cyclodextrins may enhance the sensitivity of the chromogen and the contrast between regions that regions of different color. While not wishing to be bound by theory, the present inventors believe that such additives may inhibit the crystallization of the dye and thus provide a more vivid color and also enhance detection sensitivity. That is, single dye molecules have greater sensitivity for microbes because each dye molecule is free to interact with the microbial membrane. In contrast, small crystals of dye have to first dissolve and then penetrate the membrane. Examples of suitable cyclodextrins may include, but are not limited to, hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, and hydroxyethyl-γ-cyclodextrin, which are commercially available from Cerestar International of Hammond, Ind.
Surfactants may also help enhance the sensitivity of the chromogen and the contrast between different regions. Particularly desired surfactants are nonionic surfactants, such as ethoxylated alkylphenols, ethoxylated and propoxylated fatty alcohols, ethylene oxide-propylene oxide block copolymers, ethoxylated esters of fatty (C8-C18) acids, condensation products of ethylene oxide with long chain amines or amides, condensation products of ethylene oxide with alcohols, acetylenic diols, and mixtures thereof. Various specific examples of suitable nonionic surfactants include, but are not limited to, methyl gluceth-10, PEG-20 methyl glucose distearate, PEG-20 methyl glucose sesquistearate, C11-15 pareth -20, ceteth-8, ceteth-12, dodoxynol-12, laureth-15, PEG-20 castor oil, polysorbate 20, steareth-20, polyoxyethylene-10 cetyl ether, polyoxyethylene-10 stearyl ether, polyoxyethylene-20 cetyl ether, polyoxyethylene-10 oleyl ether, polyoxyethylene -20 oleyl ether, an ethoxylated nonylphenol, ethoxylated octylphenol, ethoxylated dodecylphenol, or ethoxylated fatty (C6-C22) alcohol, including 3 to 20 ethylene oxide moieties, polyoxyethylene-20 isohexadecyl ether, polyoxyethylene-23 glycerol laurate, polyoxy-ethylene-20 glyceryl stearate, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, polyoxyethylene-20 sorbitan monoesters, polyoxyethylene-80 castor oil, polyoxyethylene-15 tridecyl ether, polyoxy-ethylene -6 tridecyl ether, laureth-2, laureth-3, laureth-4, PEG-3 castor oil, PEG 600 dioleate, PEG 400 dioleate, and mixtures thereof. Commercially available nonionic surfactants may include the SURFYNOL� range of acetylenic diol surfactants available from Air Products and Chemicals of Allentown, Pa. and the TWEEN� range of polyoxyethylene surfactants available from Fischer Scientific of Pittsburgh, Pa.
The detection composition may also contain a binder to facilitate the immobilization of the chromogen on a substrate. For example, water-soluble organic polymers may be employed as binders. One suitable class of water-soluble organic polymers includes polysaccharides and derivatives thereof. Polysaccharides are polymers containing repeated carbohydrate units, which may be cationic, anionic, nonionic, and/or amphoteric. In one particular embodiment, the polysaccharide is a nonionic, cationic, anionic, and/or amphoteric cellulosic ether. Suitable nonionic cellulosic ethers may include, but are not limited to, alkyl cellulose ethers, such as methyl cellulose and ethyl cellulose; hydroxyalkyl cellulose ethers, such as hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl hydroxybutyl cellulose, hydroxyethyl hydroxypropyl cellulose, hydroxyethyl hydroxybutyl cellulose and hydroxyethyl hydroxypropyl hydroxybutyl cellulose; alkyl hydroxyalkyl cellulose ethers, such as methyl hydroxyethyl cellulose, methyl hydroxypropyl cellulose, ethyl hydroxyethyl cellulose, ethyl hydroxypropyl cellulose, methyl ethyl hydroxyethyl cellulose and methyl ethyl hydroxypropyl cellulose; and so forth.
If desired, the detection composition may be applied to a substrate, which is subsequently contacted with the infected test sample. Suitable application techniques include printing, dipping, spraying, melt extruding, coating (e.g., solvent coating, powder coating, brush coating, etc.), and so forth. Upon application, the detection composition is dried to remove the carrier and leave a residue of the chromogen for interacting with a microbe. For example, the detection composition may be printed onto a surface of the substrate to signal the presence of microbes upon a change in color. The detection composition may cover all or only a portion of the substrate surface. In one embodiment, for example, the detection composition is printed in the form of indicia that conveys a certain message to the user. The substrate may be formed from any of a variety materials capable of being applied with the detection composition. For example, the substrate may be formed from film, paper, a nonwoven fabric, a knitted fabric, a woven fabric, foam, etc. The substrate may be incorporated into a wide variety of articles, such as strips, lateral flow devices, stickers, test kits, swab & know cards, facial tissues, diapers, throat swabs, wound dressings, and so forth. In one particular embodiment, the substrate is a facestock material commonly employed in the manufacture of labels, such as paper, polyester, polyethylene, polypropylene, polybutylene, polyamides, etc. An adhesive, such as a pressure-sensitive adhesive, heat-activated adhesive, hot melt adhesive, etc., may be employed on one or more surfaces of the facestock material to help adhere it to a surface. Suitable examples of pressure-sensitive adhesives include, for instance, acrylic-based adhesives and elastomeric adhesives. In one embodiment, the pressure-sensitive adhesive is based on copolymers of acrylic acid esters (e.g., 2-ethyl hexyl acrylate) with polar co-monomers (e.g., acrylic acid). The adhesive may have a thickness in the range of from about 0.1 to about 2 mils (2.5 to 50 microns). A release liner may also be employed that contacts the adhesive prior to use. The release liner may contain any of a variety of materials known to those of skill in the art, such as a silicone-coated paper or film substrate. Although the discussion above refers to the application of the detection composition to the substrate, it should be understood that additive(s) may also be applied separately from the chromogen-containing detection composition.
