Identification of Dermatophytes | Experiment

Identification of Dermatophytes | Experiment Abstract Dermatophytosis constitutes a group of superficial fungal infections of keratinized structures like stratum corneum of the skin, nails and hair. Despite the availability of effective antifungal agents, dermatophytic infections continue to be one of the principal infections throughout the world, especially in tropical countries like India. Identification of causative dermatophytic species in clinical settings are not only of epidemiological concerns but also important for the treatment of rather common dermatophytosis with great precision. To study the clinical variants, species of fungi causing dermatophytic infections, epidemiological factors responsible for the disease in central Mysore, molecular characterization and antifungal susceptibility tests by MIC. The prospective observational analyses of clinically suspected 600 dermatophytic infected cases were studied at the Dermatology department of K.R. Hospital and C.S.I. Holdsworth Memorial Hospital, Mysore, India. The study was conducted from November 2010 to October 2012. All the cases were evaluated and patient’s data were recorded as per pre-designed proforma. Skin scraping, hair and nail samples were collected, processed, molecular characterization and antifungal susceptibility test were done according to standard mycological protocol. Patients on topical and systemic antifungal treatment were excluded from the present study. Clinically suspected 600 patients samples were collected, among them 389 (64.8%) were skin scraping, 113 (18.8%) were hair plucking and 98 (16.3%) were nail clipping. They were processed for isolation and identification of dermatophytes from different clinical types. The specific and non specific variables effecting dermatophytosis were also analyzed. Samples were collected throughout the year, of which samples collected from July to October showed highest incidence of dermatophyte infection among patients. The number of specimen collected under the age group 21-30 years were 333 (55.5%), which was statistically significant (PMicrosporum canis and 12 were infected by Trichophyton verrucosum. Among 600 clinically suspected dermatophytes infected patients, 433 (72.2%) samples were collected from K.R. Hospital and 167 (27.8%) samples were collected from C.S.I. Holdsworth Memorial Hospital, Mysore. The various factors which were found to increase chances of acquiring dermatophytosis were by sharing bed, 343 (57.2%); not bathing daily, 362 (60.3%); and domestic animals in their house, 102 (17.0%). Partial treatment and over the counter medication was also considered as major factors influencing dermatophytic infection among patients. Less number of patients 86 (14.3%) showed family history of dermatophytosis. Most of the patients visited to the Doctor in 5th-8th week after infection, 391 (53.2%). During that period of infection, they were either taking home made medicine or over the counter medicine. Most of the cases found predominantly were tinea corporis and tinea capitis. Tinea corporis 282 (47.0%) and tinea capitis 75 (12.5%) were seen among 26-30 year old patients and 6-10 year old patients respectively. Most of the isolates belonged to three genera and eight species, where T. mentagrophytes was predominantly isolated 105 (29.7%), T. rubrum 84 (23.8%) was isolated from tinea corporis cases, T. violacium 4 (12.2%), T. verrucosum 54 (15.3%), Epidermophyton floccosum 11 (3.1%), M. canis 13 (3.6%), T. tonsurans 38 (10.8%) and T. schoenleinii 5 (1.4%) were also isolated from different cases of tinea. Dermatophytes infected culture positive, 132 patient samples (skin scrapings, hair plucking and nail clipping), were obtained for molecular characterization in the study. Nearly 87 males and 45 females were referred by Department of Dermatology, K.R. Hospital and C.S.I. Holdsworth Memorial Hospital, Mysore during two years of study period. The collected samples were analyzed by both direct microscopic examination and culture assays. Of the 132 culture positive cases, the organisms identified by phenotypic characterization were belonging to three genera and eight species viz., T. mentagrophytes 52 (39.4%), T. rubrum 30 (22.7%), T. violacium 18 (13.6%), T. verrucosum 11 (8.3%), E. floccosum 10 (7.6%), M. canis 6 (4.5%), T. tonsurans 3 (2.3%) and T. schoenleinii 2 (1.5%). The culture isolates of these organisms were further characterized by PCR. The present study showed positive result for phenotypically analyzed culture positive with 100% sensitivity and specificity by PCR. PCR targeting internal transcribed spacer (ITS) and Chitin