GENERAL CHARACTERISTICS OF FUNGI AND CANDIDA ALBICANS

Fungi are eukaryotic microorganisms. GENERAL CHARACTERISTICS OF FUNGI: Fungi are eukaryotic microorganisms. Only about 200 of the thousands of species have been identified as human pathogens. The basic morphological element of filamentous fungi is the hypha and a web of intertwined hyphae is called a mycelium. The basic form of a unicellular fungus is the yeast cell. Yeast: the basic element of the unicellular fungi. It is round to oval and 3– 10 lmin diameter. Several elongated yeast cells chained together and resembling true hyphae are called pseudohyphae. Dimorphism: some fungal species can develop either the yeast or the mycelium form depending on the environmental conditions, a property called dimorphism. Dimorphic pathogenic fungi take the form of yeast cells in the parasitic stage and appear as mycelia in the saprophytic stage.

COMPARISON BETWEEN YEASTS AND MOLDS Definition Mold is a fungi that contains multiple identical nuclei. It grows in the form of hyphae of filaments. A type of fungi that contains only a single cell. Reproduction Reproduce through hyphal formation and small spores, which can be either sexual or asexual Most reproduce asexually through mitosis. Most common form called “budding.” Habitat Typically found in damp, dark or steam-filled areas. Very common. Can be found on fruit and berries, in the stomachs of mammals and on skin, among other places. Uses Some molds are used in food production, for example, Penicillium is used in the production of cheese. Ethanol production, baking, vitamin supplements, study of cell cycle. Energy Production Secrete hydrolytic enzymes that degrade biopolymers such as starch, cellulose and lignin into simpler substances that can be absorbed. Convert carbohydrates to alcohol and carbon dioxide in anaerobic through fermentation. Also obtain carbon from hexose sugars. Health Hazards Certain types of mold can be hazardous to human health in large quantities and can cause allergic reactions and respiratory problems. Some molds also produce mycotoxins, which pose a serious risk to humans and animals. Can cause infection in individuals with compromised immune systems. The yeast candida can also cause candidiasis in humans.

Penicillium sp.

Figure 2. Typical mold Figure 1. Typical Yeasts This slide shows the typical yeast and typical mould. Yeasts and moulds are two main broad, morphological groups of fungi. Candida albicans is referred to as a yeast, but has characteristics of both morphologies (Baron, 1996). Figure 2. Typical mold Figure 1. Typical Yeasts

THE MOST FREQUENT FUNGAL DISEASES THE MOST FREQUENT FUNGAL DISEASES. Mycoses are classified clinically as follows: 1-Primary mycoses (coccidioidomycosis, histoplasmosis, blastomycoses). 2-Opportunistic mycoses (surface and deep yeast mycoses, aspergillosis, mucormycoses, phaeohyphomycoses, hyalohyphomycoses, cryptococcoses; penicilliosis, pneumocystosis). 3-Subcutaneous mycoses (sporotrichosis, chromoblastomycosis, Madura foot (mycetoma). 4-Cutaneous mycoses (pityriasis versicolor, dermatomycoses). Mycotoxicoses Some fungi produce mycotoxins, the best known of which are the aflatoxins produced by the Aspergillus species. These toxins are ingested with the food stuffs on which the fungi have been growing. Aflatoxin B1 may contribute to primary hepatic carcinoma.

Diagnosis Microscopy. Native preparation: briefly heat material under coverslip with 10% KOH. Stained preparation: stain with methylene blue, lactophenol Blue. Culturing. This is possible on universal and selective mediums. Sabouraud dextrose agar can contain selective agents (e.g., chloramphenicol and cycloheximide), this medium has an acid pH of 5.6. Biochemical tests are used mainly to identify yeasts Serology By the identification of antibodies to special fungal antigens in patient’s serum. Antigen detection. By finding of specific antigens in the diagnostic material by direct means using known antibodies, possible in some fungal infections (e.g., cryptococcosis). Cutaneous test. Cutaneous (allergy) tests with specific fungal antigens can be useful in diagnosing a number of fungal infections. Nucleic acid detection. Combined with amplification, such tests are useful for rapid detection of mycotic diseases in immunocompromised patients.