Regardless of the manner in which it is applied, the amount of the chromogen employed is effective to result in a detectable color change upon contact with bacteria. The exact quantity may vary based on a variety of factors, including the sensitivity of the chromogen, the presence of other additives in the detection composition, the desired degree of detectability (e.g., with an unaided eye), the suspected concentration of the microbe, etc. In some cases, it is desirable to only detect the presence of bacteria at concentrations that are considered pathogenic. For example, a bacteria concentration of about 1�105, in some embodiments about 1�106, and in some embodiments, about 1�108 colony forming units ("CFU") per milliliter of a test sample may be considered a threshold pathogenic concentration for bacterial conjunctivitis. Thus, the chromogen may be present in an amount sufficient to undergo a detectable color change in the presence of bacteria at a concentration of at least about 1�108 CFU per milliliter of the test sample. For example, when present on a substrate, the chromogen may constitute from about 0.001 wt. % to about 20 wt. %, in some embodiments from about 0.01 wt. % to about 10 wt. %, and in some embodiments from about 0.1 wt. % to about 5 wt. % of the dry weight of the substrate. The amount of other additives (e.g., cyclodextrins, surfactants, binders, etc.) may also vary as desired, such as from about 0.001 wt. % to about 10 wt. %, in some embodiments from about 0.01 wt. % to about 5 wt. %, and in some embodiments from about 0.025 wt. % to about 1 wt. % based on the dry weight of the substrate.
As stated above, the degree to which the chromogen changes color provides information regarding the presence of bacteria or virus to which it is exposed. For example, Reichardt's dye shows strong negative solvatochromism and may thus undergo a significant color change from blue to colorless in the presence of bacteria. In the presence of certain viruses, the color change occurs to a lesser extent if at all. Thus, when an infected test sample is placed in to contact with the dye, the color change may simply be observed to determine whether the infection is caused by bacteria. That is, if the color change occurs to a certain degree (e.g., from blue to colorless), it may be determined that the infected test sample contains bacteria. Likewise, if a color change occurs to a lesser extent (e.g., from blue to faint blue), it may be determined that the test sample contains an infectious level of a virus or that the conjunctivitis is simply due to non-infectious causes, such as allergenic conjunctivitis. In either case, however, it will become readily apparent whether or not an antibiotic is needed.
The color of a reacted test chromogen may be compared (e.g., visually or with the aid of an instrument) to the color of a control chromogen, which is formed from a compound that is the same or similar to the test chromogen with respect to its responsiveness to microbes. Multiple control chromogens may likewise be employed that correspond to different types of bacteria at the threshold pathogenic concentration. Upon comparison, one or more of the control chromogens may be selected that have a color that is the same or substantially similar to a test chromogen reacted with a test sample. The type of microbe within the test sample is then determined from the selected control chromogen(s) and the corresponding known microbe(s).
The degree to which the chromogen changes color may be determined either visually or using instrumentation. In one embodiment, color intensity is measured with an optical reader. The actual configuration and structure of the optical reader may generally vary as is readily understood by those skilled in the art. Typically, the optical reader contains an illumination source that is capable of emitting electromagnetic radiation and a detector that is capable of registering a signal (e.g., transmitted or reflected light). The illumination source may be any device known in the art that is capable of providing electromagnetic radiation, such as light in the visible or near-visible range (e.g., infrared or ultraviolet light). For example, suitable illumination sources that may be used in the present invention include, but are not limited to, light emitting diodes (LED), flashlamps, cold-cathode fluorescent lamps, electroluminescent lamps, and so forth. The illumination may be multiplexed and/or collimated. In some cases, the illumination may be pulsed to reduce any background interference. Further, illumination may be continuous or may combine continuous wave (CW) and pulsed illumination where multiple illumination beams are multiplexed (e.g., a pulsed beam is multiplexed with a CW beam), permitting signal discrimination between a signal induced by the CW source and a signal induced by the pulsed source. For example, in some embodiments, LEDs (e.g., aluminum gallium arsenide red diodes, gallium phosphide green diodes, gallium arsenide phosphide green diodes, or indium gallium nitride violet/blue/ultraviolet (UV) diodes) are used as the pulsed illumination source. One commercially available example of a suitable UV LED excitation diode suitable for use in the present invention is Model NSHU55 OE (Nichia Corporation), which emits 750 to 1000 microwatts of optical power at a forward current of 10 milliamps (3.5-3.9 volts) into a beam with a full-width at half maximum of 10 degrees, a peak wavelength of 370-375 nanometers, and a spectral half-width of 12 nanometers.
In some cases, the illumination source may provide diffuse illumination to the chromogen. For example, an array of multiple point light sources (e.g., LEDs) may simply be employed to provide relatively diffuse illumination. Another particularly desired illumination source that is capable of providing diffuse illumination in a relatively inexpensive manner is an electroluminescent (EL) device. An EL device is generally a capacitor structure that utilizes a luminescent material (e.g., phosphor particles) sandwiched between electrodes, at least one of which is transparent to allow light to escape. Application of a voltage across the electrodes generates a changing electric field within the luminescent material that causes it to emit light.
The detector may generally be any device known in the art that is capable of sensing a signal. For instance, the detector may be an electronic imaging detector that is configured for spatial discrimination. Some examples of such electronic imaging sensors include high speed, linear charge-coupled devices (CCD), charge-injection devices (CID), complementary-metal-oxide-semiconductor (CMOS) devices, and so forth. Such image detectors, for instance, are generally two-dimensional arrays of electronic light sensors, although linear imaging detectors (e.g., linear CCD detectors) that include a single line of detector pixels or light sensors, such as, for example, those used for scanning images, may also be used. Each array includes a set of known, unique positions that may be referred to as "addresses." Each address in an image detector is occupied by a sensor that covers an area (e.g., an area typically shaped as a box or a rectangle). This area is generally referred to as a "pixel" or pixel area. A detector pixel, for instance, may be a CCD, CID, or a CMOS sensor, or any other device or sensor that detects or measures light. The size of detector pixels may vary widely, and may in some cases have a diameter or length as low as 0.2 micrometers.
In other embodiments, the detector may be a light sensor that lacks spatial discrimination capabilities. For instance, examples of such light sensors may include photomultiplier devices, photodiodes, such as avalanche photodiodes or silicon photodiodes, and so forth. Silicon photodiodes are sometimes advantageous in that they are inexpensive, sensitive, capable of high-speed operation (short risetime/high bandwidth), and easily integrated into most other semiconductor technology and monolithic circuitry. In addition, silicon photodiodes are physically small, which enables them to be readily incorporated into various types of detection systems. If silicon photodiodes are used, then the wavelength range of the emitted signal may be within their range of sensitivity, which is 400 to 1100 nanometers.
