and are categorized into two groups: superficial and systemic. Superficial yeast infections can affect different parts of the human body

Transcription

1 Introduction / 1 Yeasts are unicellular fungi that cause a wide range of infections and are categorized into two groups: superficial and systemic. Superficial yeast infections can affect different parts of the human body including skin, nails, mucosal surface of the mouth, digestive tract, vagina and oesophagus and can also become chronic (Thevissen, 2005). Systemic yeast infection also known as invasive yeast infection can occur in various organs of the body such as the brain, spinal cord, eye, gall bladder, heart, lungs, kidneys and urinary bladder (Talaro and Talaro, 1996) and can be seen in individuals with reduced function of the immune system (Thevissen, 2005). The most common yeasts that infect humans are the Candida species (Talaro and Talaro, 1996). Candida albicans remains the predominant Candida species causing candidal infections accounting for over half of all cases in the world (Pfaller and Diekema, 2007). An increase in the prevalence of yeast infections caused by non- albicans Candida such as Candida glabrata, Candida krusei, Candida tropicalis and Candida parapsilosis have been reported in other parts of the world (Pfaller and Diekema, 2007). Many epidemiological studies have investigated the prevalence of yeast infections and their etiological agents in different parts of the world (Narkwa, 2010).

2 Introduction / 2 The most common type of yeast infection is Candida vulvovaginitis (Sobel, 1992). It is estimated that at least 75% women suffer from at least an attack of Candida infection during their life time (Saporiti and Gomez, 2001; Ferrer, 2000). A study conducted in Nigeria revealed that the prevalence of candidal infection in female was 40.6% (Enweani et al 2001). Taxonomic classification of Candida The relative increase in incidence of fungal infections in the past two decades has been overwhelming, affecting even the immunocompromised people. Although recently newer and less common fungal agents are being increasingly associated with infection in immuno- suppressed host. Yeasts are members of Kingdom Fungi which is the large group of eukaryotic organisms that includes moulds as well as the more familiar mushrooms. Yeast do not form a specific taxonomic or phylogenetic grouping. At present, it is estimated that only 1 % of all yeast species have been described (Kurtzman and Piskur, 2006).

3 Introduction / 3 The phylogenetic diversity of yeast is shown by their placement in the divison Ascomycota, Basidiomycota and Deuteromycota. The division Ascomycota commonly known as ascomycetes comprises Candida. Currently more than two hundred ascomycetes yeasts are included into the genus Candida, but only few species are opportunistic pathogens in humans (Hazan, 1995; Pfaller and Diekema, 2004). Majority of the medically important fungi belong to group Deuteromycota. The fungi in this group are segregated into: (a) Blastomycetes which contain yeasts that reproduce by budding (b) Coelomycetes which contain moulds that produce conidia within a cavity of fungal tissue referred to as conidioma (eg. Pycnidium) and (c) Hypomycetes which contain moulds that produce conidia without fruiting structures or with synnema or that produce sterile hyphae (Sharma, 2010).

4 Introduction / 4 Classification of Fungi Division Sexual spore type Asexual spore type Human pathogenic fungi Zygomycota Zyogte (zygospore)- due to fusion of two gametangia Sporangiospore Rhizopus sp.; Absidia sp.; Rhizomucor sp.; Mucor sp.; Conidiobolus sp.; Basidiobolus sp. Ascomycota Ascosporesspore in a sac (Ascus) Conidium Dermetophytes, Aspergillus sp.; Histoplasma capsulatum; Blastomyces dermatitidis Basidiomycota Basidiosporespore borne on the narrow stalk (sterigma) protruding from rounded structure called basidium. Conidium Cryptococcus neoformans Deuteromycota Not known Conidium / Sporangiospore Candida albicans; Coccidioides immitis; Sporothrix schenckii

5 Introduction / 5 Phylogenetic position of medically significant fungi [ Modified from Chandler, Kaplan and Ajello, (1980)] PHYLUM CLASS ORDER FAMILY GENUS / SPECIES Zygomycota Zygomycetes Entomophthors Mucorales Ascomycota Heminascomycetes Loculoascomycetes Endomycetales Myriangiales Entomophthoraceae Mucoraceae Endomycetaceae Saccharomycetace Basidobolus Absidia, Cunninghamella, Mucor,Rhizopus, Syncephalastrum Endomyces (Geotrichum sp.) Kluyveromyces Microascales Saccardinulaceae Piedraia hortae Microascaceae Pseudallescheria boydii Plectomycetes Eurotiales Eurotiaceae Emericella (Aspergillus nidulans) Sortorya (Aspergillus fumigatus) Gymnoascaceae Ajellomyces (Histoplasma capsulatum) Arthromyces (Trichophyton species) Nannizzia (Microsporum species) Basidiomycota Teliomycetes Ustilaginales Filobasdiaceae Filobasidiella (Cryptococcus neoformans)