THERAPY A limited number of anti-infective agents are available for specific treatment of fungal infections: Polyenes. These agents bind to membrane sterols and destroy the membrane structure: — Amphotericin B. Used In systemic mycoses. Fungicidal activity with frequent side effects. — Nystatin, natamycin. Only for topical use in mucosal mycoses. Azoles. These agents disrupt ergosterol biosynthesis. Their effect is mainly fungistatic with possible gastrointestinal side effects. Hepatic functional parameters should be monitored during therapy: — Ketoconazole. One of the first azoles. No longer used because of side effects. — Fluconazole. Oral or intravenous application. For the treatment of surface and systemic mycoses and cryptococcal meningitis in AIDS patients. — Itraconazole. Oral and intravenous application. Use in systemic and cutaneous mycoses and also for the treatment of aspergillosis. — Voriconazole. Oral and intravenous application. Good activity against Candida and Aspergillus

Antimetabolites. 5-Fluorocytosine. Interferes with DNA synthesis (base analog). Given by oral application in candidiasis, aspergillosis, and cryptococcosis. It is necessary to monitor the course of therapy for the development of resistance. The toxicity of amphotericin B is reduced in combination with 5-fluorocytosine. Allylamines. Terbinafine. By oral and topical application to treat dermatomycoses. Inhibition of ergosterol biosynthesis. Echinocandins. Caspofungin has been approved as a salvage therapy in refractory aspergillosis. It is useful also in oropharyngeal and esophageal candidiasis. Inhibition of the biosynthesis of glucan of the cell wall. Griseofulvin.This is an older antibiotic used in treatment of dermatomycoses. By oral application, therapy must often be continued for months.

Candidiasis At least 70% of all human Candida infections are caused by C. albicans, the rest by C. parapsilosis, C. tropicalis, C. guillermondii, C. kruzei, and a few other rare Candida species.

Morphology and culture Morphology and culture. 1-Gram staining of primary preparations reveals C. albicans to be a Gram-positive, budding, oval yeast with a diameter of approximately 5µm. 2-Gram-positive pseudohyphae are observed frequently and septate mycelia occasionally . 3-C. albicans can be grown on the usual culture mediums. 4-After 48 hours of incubation on agar mediums, round, whitish, somewhat rough-surfaced colonies form. Pathogenesis and clinical pictures. 1-Candida is a normal inhabitant of human and animal mucosa (commensal). 2-Candida infections must therefore be considered endogenous. 3-Candidiasis usually develop in persons whose immunity is compromised, most frequently in the presence of disturbed cellular immunity. 4-The mucosa are affected most often, less frequently the outer skin and inner organs (deep candidiasis). 5- In oral cavity infections, a white, adherent coating is seen on the cheek mucosa and tongue.

Medical mycology is a growing field of interest because an increased number of clinical diseases are associated with pathogenic fungi. From athlete’s foot to candidal sepsis, fungi cause a wide range of diseases in humans. More specifically, Candida albicans is a key player in causing genital yeast infections, thrush, and sepsis (Calderone, 2002). Interestingly, C. albicans is an unharmful, commensal organism in the healthy individual. However, when the environmental balance in the body has been tipped, C. albicans becomes virulent. Figure 1. Skin Smear Candida albicans www.meddean.luc.edu Candida albicans

- most common fungal pathogen worldwide Frequency - most common fungal pathogen worldwide - 4th leading causes of nosocomial infections, - significant mortality and morbidity in low birth-weight infants - affects 75% women Immunocompromised - most commonly manifested in patients with leukemia, cancer and HIV-AIDs patients. -Oral candidiasis is often a clue to acute primary infection Public Concerns - increasing resistance to drug therapies due to antibiotics and antifungals Alarmingly, the statistics show how serious infections by C. albicans can be. The CDC has ranked it as the 4th leading pathogen in causing nosocomial bloodstream infections (Naglik et. Al., 2003). Surgical and neonatal intensive care units are dense sources of spread of Candida infections (Roilides et. Al., 2004). Moreover, 75% of women will suffer from a yeast infection at least once in her life (Owen et. Al., 2004). In the U.S., vaginitis accounts for 10 million visits to the physician each year. C. albicans is also considered a sexually transmissable pathogen for one’s ability to contract it though sexual intercourse (Prescott et. Al., 2002). Many environmental factors from taking steroids to being a patient in a hospital can predispose a person to diseases that are caused by C. albicans. These factors will be highlighted later in this discussion.