Optical readers may generally employ any known detection technique, including, for instance, luminescence (e.g., fluorescence, phosphorescence, etc.), absorbance (e.g., fluorescent or non-fluorescent), diffraction, etc. In one particular embodiment of the present, the optical reader measures color intensity as a function of absorbance. In one embodiment, absorbance readings are measured using a microplate reader from Dynex Technologies of Chantilly, Virginia (Model # MRX). In another embodiment, absorbance readings are measured using a conventional test known as "CIELAB", which is discussed in Pocket Guide to Digital Printing by F. Cost, Delmar Publishers, Albany, N.Y. ISBN 0-8273-7592-1 at pages 144 and 145. This method defines three variables, L*, a*, and b*, which correspond to three characteristics of a perceived color based on the opponent theory of color perception. The three variables have the following meaning:
L*=Lightness (or luminosity), ranging from 0 to 100, where 0=dark and 100=light;
a*=Red/green axis, ranging approximately from −100 to 100; positive values are reddish and negative values are greenish; and
b*=Yellow/blue axis, ranging approximately from −100 to 100; positive values are yellowish and negative values are bluish.
Because CIELAB color space is somewhat visually uniform, a single number may be calculated that represents the difference between two colors as perceived by a human. This difference is termed ΔE and calculated by taking the square root of the sum of the squares of the three differences (ΔL*, Δa*, and Δb*) between the two colors. In CIELAB color space, each ΔE unit is approximately equal to a "just noticeable" difference between two colors. CIELAB is therefore a good measure for an objective device-independent color specification system that may be used as a reference color space for the purpose of color management and expression of changes in color. Using this test, color intensities (L*, a*, and b*) may thus be measured using, for instance, a handheld spectrophotometer from Minolta Co. Ltd. of Osaka, Japan (Model # CM2600d). This instrument utilizes the D/8 geometry conforming to CIE No. 15, ISO 7724/1, ASTME1164 and JIS Z8722-1982 (diffused illumination/8-degree viewing system. The D65 light reflected by the specimen surface at an angle of 8 degrees to the normal of the surface is received by the specimen-measuring optical system. Still another suitable optical reader is the reflectance spectrophotometer described in U.S. Patent App. Pub. No. 2003/0119202 to Kaylor, et al., which is incorporated herein in its entirety by reference thereto for all purposes. Likewise, transmission-mode detection systems may also be used in the present invention.
The above-described techniques may be implemented in a variety of ways in accordance with the present invention. For example, a substrate may be utilized that contains a detection zone that provides any number of distinct detection regions (e.g., lines, dots, etc.) so that a user may better determine the presence of one or more microbes within a test sample. Each region may contain the same test chromogen, or may contain different chromogens for reacting with different types of microbes. Some chromogens, for example, are more sensitive to gram-positive bacteria and some are more sensitive to gram-negative bacteria. The test chromogen concentration may also be selectively controlled to provide the desired level of detection sensitivity. For example, higher concentrations may provide a higher level of detection sensitivity when low microbe levels are suspected. Referring to
If desired, the substrate may also contain a control zone that is applied with a control chromogen that is the same or similar to the test chromogen. The control zone does not generally change color during testing so that it may be used for comparison. Similar to the detection zone, the control zone may also provide any number of distinct regions. For example, the control zone may contain regions corresponding to different predetermined microbe concentrations, such as described above. In addition, the regions may contain dyes that have a different sensitivity level for different types of microbes.
Referring to
To initiate the detection of microbes within a test sample, a user may directly apply the test sample to a portion of the porous membrane 23 through which it may then travel. Alternatively, the test sample may first be applied to a sampling pad (not shown) and/or conjugate pad (not shown) that are in fluid communication with the porous membrane 23. Some suitable materials that may be used to form the sampling pad and conjugate pad include, but are not limited to, nitrocellulose, cellulose, porous polyethylene pads, and glass fiber filter paper. Regardless of where it is applied, the test sample migrates to a detection zone 31 defined by the porous membrane 23 that is capable of signaling the presence of a microbe. In particular, as shown in
The test and control chromogens are sometimes applied in a manner so that they do not substantially diffuse through the matrix of the porous membrane 23. This enables a user to readily detect the color of the dyes after the desired reaction time. For instance, the chromogens may form an ionic and/or covalent bond with functional groups present on the surface of the porous membrane 23 so that they remain immobilized thereon. In one embodiment, a positively-charged dye may form an ionic bond with negatively-charged carboxyl groups present on the surface of some porous membranes (e.g., nitrocellulose). Alternatively, certain components may be added to a chromogen solution that substantially inhibit the diffusion of the chromogen into the matrix of the porous membrane 23. In other cases, immobilization may not be required, and the chromogen may instead diffuse into the matrix of the porous membrane 23 for reaction with the test sample.
As a result of the present invention, it has been discovered that bacterial infection may be readily detected through the use of a chromogen that undergoes a change in color in the presence of bacteria at a threshold pathogenic concentration. The color change is rapid and may be detected within a relatively short period of time. For example, the chromogen may undergo a detectable color change in less than about 30 minutes, in some embodiments less than about 10 minutes, in some embodiments less than about 5 minutes, in some embodiments less than about 3 minutes, in some embodiments less than about 1 minute, and in some embodiments, less than about 30 seconds. In this manner, the chromogen may provide a "real-time" indication of the presence or absence of bacterial conjunctivitis.
The present invention may be better understood with reference to the following examples.
All reagents and solvents used in the Examples were obtained from Sigma-Aldrich Chemical Co., Inc. of St. Louis, Mo. unless otherwise noted and were used without further purification. All viruses were obtained from Gibraltar Laboratories, Inc. of Fairfield, N.J. Reichardt's dye was also obtained from Sigma-Aldrich Chemical Co, Inc.
The ability to form merocyanine dyes for use in the present invention was demonstrated. For example, the merocyanine dye shown in
Other merocyanine dyes were also synthesized in a similar manner by selecting an alkyl group of the alkyl iodide to correspond with the desired respective "R" group for the dye. The following Table 1 shows the compounds and the yields obtained for dye structure 13 for three different R groups.