6 Introduction / 6 Deuteromycota Blastomycetes Hyphomycetes Ceolomycetes Moniliales Sphaeropsidales Cryptococcaceae Moniliaceae Dematiaceae Tuberculariaceae Candida Acremonium Aspergillus Blastomyces Chrysosporium Coccidioides Epidermophyton Geotrichum Gliocladium Histoplasma Microsporum Paecilomyces Paracoccidioides Penicillum Sepedonium Scopulariopsis Sporothrix Trichoderma Trichophyton Alternaria Aureobasidium Cladosporium Curvularia Drechslera Exophiala Fonseacaea Heliminthosporium Madurella Nigrospora Phialophora Rhinocladiella Stemphylium Ulocladium Wangiella Epicoccum Fusarium Phoma

7 Introduction / 7 Candida is a unicellular fungal cell encased in cell wall made up of chitin. Cells of Candida possesses membrane- bound organelles such as vacuoles, Golgi apparatus, mitochondria as well as ribosomes. They have no flagella. Generally, yeast cells are larger than bacteria and are commonly spherical to egg shaped. The size of yeast cells vary greatly depending on the species, typically measuring 3-4 µm in diameter, although some yeasts can reach over 40 µm (Walker et al 2002). Like all other fungi Candida lack chloroplast and are therefore heterotrophic organisms that require preformed organic compounds as energy source (Prescott et al 1996). Candida is capable of adapting itself to a wide range of environmental conditions. In vivo it can grow on the relatively acidic vaginal mucosa (ph 4.5) and in the blood (ph 7.4), whereas in vitro it is able to grow in the ph range between 2.5 and 10 and at temperature ranging from 5ºC to 46 ºC (Hubbard et al 1986; Odds, 1988). Optimum growth of C.albicans has been observed under aerobic conditions with maximal achieved doubling time just under one hour (Odds, 1988). The most common mode of vegetative growth in Candida is asexual reproduction by budding. In budding, the parent yeast cell forms a protuberance called a bud on its outer surface. As the bud

8 Introduction / 8 elongates, the parent cell s nucleus divides into two and one nucleus migrates into the bud. Cell wall material is then formed between the bud and the parent cell. The bud eventually breaks away from the parent cell (Kurtzman, 1994). Yeast species that cause disease in humans are mainly members of the genera Candida and Cryptococcus. The Candida includes about 150 different species. More than 17 different species of Candida have been reported to be etiologic agents of yeast infection in humans (Hazan, 1995; Pfaller and Diekema, 2004). The pathogenic yeasts of Candida in descending order of virulence for humans are: Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii and Candida lusitaniae (Hurley et al 1987). More than 90% of yeast infections due to Candida are attributed to the species- Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei. Others are Candida lusitaniae and Candida dubliniensis (Pfaller and Diekema, 2004). Infection caused by Candida species are in general referred to as candidiasis. Candidiasis or thrush a fungal infection (mycosis) of any of the Candida species affects both sexes, all age groups with world wide

9 Introduction / 9 distribution. Candidiasis includes diverse group of infections caused by Candida albicans or by other members of genus Candida. It is a member of the normal flora of skin, oral cavity, gastro intestinal and genitals of humans (Talaro and Talaro, 1996). Clinical Classification of Candidiasis (Sharma, 2010) I. Infectious Diseases A. Mucocutaneous Manifestations Oral: thrush, stomatitis, glossitis, cheilitis Alimentary: esophagitis, gastritis Genitals: Vulvovaginitis, balanitis, balanoposthitis Chronic mucocutaneous candidiasis Ocular candidiasis B. Cutaneous Manifestations Intertriginous and generalized Paraonychia and onychomycosis Candidal granuloma C. Systemic Manifestation Urinary tract Endocarditis

10 Introduction / 10 Pulmonary candidiasis Meningitis Candidemia Dissemination Arthritis Osteomyelitis Endophthalmiti II. Allergic Diseases Candidid Eczema Asthma Gastritis The Candida species produce various infectious manifestations among the human beings depending upon their immune status and underlying predisposing factors. There are various sites involved in candidiasis. Oral candidiasis also known as oral thrush is the most common form of disease produced by colonization of Candida species. The oropharyngeal candidiasis occurs in infants, individuals with