synthase 1 was sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA respectively. From the same source (houses) 10 isolates from patients and 10 domestic animals were subjected to both PCR and RFLP. The product after agarosegel electrophoresis, restriction enzyme cutting patern was found to be infected by same T. verrucosum with 100% sensitivity and specificity. This indicates that T. verrucosum infection is from animal source. The dermatophyte specific primer based PCR-RFLP which targets the internal transcribed spacer and chitin synthase 1 region are useful in the direct identification of der ­matophytosis from clinical specimens. The application of the Hae III, Hinf I and Mva I restriction enzymes by using the ITS amplicons and chitin synthase 1 region are constant, stable and reproducible show variation within the species, so these sequences have been widely used to develop rapid procedures for the identification of fungal species by PCR-RFLP analysis. The PCR-RFLP method, on using the dermatophyte specific primer with restriction enzymes Mva I, Hae III and Hinf I, showed species differentiation among the T. rubrum and T. mentagrophytes isolates. Since direct microscopy and culture have limitations, performing a direct PCR on the clinical speci ­mens can augment the diagnosis of more dermatophytic cases. PCR confirmed 132 dermatophytic samples were taken for antifungal sensitivity test. In the current study, among 132 isolates of dermatophytes some are sensitive and some are moving towards resistance, but high MIC value indicated that it has slowly acquired adaptation towards the drug. This indicates in near future it will develop drug resistance against the antifungal agents. Twenty three isolates (14.4%) were showing high MIC value for fluconazole of which T. mentagrophytes (8), T. rubrum (5) and T. verrucosum (10) and M. canis (3) had MIC50 of 16  µg ml-1. Second most frequently used drug next to fluconazole is ketoconazole, which had MIC50 of 0.125  µg ml-1 for most of the isolates. Griseofulvin, itraconazole and terbinafine showed similar results of 0.03-0.06  µg ml-1. The present study showed drug concentration inhibition of some fungi against 50% of isolates and 90% of isolates and their geometric mean of five drugs against 132 isolates. MIC50 and MIC90 were not determined because of small number of samples i.e., C. parapsilosis ATCC-22019 were within the value standardized by CLSI guidelines. The study highlighted tinea corporis as the most common clinical pattern of dermatophytosis followed by tinea capitis in Mysore. The predominant causative fungal species isolated were T. mentagrophytes, T. rubrum, T. violacium, T. verrucosum, E. floccosum, M. canis, T. tonsurans and T. schoenleinii confirmed by PCR with 100% sensitivity and specificity. Trichophyton verrucosum was isolated and confirmed by PCR and RFLP from human and their domestic animals (from animals to human). PCR-RFLP provides the best technique for dermatophytic identification at species level. MIC was done to see susceptibility and resistance pattern for clinician as well as for lab workers where terbinafine was 100% sensitive drug against dermatophytes. Clinical evaluation and fungal species identification are mandatory for proper management of dermatophytosis rather than empirical treatment. Need for the study In the medical science dermatophytes are constant parasites of human beings, and has survived several generations of therapeutic regimens, ranging from single dose antifungal to several dose drugs like griseofulvin, tolnaftate and early imidazole. There are some dermatophytes which were abundant once upon a time but now it is rare, it may be due to geographical restrictions, indicate that economy, geography, type of the species and environmental factors also interferes with the dermatophytic survival and distribution. It is equally important to review anthropophilic dermatophyte as well as zoophilic dermatophytes, which may increase chances of infection in the near future, as immunocompromised diseases are increasing day by day. The dermatophytes are oldest parasites of human and animals. Good and specified knowledge about these agents are still far from human understanding. The medical mycologists have done considerable work, but the studies were mainly devoted to the reactions of the host rather than the study of the parasites. The rates of publication on dermatophytes are limited as compared to the extensive work being done in the field of bacteriology and virology. As we know, these dermatophytic infections are not usually life threatening, but offer an interesting approach to the variety of fundamental problems in