Alkyl Derivatives Synthesized and the Yields Obtained
"R" Group
Yield (%)
Adenovirus type 2 pool (ATCC No. VR-846) was propagated in Vero monkey kidney cells and fed with Dulbecco's Modified Eagle's Medium (DMEM), supplemented with fetal calf serum to a concentration of 5% and incubated at 37� C.�1� C. in its presence of 5% CO2 for 6 days. Viral propagation was detected by microscopic observation of infected cell sheets for cellular disintegration (cytopathic effect, CPE) such as rounding, crenation, lysis, pyknosis, etc. as observed in at least 50% of the cell sheet. Cytotoxicity was measured as the extent of cellular disintegration as produced by the agent alone without the virus. Virus was titrated using ten-fold serial dilutions in DMEM, 4 replicates MA 104 cultures per dilution, each replicate inoculated with 0.1 milliliter of virus dilution. The extent of viral replication was calculated as the tissue culture infectious dose-50% (TCID 50) as determined by the method of Reed and Muench. The virus titer was calculated to be 10−6.5.
Haemophilus influenzae (ATCC No. 8149) and Streptococcus pneumoniae (ATCC No. 33400) were grown on Chocolate Agar and fed with Trypticase Soy Bean Broth (TSB). Streptococcus pyogenes (ATCC No. 49399) was grown on 5% sheep red blood agar and also fed with TSB media. The concentration of these bacteria (in TSB) was found to be 108 CFU/ml. Dilutions of the bacteria (107, 106 CFU/ml) were created by serially diluted using saline.
Labels were dip-coated with Reichardt's dye (80 milligrams per 10 milliliters in acetonitrile) and allowed to dry to yield a finished product of indicator-coated stickers. 50 microliters of undiluted virus in media was dropped onto a label and allowed to stand for 3 minutes before removing the droplet with a cotton swab. An aliquot of media alone was used as a control. The resulting de-colorization from the Adenovirus droplet was faint and barely above the de-colorization observed for the control media. Aliquots (50 microliters) of 107 and 106 CFU/mL bacteria were also dropped onto stickers, along with drops of the saline control. Drops of 108 CFU/ml bacteria were also dropped onto the stickers, but TSB was used as the control media for these samples. Haemophilus influenzae and Streptococcus pyogenes exhibited de-colorization above media controls for all CFU/mL levels tested. The de-colorization observed for Streptococcus pneumoniae only registered above the control for the 108 CFU/ml concentration. It is of note that the de-colorization observed for all bacteria at the 108 CFU/ml concentration was significantly greater than that observed for the Adenovirus, as was the de-colorization seen for the lesser concentrations of bacteria.
A cellulosic towel was used as a substrate onto which P. aeruginosa was pipetted onto the towel (100 μl). Reichardt's dye solution (160 mg in 10 ml of acetonitrile) was then added in 10 μl aliquots to the spot and the numbers of drops needed to establish a persistent color were counted. The amount of dye required to maintain a persistent purple color was 80 microliters.
A paper-based substrate (Neenah Bond™) (available from Neenah Paper, Inc. of Alpharetta, Ga.) and a label (available from Avery-Dennison) were initially coated with a Reichardt's dye solution (80 milligrams/10 milliliters acetonitrile) and hung to dry. An indicator strip coated with Reichardt's dye was exposed with decreasing amounts of P. aeruginosa aliquots. A hand-held spectrophotometer was used to after application of each aliquot to determine "Delta E" value (calculated using L*, A*, and B* values) for each CFU/mL concentration. The results are set forth below in Table 3 (for paper) and Table 4 (for label).
Results for Paper Substrates
log CFU/ml
Delta E (P. aeruginosa)
Results for Label Substrates
log CFU/ml
Delta E (P. aeruginosa)
From the above, standard detection curves were generated as shown in
Results for Paper Substrates
P. aeruginosa
log CFU/ml (actual)
Delta E
log CFU/ml (estimated)
Results for Label Substrates
P. aeruginosa
log CFU/ml (actual)
Delta E
log CFU/ml (estimated)
5 to 6
3 to 6
As can be seen from the numerical data, the unknown concentrations were predicted by determining to which known Delta E value the Delta E value of the unknown was closest. Although a few of the results were not completely accurate, the present inventors believe that improving the uniformity of the coating would further enhance detection accuracy.
Herpes Simplex Virus 1 (HSV-1) was prepared and inoculated into MA-104 embryonic monkey kidney cells propagated and fed with Dulbecco's Modified Eagle's Medium (DMEM), supplemented with fetal calf serum to a concentration of 5% and incubated at 37� C.�1� C. in its presence of 5% CO2 for 6 days. Viral propagation was detected by microscopic observation of infected cell sheets for cellular disintegration (cytopathic effect, CPE), such as rounding, crenation, lysis, pyknosis, etc., as observed in at least 50% of the cell sheet. Cytotoxicity was measured as the extent of cellular disintegration as produced by the agent alone without the virus. Virus was titrated using ten-fold serial dilutions in DMEM, 4 replicates MA 104 cultures per dilution, each replicate inoculated with 0.1 milliliters of virus dilution. The extent of viral replication was calculated as the tissue culture infectious dose-50% (TCID 50) as determined by the method of Reed and Muench. Reichardt's dye-coated stickers (160 milligrams/10 milliliters acetonitrile, 80 milligrams/10 milliliters acetonitrile, 40 milligrams/10 milliliters acetonitrile, and 20 milligrams/10 milliliters acetonitrile) were used as a test surface. 50 microliters of undiluted virus (TCID50 10−7 HSV-1/mL) in media was dropped onto each sticker and allowed to stand for 3 minutes before removing the droplet with a cotton swab. The subsequent de-colorization observed was compared to that observed for a virus-free control media and also to a Salmonella (108 CFU/mL) positive control. Though the de-colorization was not as strong as that observed for Salmonella bacteria, exposure to undiluted HSV-1 virus led to some de-colorization of the sticker.
While the invention has been described in detail with respect to the specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present invention should be assessed as that of the appended claims and any equivalents thereto.
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|Citing Patent
|Filing date
|Publication date
|Aug 6, 2010
|Apr 28, 2015
|Kimberly-Clark Worldwide, Inc.