Biology of Candida albicans Commensal Pathogen Morphogenesis Unicellular yeast (harmeless) Filamentous (pathogenic) Principal Cell Wall Polymers Gluccan and Mannan Strict aerobe, favors moist surfaces Commensally found in gut, genitals, and lungs Body Temp 37º C, neutral pH The genus Candida includes approximately 154 species of which Candida albicans is the most frequently (50%) isolated in human fungal infections. C. albicans is also the most abundant and significant of all the Candida related species. Its natural habit is found mainly contained in animals and humans, and on average, colonizes 50% of healthy individuals (Henderson, 2005). However, C. albicans and related species are frequently recovered from hospitals, foods, counter tops, medical equipment and so on. It is part of the normal human microflora of mainly skin and mucosal membranes of gastrointestinal, genitourinary, and respiratory tracts (Hidalgo, 2005). A healthy host is typically extremely resistant to the potentially pathogenic effects of C. albicans. However, when slight alterations of the host’s environment occurs, a harmless commensal organism can turn into agents of severely inflicting illnesses (Naglik et. Al., 2003). A host may become highly susceptible to C. albicans when the following defense mechanisms are challenged: intact mucocutaneous barriers, phagocytic cells, polymorphonuclear leukocytes, monocytic cells, complement system, immunoglobulins, cell-mediated immunity, and mucocutaneous protective bacterial flora (Hidalgo, 2005). There are many risk factors that can stage the scene for susceptibility. These include granulocytopenia, bone-marrow transplation, organ transplanation, general and invasive surgical procedures, catheters, chemotherapy, radiation therapy, the use of corticosteroids, oral contraceptives, broad-spectrum antibiotics, prolonged hospitalization, trauma, pregnancy, sexual intercourse, and premature low-weight births (Hidalgo, 2005). Biological characteristics Candida albicans is a yeast-like fungus that has the capability to produce blastoconidia, pseudohyphae, and true hyphae (Hidalgo, 2003). Only Candida albicans and one other Candida species (C. dubliniensis) are capable of germ-tube production. Germ-tube production occurs at the beginning of true hyphae formation. In lab diagnostics, this feature is key to identifying a sample with strains of C. albicans (Larone, 1995). C. albicans can also be recognized for its production of a typical asexual spore called a chlamydoconidium (Larone, 1995). Exhibiting structural dimorphism is a key biological feature of C. albicans. That is why C. albicans considered “yeast-like” because it can take on the form of yeast, reproducing by budding, or mould, reproducing by hyphal elongation (Gow, 2002). Its ability to morphogenosize into various forms enables its survival in the host as a colonizer (Calderone, 2002). Substances such as biotin, cysteine, serum transferrin, and zinc stimulate dimorphism (Baron, 1996). Bud yeast formation is also favored in environments where pH and temperature are low (Baron, 2002). For instance, C. albicans is a harmless commensal in the vagina where the pH is low and exists as yeast. In its normal environment of the mucosal membranes of humans and animals, C. albicans grows as yeast. When the environment is perturbed however, C. albicans demonstrates hyphal growth (Kwon-Chung & Bennett, 1992). This is seen when the number of beneficial bacteria in the vagina declines. Beneficial bacteria, including species of Lactobacilli, abundantly populate the vagina and secrete lactic acid, which keeps the pH low. When these are wiped out, the pH level is elevated and changes the environment to an ideal environment for C. albicans to grow and multiply (Kwon-Chung & Bennett, 1992). The cell wall is significant for protection, and it also represents the primary way that C. albicans is able to interact with its host. A number of cell-wall proteins are necessary to ensure the proper binding and adherence of the organism to its host, which is a significant factor of virulence. Carbohydrates make up about 80-90% of the cell wall of C. albicans, and the majority of these carbohydrates are glucan and mannan polymers (Chauhan et. Al., 2002). It has been determined that mannan is a major antigen of Candida species, and the different serological concentrations aid as a tool in identifying certain ones (Suzuki, 2002). C. albicans, in particular, contains around 20% of mannan in its cell wall (Baron, 1996). 50-70% of the carbohydrates are glucans, which have been suggested to impede the antifungal amphotericin B from gaining access to the organism plasma membrane (Baron, 1996). The cell wall matrix is layered, which seems to serve a functional role in providing the cell with a rigid structure against osmotic and environmental threats (Chauhan et. Al., 2002). Figure 1. Yeast in Oral Scraping A sample of an oral scraping contains yeast cells and pseudohyphae (www.doctorfungus.org) Rapid Multiplication & Spread

Diseases by C. albicans Thrush Esophagitis Cutaneous Candidiasis Genital Yeast Infections Deep Candidiasis The common diseases that C. albicans can cause are oropharyngeal candidiasis, which includes thrush and esophagitis, cutaneous candidiasis, genital yeast infections, and deep candidiasis. As an overview, thrush and esophagitis are infections concering the mouth and throat. Patients infected with HIV or who have cancer have these types being the most frequent manifestations of mucotaneous lesions (Ruhnke, 2002). Cutaneous candidiasis is infection of the skin, scalp, and nails by C. albicans. An example is diaper rash that often occurs in newborn infants. Genital yeast infections, especially those women, are extremely frequent, but can affect men, too. Several lifestyle factors can predispose a man or woman to a genital yeast infection. Deep candidiasis is also known as invasive candidiasis and afflicts mainly people who are severely immunocompromised. Colonies of C. albicans invade the bloodstream and can spread to many areas of the body destroying tissue and leading to organ failure. It is one type of sepsis that carries a significant mortality and morbidity rate particularly in intensive care units where nosocomial infections are rampant (Marr, 2004). Recall that C. albicans is the fourth leading cause of nosocomial infections in the United States (Marr, 2004).