human, animals and fungal biology. The obvious reasons apparently disregard to the study of Medial Mycology Most of the fungal diseases are not fatal and hence do not attract much attention of the investigators. The development of the infection is very slow and persists for years without any serious damage to the host. Carrier among human and animals is very common and survives for decades without harming the host. Methods of isolation and identification are relatively very complicated and require long time to obtain positive results. Most of them are sensitive to the limited antifungal drugs except few and drug resistance is very slow and limited. Sources of infection are mostly human, animals and soil. Factors that attract to do research on dermatophytes Increasing incidence of immunocompromised diseases in and around the world. Changing patterns of dermatophytic infections with change in environment. Development of resistant species among the dermatophytes. Carrier harbours pathogenic species for many years to produce disease under favourable condition. Formulate treatment of choice according to the new changing pattern of drug resistant dermatophytes. Fungal infections of skin, hair and nail are common in hot and humid climate. Humans acquire various dermatophytic infections due to poor health hygienic conditions. The present investigation was aimed to study molecular level identification of tinea by PCR and its antifungal susceptibility test. Objectives of the study To study the incidence of dermatophytic infections and their clinical variants To identify the sources of infection by PCR for dermatophytes To determine the in vitro antifungal susceptibility test Slide culture For visualization of the normal colonial characteristics, arrangement and morphology of spores and mycelial appendages, slide culture was done. SDA media without antibiotics or cornmeal agar with 1% glucose was used. Lactophenol cotton blue was used as the mounting fluid for easy visualization of hyphae and spores (WHO, 1986). Advantages It is arapid method of preparing fungal colonies for examination and identification. Permits fungi to be studied virtually in situ with as little disturbance as possible fungi were identified mostly by close examination of its morphology and the characteristics it possess. We were growing the fungi directly on the slide on a thin film of agar. By doing this, there was no need to remove a portion of the fungus from a culture plate and transfer it to the slide. So there wasless chances for the features that were key to identification, notably the spore-bearing structures, without any damage. A modified method adapted from Roberts et al. (2003) and described here fulfills those requirements. Sterile 1.5% water agar (7 to 8 ml) was poured into sterile 60-mm plastic petri plates and allowed to solidify. A sterile 22-mm2 cover glass was centered on the agar. The desired nutrient agar medium (10 ml) was poured into a second 60-mm petri dish, allowed to solidify, and cut with a sterile stainless steel spatula into blocks approximately 5 to 8 mm2. One block was aseptically removed and placed on the cover glass. Inoculation of the agar block on one or more sides with fungal hyphae or conidia was followed by placement of a second sterile cover glass on top of it. After the petridish lid was replaced, the completed modified slide culture was incubated at the desired temperature until adequate growth and conidiogenesis had occurred. Each cover glass was used to prepare a semi permanent mount on a standard microscope slide 3 by 1 inch (7.62 by 2.54 cm). The top cover glass was lifte d off with forceps and wetted on the specimen side with a drop of ethanol (70 to 90%). One drop of fungus mounting medium (e.g., lactophenol cotton blue) was applied to the specimen, and the cover glass was lowered gently onto the slide, specimen side down. The bottom cover glass was lifted from the water agar and similarly mounted on a second slide. The nutrient agar block adhered to one of the cover glasses during the cover glass mounting procedure and was carefully lifted off with a sterile dissecting needle before the application of alcohol and mounting medium. Heat fixation of the cover glasses before mounting as described by McGinnis may improve the stability of the conidium-bearing structures (James, 2011). Lactophenol Cotton Blue (LPCB) The Lactophenol Cotton Blue wet mount preparation was the most widely used method of staining and observing fungi and was simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. It was used constantly as a mounting fluid when examining culture of fungi microscopically (Leck, 2012). Materials Lactic acid †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 20 ml Phenol (crystals†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 20 gm Glycerine†¦.