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|Apr 30, 2009
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|U.S. Classification
|424/9.6, 435/31, 435/29, 435/34
|International Classification
|Cooperative Classification
|European Classification
|May 8, 2006
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Fairgrounds horse track, stables closing for winter
The Alameda County Agricultural Fair Association has decided to close down its horse training operation for the winter beginning... Last comment 23 hours ago | 7 comments
Giants shortstop, Foothill grad Brandon Crawford gives back to local schools
San Francisco Giants shortstop and Foothill High School graduate Brandon Crawford, along with Wells Fargo, recently awarded... Last comment 10 hours ago | 3 comments
Changes to FAFSA make it easier to apply for student aid
Do you have a high school senior who is knee deep in the college admissions process? Read the full story here -... Last comment on Nov 28, 2016 at 4:44 am | 2 comments
Tim Talk
by Tim Hunt
Pleasanton school enrollment stable as demographics continue to shift
Dec 1, 2016 | 1 comment
The Observer
by Roz Rogoff
Suspicious Stein, Watchdog Warren, and a Basket of Deplorables
Nov 26, 2016 | 20 comments
Doing College
by Elizabeth LaScala
What's Your Major and Other Pesky Questions Colleges Ask
Nov 9, 2016 | 3 comments
Parade opens holiday season tomorrow
Pleasanton's annual Hometown Holiday parade and tree-lighting ceremony will be held starting at 5 p.m. tomorrow on Main Street. Friday, 7:18 AM
Mayor, 2 Council members to be sworn in to new terms Tuesday
Fundraiser in Pleasanton tomorrow morning for ill teen
Tassajara Road will re-open Dec. 16 after paving delayed by November rain
Fairgrounds horse track, stables closing for winter
'A Rocky Mountain Christmas' at Firehouse Arts Center Sunday
Red Cross urges blood, platelet donations in Pleasanton this holiday season
Rules Don't Apply
"Never check an interesting fact." With these words, Warren Beatty returns to the silver screen, after a 15-year absence, in his film "Rules Don't Apply." They are, we're told, the words of Howard Hughes, the mercurial movie producer and aviation ind...... Read the full review
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Is this attraction pet friendly?
Would this be a good sunny day activity?
Would this be a good hot day activity?
Would this be a good cold day activity?
Is this an outdoor attraction or activity?
Is this attraction suitable for adults only?
Is this attraction suitable for all ages?
Does this attraction have good scenic or skyline views?
Is this attraction good for couples?
Does this attraction require above average amounts of physical activity (long walks, climbs, stairs or hikes)?
Thanks for helping!
We had been to Ancient Corinth a few times in the past to shop and dine; this past summer we decided to see the ruins of what was once one of the largest and most important cities...
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We had been to Ancient Corinth a few times in the past to shop and dine; this past summer we decided to see the ruins of what was once one of the largest and most important cities in ancient Greece with a population of 90,000 in 400 B.C.
The feature attracation is definitely the Temple of Apollo with 7 columns...
We arrived in ancient Corinth early afternoon and parked free in the public parking lot half a block below the town's narrow main street. I should mention first that the public restrooms next to the parking lot are despicable. There is intentionally no toilet paper in the ladies room, no paper towels for drying hands, no air dryers, and there...
Great place to visit, train journey from Athens to Corinth was interesting and very scenic in places,
Took a taxi from the station to Ancient Corinth as the bus does not go to the new station.
The sites were very interesting, lots of space to stroll round and the museum was good.
Really enjoyed my visit.
Small row of shops...
It is very difficult to grasp how dramatically the land scape and ocean defined the landscape thousands of years ago without driving out this distance and looking to where the sea currently lies. Small museum and large amounts of well-preserved architecture.
We took a day trip from Athens and went to Corinth, Mycenae and Epidaurus with a driver and guide. This is the way to see the area outside of Athens. We were here with no buses and few people.
This site is just over the fence from the parking lot and on flat land so it is very easy to...
Extensive archaeological site with ruins of Greek temples. Where St. Paul preached to the Corinthians. Easy access to ruins. Museum contains artifacts from several centuries BC. Museum staff is very knowledgable.
This site is better than most because it has decent signage (the lack of, being a problem with most Greek sites). Corinth was a massive city in biblical times which makes Paul's journeys and teachings here all the more impressive. I've added a picture of the Bema (judgment seat) where he was brought before Gallio of Achaia, in Acts 18:...
We visited this place before and were not too impressed. However, on our recent trip we visited it again with a guide who was able to put this place in perspective as to the places and persons mentioned in the Bible which really helped us to appreciate the historic significance of this place. Standing in front of the judgment seat...
This is one of those must visit sites of ancient Greece. The ruins are fairly compact, and have nice vistas overlooking the sea. The ruins have quite a rich history - from the ancient Greeks to early Christian times (you can see the platform where St. Paul preached to the Corinthians). Apparently the site is popular with touring Christian groups....
This was so fascinating because we have read about this place in the Bible. Loved seeing the Bema seat. The museum fills in the gaps. This is a must see place on a Biblical Greece tour.
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Visiting by cruise ship and wondered the cost of a taxi for 4 to Corinth and surrounding area.
We will be sailing to Corinth arriving 11th Sept, and then planning to visit Ancient Corinth on Sat 12th before heading to Athens Airport late afternoon. Could you recommend a guide for the Ancient sites and please could anybody help me with train times from Corinth to the Airport and booking /price details. Alternatively we could maybe get a taxi from the Harbour to Athens Venizelos?
Thanks very much.
Are there tours or guide available at Corinth or is it all self guided?