11 Introduction / 11 diabetes mellitus, those receiving antibacterial antibiotics and patients infected with HIV. Vaginal candidiasis is one of the most common infections seen in general practice. This affects primarily young and middle aged females. Chronic mucocutaneous candidiasis is common in the patients with deficient cell- mediated immunity (Sharma, 2010). The cutaneous manifestations of candidiasis are intertrigo (inflammatory lesion of the skin folds), paronychia (inflammation of the nail fold), diaper dermatitis (diaper rash in infants) etc (Sharma, 2010). Systemic Candida infections include urinary tract candidiasis, candiduria, pulmonary candidiasis, candidemia and septicemia as well as nosocominal candidiasis (Sharma, 2010). Bronchial asthma caused by Candida species has also been reported (Sharma, 2010). The cutaneous allergies such as urticaria and eczema caused due to Candida species have been well documented (Sharma, 2010). Several factors contribute to pathogenesis of Candida species. These factors are: (a) ability to bind to the host tissue and (b) production of proteolytic enzymes which facilitates tissue penetration as well as invasion (Collier et al 1998). Another virulence factor is the reversible transition between unicellular yeast and filamentous forms (Collier et al 1998). Candida species are mostly found as commensals in

12 Introduction / 12 humans and other warm-blooded animals. However, sometimes these yeasts can become pathogenic when they sprout hyphal outgrowths (Collier et al 1998). Human infections by Candida species are usually endogenous (Talaro and Talaro, 1996). This emerges from the fact that Candida species are frequently part of the normal flora of the mouth, throat, large intestine, vagina and skin of humans. Infections are usually produced in the patients whose immune systems are compromised and in those taking broad spectrum antibiotics (Talaro and Talaro, 1996). Candida can be acquired from exogenous sources through the contaminated hands of health care workers, contaminated infusates and biomaterials (Khatib and Clark, 1995). Person to person transmission of Candida infection is uncommon (Talaro and Talaro, 1996). It is seen primarily in oral thrush of newborns whose mothers have vaginal infections (Talaro and Talaro, 1996). Sexual transmission from patients with vaginitis to their sexual partners has been reported (Murray et al 2002). A presumptive diagnosis of Candida infection is made if budding yeast cells and pseudohyphae are found in specimens from localized infections during microscopic examinations of slides prepared from the specimens. Specimens are also cultured on Sabouraud dextrose agar

13 Introduction / 13 (SDA) plates and incubated at room temperature (25-28 C) for several days during which colonies of the yeast develop. Identification of various species of the yeasts are done using cultural characteristics, the germ tube test and biochemical tests (Narkwa, 2010). In clinical settings, yeast infections are commonly treated with antifungal drugs. The choice of antifungal agent depends on the site of infection and the immune status of the patients. For example, the antifungal drugs commonly used to treat superficial yeast infections are topical clotrimazole, topical nystatin, fluconazole, itraconazole and topical ketoconazole (Moosa et al 2004). Since 1950 s, antifungal drug discovery has identified three classes of natural products : (a) griseofulvin (b) polyenes and (c) echinocandins and four classes of synthetic chemicals:(a) allylamines (b) azoles (c) flucytosine and (d) phenylmorpholines with clinical value against fungal infections (Odds, 2003). (a) Griseofulvin was tested as an antifungal agent in humans in the 1959 s and has a selective inhibitory activity against fungi. It is a secondary metabolite of the fungus Penicillium griseofulvum. Griseofulvin inhibits fungal mitosis by interfering with polymerized

14 Introduction / 14 microtubules and spindle formation in dividing cells hence it is a fungistatic and not a fungicidal drug. Its activity spectrum is limited to the dermatophytes which causes ringworm infections of skin, nails and hair. Griseofulvin has proved its worth over many years, particularly in the successful treatment of scalp ringworms (Odds, 2003). (b) Polyenes are nature s most frequently produced chemical structure. Natural products with conjugated double bonds (poly -enes ) include terpenes, vitamin A and many essential oils in addition to molecules such as amphotericin B and nystatin which are used as antifungal agents for human diseases. These antifungal molecules, produced by streptomyces bacteria, bind to sterols in cell membranes and cause leakage of the cellular constituents (Odds, 2003). The following four polyene antibiotics are clinically important in the management of fungal infections: (i) nystatine (ii) amphotericin B (iii) pimaricin and (iv) hamycin. Pimaricin is obtained from Streptomyces natalensis. It shows efficacy against Fusarium solani and Aspergillus species. Hamycin is isolated from Streptomyces pimprina. Nystatin can only be used as a topical agent in the form of creams, pessaries, etc. However,

15 Introduction / 15 amphotericin B is formulated for intravenous (i.v.) use. Its broad antifungal spectrum makes it an ideal choice for immediate use when the exact cause of a probable fungal infection has not been established (Odds, 2003). The problem with intravenous amphotericin B treatment is that the low selectivity of the antifungal action of this molecule often results in serious toxic effects, particularly kidney damage (Sharma, 2010). (c) Echinocandins are natural products: secondary metabolites of fungi. Echinocandin B was isolated in the 1970 s from a culture of Aspergillus nidulans var. echinulatus. Ironically, in the present day semi-synthetic offspring are being developed for the treatment of Aspergillus infections (Odds, 2003). Fungal cell wall polysaccharides have long been regarded as ideal targets for antifungal activity. The echinocandins and papulacandins both inhibit synthesis of -1,3-glucan polymers and are therefore fungicidal for species where the wall is dependent on such glucans for their structural integrity. Nikkomycins inhibit synthesis of chitin in fungal walls, while pradimicins and benanomicins bind to cell wall mannoproteins and induce fatal permeability changes in the fungal cell