†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 40 ml Water (distilled)†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ 20 ml Mixed the above gently and added 50 mg the dye,  cotton blue. Procedure Placed a drop of 70% alcohol on a microscope slide. Immersed the specimen / material in the drop of alcohol. Added one or at most two drops of the lactophenol cotton blue mount and stained before the alcohol dries out. Holding the cover slip between forefinger and thumb, touched one edge of the drop of mount with the cover slip edge, and lowered gently, avoiding air bubbles. The preparation was ready for examination. Hair penetration test Many isolates of T. rubrum and T. mentagrophytes are difficult to distinguish between on the basis of colony morphology and microscopic appearance. This in vitro test is the best method to distinguish between these two dermatophytes: Procedure Obtained a small amount of juvenile human hair and washed off fats and oils (kept hair in flask or beaker with 50 to 100 ml of ether, acetone or alcohol, shacked for 2 to 5 minutes and pour off the liquid). With scissors, a scalpel or a razor blade cut the hair into segments approximately 1 cm long. Put the hair in a glass petri plates, place on the lid and autoclaved for 10 to 15 minutes. This maintained sterility in the petri plates and can be used for 10 years. Calcofluor White Stain (CFW) It is a fluorescent stain for rapid detection of yeasts, fungi and parasitic organisms. Calcofluor White stain is a non-specific fluorochrome that binds to cellucose and chitin in cell walls (Chattaway and Barlow, 1954; Green et al., 1983; Hageage and Harrington, 2005). Composition: Calcofluor White M2R 1g/l and Evans blue 0.5g/l. Directions: Put the sample to be examined onto a clean glass slide. Add one drop of Calcofluor White Stain and one drop of 10% Potassium Hydroxide Place a cover slip over the specimen and let stand for 1 minute. Examine the slide under UV light at 100X to 400X magnification. Calcofluor White Stain was a non-specific fluorochrome that binds with cellulose and chitin contained in the cell walls of fungi and other organisms. The staining procedure with Calcofluor White Stain is a rapid method for the detection of many yeasts and pathogenic fungi like Microsporidium, Acanthamoeba, Pneumocystis, Naegleria, and Balamuthia species. Evans blue present in the stain act as a counter stain and diminishes background fluorescence of tissues and cells when using blue light excitation (not UV). A range of 300 to 440 nm (Emmax 433nm; 0.1 M phosphate pH 7.0; cellulose) can be taken for emission wave length and the excitation occurs around 355nm. Fungal or parasitic organisms appear fluorescent bright green to blue, while other material is reddish-orange fluorescent. Attentions to be taken as cotton fibers will fluoresce strongly as well amoebic cysts are fluorescent. One drop of 10% potassium hydroxide solution can be added for better visualization of fungal elements.

Existentialism Essay -- essays research papers

Jean-Paul Sartre says "man is nothing else but what he makes of himself" (762). This existentialist view depicts the idea that one is not based on the essence of a soul, but rather, based on decisions made throughout life. Sartre also believes that every man is responsible for all men. One may choose his marriage partner, however, in choosing to marry, one chooses monogamy. Decisions that individuals make will collectively create a set of principles and beliefs for all of man. Many people believe that a person’s decisions are a reflection of his soul and personality. However, it is more likely that the conclusion a person reaches is due to the influence of different ideas, as well as the circumstances surrounding the choices. One does not have an innate "essence", but instead, the choices that a person makes constantly shape his character, which in turn creates his essence. Decision-making is based on many different conditions and controlling factors that exist at the time of conflict. People take into consideration the thoughts and opinions of others, hindering the idea of an individual’s essence. If an essence really existed, another person’s thoughts would not affect someone else’s. Instead, a person makes choices from birth and the different decisions that one chooses form a pattern and creates one’s character. Sartre also says, "Man is nothing else than his plan; he exists only to the extent that he fulfills himself; he is therefore nothing ...