We will be staying in Athens in mid May for one week and want to take a trip to Corinth (in the footsteps of Paul).....Does anyone have a good tour company that they could recommend....we would like to go on a small group tour? Many Tx,
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Sacramento Kings head coach Michael Malone applauds in the first half of an NBA basketball game against the New Orleans Pelicans in New Orleans, Monday, March 31, 2014. (AP Photo/Gerald Herbert)
Sacramento Kings head Coach Michael Malone directs his players against the New York Knicks during the second half of an NBA basketball game in Sacramento, Calif., on Wednesday, March 26, 2014.The Knicks won 107-99.(AP Photo/Steve Yeater)
In this photo taken Friday, March 21, 2014, Sacramento Kings forward Royce White walks past Kings head coach Michael Malone to enter his first NBA basketball game in the closing moments of the Kings 99-79 loss to the San Antonio Spurs in Sacramento, Calif., White was drafted by the Houston Rockets with the No. 16 pick in the 2012 draft but spent his rookie season in a dispute with the team over how to treat his anxiety disorder which includes a fear of flying. Not only must he prove he can still play, White, who is on his second 10 day contract with Kings, has to show he can handle the rigors of the league's schedule including his first road trip this week.(AP Photo/Rich Pedroncelli)
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An Introduction to Social Entrepreneurship
Voices, Preconditions, Contexts
Edited by Rafeal Ziegler
Chapter 1: Introduction: Voices, Preconditions, Contexts
Rafael Ziegler
Rafael Ziegler The danger of an uncritical and exclusive promotion of a free and market-based (social) system is evident. There are domains where the state has a duty to take action and to ensure the basic security of its citizens. But without a permanent input to the state system of new ideas from outside, even this legitimate domain can do more harm than good. (Judy Korn, Chapter 3 in this volume, p. 51) Social entrepreneurship has become a source of hope, but we are like water-tap users who know little about the origin of the source. Social entrepreneurship is said to be made of ideas that are tried out rather than proclaimed, ideas that are pushed through by initiatives belonging to individuals rather than multinational mega-organisations, ideas that are proposed in languages that are culturally diverse and not necessarily professionally polished, ideas that speak of pervasive social inequalities and exclusions, of ecological problems and risks, and ideas that do not speak of these issues as inevitable predicaments but as challenges that call for societal transformations. Who or what makes all this possible? Social entrepreneurs are said to have innovative solutions to pressing social problems; they are characterised as ambitious and persistent; they are said not to rely on business and government for the realisation of their ideas, and to aim at wide-scale, systemic change.1 These social entrepreneurs are promoted by support organisations, the media, companies and policy-makers. They have become increasingly familiar, branded and politicised actors. How has this happened...


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18 Homemade Christmas Decorations: How to Make Christmas Decorations
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Latest Comments
"This is perfect!! I can't wait to get started on my own cute little kitchen herb garden...when I get my own place, that is"
"Very intresting of what projects can be done with tp. We use them a lot for projects with children and this one will be this week project. Though, i think the colors can be more traditional with green and red or maybe golden colors with the green tree. I am sure the children will chose the traditional, but personally i like this one. Can be placed in any wall and every season."
"How cute are these fun and colorful little birds! I really like how easy they are to make. Can you really make a flamingo like mulberrygalprinces s says you can? That would be so cute to make. I love the idea of making several kinds of birds. My stepdad loves birds, so I can see making all kinds of these for him to add to the christmas tree or just to decorate the house."
"What a fun and festive way to use up a pile of paper clips that you never use! I love that you made them in various colors. I think it is so neat to use magnets on them too. You could use them to hang pictures from on a refrigerator or file cabinet."
Holiday Ornament Tree
By: The Krylon Design Team
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This holiday ornament tree from Krylon is great for a contemporary setting or small spaces. Feel free to paint the ornaments whatever color you'd like! This tree is perfect for elegant Christmas table decorations or bringing some cheer to your office or desk!
See this project and more glittery crafts in our free eBook, How to Create 14 Sparkly Craft Projects Using Krylon Glitter Blast.
- Metal ornament tree
- 10 - 12 small ornaments
- 1 can Krylon® Indoor/Outdoor Primer
- 1 can Krylon® Brushed Metallic Paint, Oil Rubbed Bronze
- 1 can Krylon® Glitter Blast™ Glitter Paint, Sparkling Water
- 1 can Krylon® Glitter Blast™ Glitter Paint, Silver Flash
- Masking tape
- String or fishing line
- Drop cloth
- Clean all objects to be painted, making sure the surfaces are free of dirt and dust.
- Mask off ornament hangers.
- Prime the Ornament Tree. Apply several light coats of Krylon® Indoor/Outdoor Primer. Spray from side to side in a sweeping motion beginning off the surface and ending off of the other side.
- Paint ornament tree with Krylon® Brushed Metallic Oil Rubbed Bronze. Apply multiple thin coats to avoid runs and drips. Wait approximately 1 to 2 minutes between coats. Let dry.
- Spray ornaments with Krylon® Glitter Blast™. (We sprayed half with Glitter Blast™ Silver Flash and half with Glitter Blast™ Sparkling Waters.) Apply multiple thin coats. Wait approximately 1 to 2 minutes between coats, shaking can before each coat. Let dry.
- Remove masking tape or replace ornament hangers after Glitter Blast™ is dry to handle, approximately 24 hours.
Tip: Make it easier to spray all sides of the ornaments by creating a makeshift closeline and hang ornaments from string or fishing line.
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Empowering your lifestyle choices
Recipes, Menu Plan, Grocery Lists, Photos
Starts Oct 6, 2016
December 6, 2016
The group will meet weekly over a 90 minute video call. Each call will be facilitated by Deb Gleason, Certified Holistic Nutritionist, NLP Master Practitioner, and Wellness Coach. In each session we will collectively explore specific themes. In the safety of our small group we will share our emerging thoughts, feelings, struggles, and successes.
Coming Soon to Vernon, BC
The class includes:
Detailed tutorial of the brewing process.
Tutorial on second fermentation to add flavors.
Kombucha Tasting
Printed instructions and recipes.
Kombucha Starter Kit (healthy Scoby and concentrated starter tea).
Follow-up support via email to ensure your brewing success.
Get in the kitchen with Deb and learn to make delicious new dishes, sit down to taste what you've made and take home the recipes and leftovers. Classes are always Vegan & Gluten-Free.
The menu will include:
All Dressed Kale Chips
Super Slaw
Fishless Fillets
Handmade Tartar Sauce
Roasted Potato Wedge Poutine
Nutty Caramel Apples
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This activity has GetYourGuide's stamp of approval! Exceptional customer service, knowledgeable staff, and a great time guaranteed.
Barcelona: 1.5-Hour Photography Walk
Duration: 1.5 hours
Duration: 1.5 hours
From $44
From $13 per personBook now
Meeting pont: Arc de Triomf. Your guide will be holding a red umbrella.
From $13 per personBook now
Product ID: 3027


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Somalia's government says it is in talks with some of the world's biggest energy companies in an attempt to restart oil exploration in the war-torn east African country.
Somali officials have discussed having BP, Exxon Mobil (XOM), Royal Dutch Shell (RDS.A, [RDS.B]]), Chevron (CVX), ConocoPhillips (COP) and Eni (E) return after more than two decades of violence drove away major oil producers.