16 Introduction / 16 envelope. Of all these agents, only the echinocandins have been successfully developed for clinical use (Vanden, 2002). The first semi-synthetic echinocandin developed for human use was cilofungin. This agent was limited by a relatively narrow antifungal spectrum and toxic effects. Caspofungin, anidulafungin and micafungin have an antifungal spectrum that includes most Candida spp. several Aspergillus spp. and Pneumocystis carinii (Odds, 2003). (d) Allylamines class of antifungal agents is very well known and widely represented among plant fungicides. Naftifine was developed as an allylamine agent for topical use. Its success and antifungal potency led to research for variants with systemic bioavailability. The only single compound terbinafine has emerged to have efficacy and safety needed for a pharmaceutical agent. Terbinafine has been a very valuable antifungal for dermatophyte infections. It is widely used in treatment for nail infections. Like the azoles, the allylamines act to block fungal ergosterol synthesis, but they act much earlier in the biosynthetic pathway (Odds, 2003). Several laboratories have tried to design novel allylamines for the clinic but no success was achieved for more than 20 years (Odds, 2003). Currently, there are no vaccines available for

17 Introduction / 17 preventing yeast infections hence, the only clinical recourse to combat yeast infection is the use of antifungal agents. (e) Imidazoles and Triazoles - Imidazoles and triazoles are the group of compounds collectively known as azoles. Depending upon presence of two or three nitrogen in the azole ring, they are classified as imidazole and triazoles, respectively. Azoles causes disorganization of the fungal membrane by targeting the fungal cytochrome P-450, which inhibits the 14α- demethylation of lanosterol in the ergosterol biosynthesis pathway (Vanden et al 1995). Depletion of erosterol causes an accumulation of several 14 α-methyl-sterols thereby altering permeability and fluidity of the fungal membrane with secondary consequences for membrane bound enzymes (Marichal and Vanden, 1995). Lack of ergosterol in a fungal membrane seriously cripples the fungus and leaves it unable to grow and develop in the normal way (Odds, 2003). More than any other antifungal class, the azoles have been steadily refined and improved upon over the course of almost 50 years. The earliest azole for clinical use, chlormidazole, was really not a very good pharmaceutical but the ease with which variants on the

18 Introduction / 18 chlormidazole chemical structure could be synthesized and tested led to steady progress with azole antifungal agents. The following imidazoles commonly in use are miconazole, clotrimazole, ketoconazole, econazole, tioconazole, sulconazole, isoconazole, bifonazole, oxiconazole and fenticonazole. Triazole includes fluconazole, itraconazole, voriconazole, teraconazole, posaconazole and ravuconazole. These agents are used systemically and are safe as well as effective as compared to previously available imidazoles. They are preferably used for severe and disseminated fungal infections (Sharma, 2010). (f) Flucytosine is a unique antifungal agent. It has no siblings or progeny in its antifungal class. The compound was initially made as a potential anti-cancer drug. Its antifungal activity was picked up in separate screening research and developed to treat clinically difficult infections such as meningitis caused by Cryptococcus neoformans. Its antifungal specificity comes from the fact that fungi, but not human cells, possess the enzymes needed to take up flucytosine and convert it internally to 5-fluorouracil, a compound that is highly toxic to all eukaryotic systems. Fluorouracil becomes incorporated in fungal DNA

19 Introduction / 19 and RNA and blocks synthesis of both these vital molecules preventing cell proliferation (Vermes et al 2000). Several antifungal agents are used to treat systemic fungal infections. Fluconazole is a triazole agent with a broad therapeutic range with little toxicity and is established as a first line antifungal for the treatment of candidiasis (Sheehan et al 1999). Ketoconazole is effective against a wide spectrum of fungi and yeasts, including Candida sp. (Martin, 1990). Inappropriate management of yeast infections particularly candidal infection may result in its progression leading to complicated disease state and enhance the determination of susceptibility patterns of Candida against the antifungal agents. Numerous studies have examined bacterial susceptibility but knowledge about the antifungal susceptibility of medically important yeasts is scant. Surveillance for antifungal susceptibility of Candida to available antifungal agents would provide physicians with information needed to prescribe proper antifungal agents for treating yeast infections which is essential for improved patient management. Hence, the study was planned to evaluate the sensitive patterns of clinically significant isolates of Candida against fluconazole and ketoconazole and to

20 Introduction / 20 determine the most sensitive antifungal drug fluconazole Vs ketoconazole.