Ethics and Values in Modern Media

Ethics and Values in Modern Day Media â€Å"The public has an insatiable curiosity to know everything. Except what is worth knowing. Journalism, conscious of this, and having tradesman-like habits, supplies their demands. † — Oscar Wilde. Few would doubt that the media is one of the few platforms that affords people the ability to inform, entertain, and influence society. The privilege of having such voice and influence comes with great responsibility. It is crucial that those in a position of such power behave in a responsible and ethical manner. Journalist across all mediums are held to a strong ethical standard in their profession as their work can to completely change the publics view of any given topic or event. It is obvious that technology is having a tremendous impact on all forms of media. While past generations waited for the evening news or the morning paper, people today are reaching for the internet at any time of the day or night to get the latest information. While this instant access has helped curb the public’s insatiable curiosity for information, it has also raised a whole new set of questions regarding the ethics and values in the media industry. One of the most compelling questions to arise out of this new era is whether the media industry has forsaken its core ethics and values for its own gain. Values are very personal choices which are compromised of the individual experiences in life. They are choices that help one prioritize their life. Values can be influenced by a number of factors such as family, upbringing, religion, and society. They help one define and project character attributes such as loyalty, compassion and honesty. Values are instrumental in how we behave and the choices we make. Ethics are professional rules that define how different groups of people should behave. There is a distinct sense of right and wrong with little room for â€Å"grey† or questionable behavior. Ethics guide and dictate what is professional and acceptable to each given profession. To behave ethically is to behave in a manner consistent with what is right and or moral for the profession. Values and ethics are forever at a crossroads because today’s society pushes towards personal wealth. The stakes are constantly raised as far as what it takes to gain that wealth. (,2010). It is not uncommon for people to share private information, set someone up, edit what a person has said or completely alter a picture someone has taken for their gain. It is in situations like this that values are tested and ethics are ignored. Professional values and ethics are commonly derived from government laws and regulations, state licensing boards, and trade associations. A professions code of conduct will â€Å"contain provisions that are designed to advise, guide, and regulate behavior on the job. † (Madsen & Shafritz, 1990, p. 205). There are also federal, state and local laws which regulate and enforce most professional industries. According to The United States Department of Labor (n. d), Government agencies such as OSHA (Occupational Safety and Health Administration) were created to â€Å"assure safe and healthful working conditions for working men and women; by authorizing enforcement of the standards developed under the Act; by assisting and encouraging the States in their efforts to assure safe and healthful working conditions†¦Ã¢â‚¬  . Professional associations such as the Society of Professional Journalist also have their own values and ethics which are set forth to guide their members. Conscientious journalists from all media and specialties strive to serve the public with thoroughness and honesty. Professional integrity is the cornerstone of a journalist's credibility. (Society of Professional Journalist, 1996). Each association clearly outlines its expectations in hopes that all members will abide by them and act in a professional manner. One of the biggest sectors of media affected by ethical conflicts is the advertising market. â€Å"For some business ethicists, advertising is one of the most crucial issues in corporate ethics because its pervasiveness and because of its power to mold consumer thinking and behavior. For example, many have argued that advertising is a form of a behavior control, given its subtle psychological persuasion and its repetitiveness. One such argument holds that advertising creates in the mind of the consumer a need – not just want – for that which is advertised. When the consumer in question happens to be a child subjected to a barrage of TV ads about toys, sugared cereals, candy, and so on, then the ethics of advertising taken as a form of behavior control is even more crucial. † (Madsen & Shafritz, 1990). Photographers also face backlash as women’s advocacy groups rail against the use of airbrushed models in print media. â€Å"You have to accept that fashion is fantasy. It's wearable art,† says Andrew Matusik, a New York fashion photographer and the owner of Digital Retouch, which specializes in celebrity and fashion retouching. â€Å"It's all about creating drama. † (Bennett, 2008). It is widespread knowledge that any photographer with