Shell says talks with the government "are of a preliminary and exploratory nature"; BP and Eni confirm having some discussions.
Somalia last year transitioned to its first permanent government in years and is trying to rebuild the economy with oil as a cornerstone.


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Möchtest du als deine Standardwährung festlegen oder nur für diese Sitzung behalten?
Separate living room, huge luxury bath with amazing water pressure, fabulous bed, & lovely balcony. Some ocean views better than others. Several great restaurants on site, great daily breakfast buffet, three swimming pools( one is for over 18) , many areas to sunbath, ocean front with lounge chairs, beautiful, dramatic lobby, concierge service, valet. Cost. In house laundry lost our clothes several times, attendant on beach was rude about loung chairs-several times, front desk did not send up bellman when asked. Toilet ran for days, but after the first two repair calls didn't work, they upgraded us to a better room.
Have stayed here multiple times and enjoyed each visit. You can't beat the rooms (all suites). The suites are always clean and spacious. Upgrade to partial or full ocean view and you won't be disappointed.
Everything about this place. It is just a wonderful place to stay regardless of the purpose of your stay.
Restaurants are excellent as well as pool side food and drink service. Pool side menu was more expensive than expected. An outdoor convention of Trial Lawyers Assoc. was held in the open grass area outside our lanai, both morning and night for two days. Loud speakers, music and announcements heard inside our room even with the sliders closed.
Service was personalized and discreet. They took time to call me by name. Ambiance was family friendly yet upscale. Grounds are well kept Food and drink are very pricey. Property seems a little dated, could use an update.
great rooms. felt corporate and rushed. has great potential but was difficult to "relax" when outside the room. green space is completed covered with lawn chairs, making it feel cramped.
I've stayed at many 4 and 5 star hotels. This was my first time at the Fairmont Kealani. I thought the rooms were nice and spacious. I liked that the pool side was relaxed and accommodating, opened 24 hours. I would definitely come back again. At the time there was only one hot tub...would have been nice if there were more. Also pool side gets busy early so reserve your spot by 8am.
Beautiful property, great beach and pool. Huge suites with everything you need. Musty smell in room but hotel changed filters right away to ameliorate the problem. On site restaurants are expensive.
view, location on beach, staff, overall service. daily resort fee charging for many things you may not use, we were given a discounted upgrade to a room with a view, but it was right over the meeting facilities and two days of our stay major events were just below our window and were noisy and disruptive and when we exited the elevator they were right there in our way., the cell phone coverage was bad, the room service kitchen did not know what a margherita pizza was supposed to have on it.
Beautiful property with great service. Vacationed there with a 1 and 11 year old. Suite room worked out great and we enjoyed our large lanai. Highly recommend!
Location! Location! Location! We spent a week at the Wailea Beach Villas. The complex is nestled between the Grand Wailea and the Marriot. It is a Wailea Beach front complex bordered in the back by the Shops of Wailea. The 3 bedroom penthouse unit was spectacular with gorgeous ocean views. It is really spacious and appointed with high-end everything and sophisticated, comfortable furnishings. 4 Flat screen TVs, stereo surround sound. There is even a BBQ on the solar-shaded lanai! ?Walk-in? snorkeling on Wailea Beach is not to be missed!
We had a great stay at Wailea Beach Villas. We were met with a lei greeting by the bellman and given a thorough tour of the condo explaining everything: TVs, surround-sound, A/C, phones, WiFi, lighted fans, solar shaded lanai, BBQ. The WBV 3 bedroom penthouse condo (J205) was beyond our expectations from the ocean view, fantastic decor, classic artwork - plus guide books, best selling novels, board games and DVDs. The location couldn't be better - right on Wailea Beach. We snorkeled daily directly off the beach ? it was terrific with clear water, tropical fish, coral and turtles.
Our family just loved, loved, loved our condo at Wailea Beach Villas! We rented a 3 bedroom unit which on the corner, so it had a huge, beautiful lanai on 2 sides. Everything about this place made us 'oooh' and 'ahhh'....the beds, the kitchen and cookware, the huge screen tv, the beautiful, spacious bathrooms, the view of the ocean. The walk down to the beach was short and beautiful, and at the bottom the super friendly staff happily schleped beach chairs and towels down to the beach for us. Since Wailea faces west, we saw spectacular sunsets, and February is in the heart of whale season, so we saw many whales spouting and breaching the surface as we watched from the beach! Bring binoculars, by the way. Speaking of the beach, you literally share the same gorgeous, clean beach as the Grand Wailea hotel. We were thrilled to have paid even less for our gorgeous condo than for a couple of fancy hotel rooms. We made a quick trip to the local Safeway and saved a bundle eating in. One more pe...
Exquisite new condo on Wailea Beach next to the Grand Wailea Resort and beach-side of the Shops of Wailea. Spectacular views! Very spacious 3 bedroom / 3 bath penthouse. Gourmet kitchen. Breakfast bar. Dining room. Luxurious master suite. Beautiful baths. Lanai with Viking BBQ. Surround Sound. Plasma TVs. Beautiful furnishings & accessories. Classic art work. WiFi. Indoor laundry. Private elevator entry. Full service front desk. Room service available from Longhis. Loved Wailea Beach Villas ? can?t wait to go back!
I stayed here with my fiance's whole family and it was heaven on earth. You know it's nice when your mother in law asks you to please not blog about exactly how nice it was! \n\nThe floors were stone, the whole balcony opened up so that you can enjoy the perfect Maui weather all day long, the kitchen was good enough for Thomas Keller, and the bed as divine. Plus, it was really great for a large party. There were 7 of us and I never felt crowded. And the location couldn't be beat. It's right next door to the Grand Wailea so we'd often go over there to use the spa or dine and it's on this boardwalk that goes down the shore for quite a ways. It's nice to just stroll.\n\nAnd really, it was more relaxing than a hotel because it felt like you were staying in a very very posh home. \n\nOh, did I mention the GRAND PIANO in the living room? Um, yeah. That pretty much sums it up.
The room was old. Needs updating
Location. The view. The beautiful park like gardens. The bathroom was very nice. The hotel messed up our booking. The famous water elevator didn't work. Cleaning happened very late and one day only after asking. The doors so are thin that everyone walking by sounds like they're inside the room. Too few pool chairs. 7 waterslides - 5 of them are a joke. The coffee shop has a pathetic supply of goods. The gym is average.
Landscaping and Christmas decorations were done well; bell hop, valet, housekeeping, and restaurant staff were very pleasant. The environment was very busy (family groups and children were the major customers, resulting in lots of little ones running about the resort). It's not a very relaxing atmosphere for folks looking for a quiet getaway from the busy aspects of everyday life. Attention to cleanliness was subpar to expectations (missing tissue box; (not requested) playpen was present in box on floor in main room; balcony rails had dirt residue throughout; light switches were dirty for misaligned--due for replacement). We asked valet to retrieve our vehicle, but valet staff did not alert us of our car's arrival resulting in a 15-min wait until we finally asked what had taken so long. Greeting staff in lobby failed to provide lei to my fellow traveller upon her arrival despite it being a customary practice for all guests.
It was so beautiful. They put flowers on the bed and the room was beautiful. We ordered room service in the morning and the food was amazing! I can't wait to go back to this beautiful resort.
Staff was all very friendly and helpful. Our room was beautiful and very comfortable. I love the lotions,linens and towels. Pools and beach are marvelous. Breakfast buffet is delicious. I enjoyed the visiting jewelers and artists Perfect vacation
Beautiful views and the most comfortable bed ever! Nice space. Although my friend was disappointed that there is no poolside bar, we decided we loved it - you can keep a bottle of wine in the fridge - small kitchenette probably a must with kids. Really nice pool, large enough for more relaxed holiday laps,but great poolside beds, many with sunroofs. Restaurant only open for dinner could be a downside, but we discovered great places to eat on the island!
The hotel Wailea has beautiful gardens and is spread out so you barely notice the other guests staying at the hotel. The view from our balcony was amazing and we saw a number of hump back whales. We were suprised by the size of our villa and the bed was the most comfortable bed I have ever slept it! We thought the pool lacked atmopshere- there was no bar or food service. We also though that room service should be available as the Italian Restaurant was way over priced ($38 for spaghettii bolognaise) and if you didn't feel like going out you would starve. The rooms need new carpet and couches and the kitchen had only two plates and a knife and fork- Not very practicle. I think this hotel has alot of potential but is just not there yet.
Location and view. No outdoor Jacuzzi and the pool water temperature was cold.
Our suite was lovely and a most comfortable place to relax. We didn't have a reservation at the hotel's restaurant but were able to get in, and the food was excellent, and our garden table a lovely setting.
The staff were great. The surroundings very relaxing. The location was fine. The rate was value for money
Everything, wonderful relaxing atmosphere, quiet with beautiful surrounding gardens. Perfect choice. This was our sixth visit to Maui as we have family living on the Island. Recommend this hotel. Unfortunately the only complaint was our condo had no cooking or eating irons, our request was met by we would need to wait 2 days as we were on a list, they didnt arrive for a week and that was only after having to complain. Also the beach arrangements with there sister hotel was poor we were informed that only 20 beach chairs are available for our hotel and no towel were available for our use so we had to wait for 20 minuets for towels and one chair. We were told it was always a problem. We didnt bother trying to to use the facility again once was enough.
It was very quite and peaceful. Very easy with the kids. Staff was very kind and responsive. Rooms with the view were excellent. Beautiful sunsets. The rooms need a bit of a makeover especially bathroom counter and toilet. Beds were too soft to the point that we still have back aches.
Die Mitarbeiter waren sehr freundlich. Der Strand sowie unser Zimmer waren sehr schön. Das Badezimmer ebenfalls, bis auf die Dusche/Badewanne. Diese ist, wie in den USA üblich, auch in Luxushotels meist nur eine kleine, lieblose Plastik-Nasszelle mit Duschvorhang. Der Golfplatz ist ebenfalls sehr schön und gepflegt. Das Hotel ist alt und sehr abgewohnt. Das Maui Prince wurde angeblich als erstes der großen Luxushotels am Strand von Makena gebaut. Offensichtlich ist seit damals aber nichts je renoviert worden. Der Schwimmbadrand z.B. ist rundherum abgebröckelt, die Atmosphäre am Pool ist langweilig. Es gibt nur einen relativ kleinen runden Pool für das gesamte Haus, sowie einen weiteren nichtssagenden Pool für Kinder. Sogar der schöne japanische Innenhof verströmt eine absolut langweilige, uninteressante Atmosphäre. Die gesamte Anlage wäre dringend zu restaurieren. Die Hotels rundherum bieten im Vergleich zum Maui Prince viel mehr zum vergleichbaren Preis!


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- Long Lasting Marble Countertops in Port Jervis, NY
- Professionally Installed Marble Countertops In Port Jervis, NY
- Free Estimates on Marble Countertops in Port Jervis, NY
Marble Countertops in Port Jervis, NY
The marble countertops Port Jervis, NY has to offer are a great choice to make if you want to remodel your kitchen for a number of reasons. These countertops are known for being incredibly durable and long lasting so you can count on them to last a very long time. You will find that Port Jervis marble countertops also have a unique aesthetic that you will not find with any other kind, which is one of the many reasons why they have become so popular among homeowners.
Avg Costs for Marble Countertops
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$3,418.31 - $4,934.16
Find the Best Quality Marble Countertops - Port Jervis, NY
One of the best things about Port Jervis marble countertops is that they are available in a number of different colors and patterns. If you are going to be remodeling your kitchen, you will find that marble countertops are a great overall investment to make. Another great thing about marble countertops for Port Jervis, NY homes is that they offer a very unique aesthetic and they tend to last a very long time. It is important that you choose countertops that are made of a strong material so they last for many years. These days more and more people are choosing countertops because they are resistant to scratches as well as stains and heat, making them perfect for the kitchen area of any house.
Marble Countertops Companies
Thompson Creative LLC
3 Myrtle Dr
Montague, NJ 07827


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Armani Newborn
Boys Pale Blue Velour Babysuit
Smart pale blue babysuit by Armani Baby, made with in soft cotton velour. Designed to look like a separate top and trousers, with a logo printed shirt collar and hem. There is an eagle logo appliqué on the chest and designer print. It has popper fastening at the back and between the legs to help with dressing and nappy changing.
- Product number 136170
- 80% cotton, 20% polyester (soft, velour feel)
- Machine wash (30*C)
- Popper fastening at the back & between the legs
- Comes with a muslin bag
- 1 piece