any basic graphic design skills can and will alter an image to suit his client’s needs. As technology evolves so do the ethical standards which advertisers and photographers are held to. Another form of media mired in controversy is reality based investigative news shows. One such show, Dateline’s â€Å"To Catch a Predator† purported to take child sex predators off the street as they taped a series of sting operations set up by various law enforcement agencies. The show faced a firestorm of criticism when a Dallas, Texas man committed suicide moments before he was to be arrested on charges of engaging in sexually explicit chat with a minor. The man’s family sued the network and won a substantial award. The presiding judge ruled â€Å"that the network crossed the line from responsible journalism to irresponsible and reckless intrusion into law enforcement. † (Shea, 2008). Although the show was very successful for the network, it was cancelled shortly after the lawsuit. While it is widely believed that being ethical and sticking to one’s values will lead to an honorable and decent career, the examples illustrated above show that more and more people are willing to push the envelope of professional values and ethics. Personal gain has taken a back seat to deceny and commen sense in many professions. This leaves us with an urgent and pressing question-what are professional ethics and values worth today? References: Values and Ethics. (n. d. ). Strategic Leadership and Decision Making. Retrieved from http://www. au. af. mil/au/awc/awcgate/ndu/strat-ldr-dm/pt4ch15. html Values, Morals and Ethics. (n. d. ). Changing minds and persuasion — How we change what others think, believe, feel and do. Retrieved from http://changingminds. org/explanations/values/values_morals_ethics. tm United States Department of Labor. (n. d). OSHA. Retrieved from http://www. osha. gov/oshinfo/mission. html Madsen, P. , & Shafritz, J. M. (1990). Essentials of Business Ethics. New York, New York: Penguin Group Society of Professional Journalist. (1996). Code of Ethics. Retrieved from http://www. spj. org/ethicscode. asp Bennett, J. (2008, May). Picture Perfect. Newsweek, (), . Shea, D. (2008). The Huffington Post. Retr ieved from http://www. huffingtonpost. com/2008/06/25/nbc-settles-to-catch-a-pr_n_109261. html

All religious systems are based on certain beliefs. Some...

All religious systems are based on certain beliefs. Some are extreme while others appear to be the same in some circles. When it comes to Christians, Judaist, and Muslims, what are their beliefs regarding the creation? Are they monotheistic or polytheistic religions and are they worshiping the same God? What are their beliefs regarding end of time prophecies, hell, paradise etc.? These are questions this paper will attempt to answer to bring to light the major similarities among these religious groups previously mentioned. This discussion will not take place in any particular order. According to Miriam Webster Historic eschatologies are based on datable events that are seen as fundamental to the progress of history. Judaism,†¦show more content†¦Needless to even attempt to say that the Judaists believe contrary since this account is found in one of the book they hold dear. The general belief is that Yahweh has created the solar system, earth, trees, sweet and salty waters e tc. Muslims are not far removed from this perspective. If they believe in restoration, which simply means renewal, repair, rebuilding, and so on, there must have been a degraded system that Allah created good and becomes corrupt over time or due to humankind wickedness. The author purposely uses different names that are used by each religious group. As one realizes, this supreme ruler, creator of all thing is another commonality among members of the three religions. In other word, they are all monotheists. They all make connection with their â€Å"Almighty† by means of prayers, fasts, and sacred scriptures. For the Muslims, the Quran is where they learn about Allah through the teaching of Prophet Muhammad. The Judaists, pay particular attention to teachings of Prophet Moses in the Torah. Christians, on the other hand, while focusing on Jesus teachings through the apostles written in the New Testament, often refer to the book of Old Testament to understand certain prophecies o r interpret certain biblical principles. Another great point worth mentioning is that all three religions are from the lineage of Abraham. Christians are believed to be spiritual descendants of Abraham through Isaac and through Christ of whom they think theyShow MoreRelatedThe Humanities And Its Impact On Education Essay1750 Words   |  7 Pagesinstitutions are cutting the humanities from curricula. According to Nussbaum, our nation and those like it are economically hungry and are pushing technical careers instead. She disputes that a decrease in the humanities will cause a loss in free thinking, and therefore a loss of democracy. Her argument is based off of her theory that without the humanities, citizens become soulless, mechanical, and profit-making machines. 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