Oropharyngeal Thrush * Pseudomembranous Angular cheilitis Figure 1. Angular chelitis (www.emed.com) In 400 B.C., Hippocrates described oral thrush as oral ulcers. It was not until 1839 when Langenbeck detected fungi in the oral cavity. Then in 1846, Berg connected that the fungi caused oral thrush. To demonstrate, he infected healthy infants with samples he had taken from the oral lesions and saw the manifestation of thrush (Ruhnke, 2002). Although that would be considered an unethical approach to studying science in today’s society, it was a common practice to understand pathogenecity that lasted until the middle of the 20th century (Ruhnke, 2002). Thrush is the common name for an oral infection that is caused by Candida albicans. The areas of the mouth that are affected are the moist surfaces around the lips, inside the cheeks, and on the tongue and palate. Thrush in patients with cancer and AIDS is frequently observed. Other patients who are at-risk for developing thrush are elderly people, people who have diabetes, or those who have irritation from wearing dentures. There are three types of oral thrush that are clinically classified: pseudomembranous, atrophic, and angular chelitis (Samaranayake et. al., 1990). White, thick plaques that spot the sites of the buccal, mucosa, tongue, palata, and uvula characterize pseudomembraneous candidiasis. When these plaques are removed, they leave an erythematous bleeding surface. Symptoms include burning, pain, and changes in taste. In atrophic candidiasis, there is diffuse erythema that affects mainly the palate and the tongue and result in soreness. Oftentimes, this is denture-induced. Finally, in angular chelitis, the corners of the mouth show signs of cracking and inflammation and associated with the feeling of pain, burning, and soreness (Samarnayake et. al., 1990). When Candida infections spread to the esophagus, a condition known as esophagitis occurs. Symptoms related to this disease may be dysphagia, odynophagia, chest pain, and possible fever (Mildvan, 1995). Candidal esophagitis is the most frequent candidal disease in patients with HIV-AIDS and with oral candidiasis, contributes to an incident rate as high as 50-90% (Ruhnke, 2002). In many clinical cases, esophagitis is a marker that the HIV-infected patient is becoming significantly immunocompromised and is developing AIDs (HSTAT, 2005). Treatment for most oropharyngeal candidiasis typically involves topical antifungal agents (nystatin and clotrimazole) for thrush (Intelihealth). For more severe cases including esophagitis, treatment would require the administration of ketoconazoles or fluconazoles, which can be taken orally (Intelihealth). Flucanzole has proven to be the most effective medication in patients with HIV/AIDS (Intelihealth). Figure 2. Oral Thrush, atrophic (www.mycolog.com) Figure 3. Oral Thrush, pseudomembranous (www.emed.com)

Pathogenesis Host Recognition: Adhesins Enzymes: Hydrolases: Phosphoplipases, Lipases, Proteinases Morphogenesis: Yeast form to Filamentous hyphae/pseudohyphae Without its virulence factors, Candida albicans would not survive in the human body. The major mechanisms of its pathogenesis come in the form of host recognition, the production of enzymes, the ability to assume different morphologies, and the capacity to switch phenotypically (Calderone & Gow, 2002). Host Recognition Host recognition involves key players called adhesins (Calderone & Gow, 2002), which also contribute to colonization. In studies where genes that encoded for adhesions were deleted, C. albicans demonstrated the inability to adhere onto the host and as a result, infection did not proceed (Calderon & Gow, 2002). Thus, adhesion is a major virulence determinant of C. albicans. What prompted these studies of adherence factors were investigations led by King et. al. in the 1980s. An assay was developed to measure the adherence of several species in Candida by using radiolabeled Candida species that were added to human buccal or vaginal exfoliated cells in a suspension. Filters removed non-adhering yeasts and adherence was measured by the amount of radiolabeled yeast cells left. The studies revealed that C. albicans adhered most significantly and to the greatest extent (Calderone & Gow, 2002). Adhesins are either polysaccharide or glycoprotein, but are more abundant as glycoproteins (Calderone & Gow, 2002). Types of adhesins include MP66 whose ligand is Asialoglcospingolipid, MP-hemed whose ligand is fibronectin, and Ala1p whose ligand is also fibronectin (Cormack et. al., 1999). Not only do adhesins allow C. albicans to bind to human epithelial cells, but also to human proteins and internal tissues (Calderone & Gow, 2002). The adherence onto plastic surfaces by C. albicans is yet another growing problem that exacerbates the spread of candidiasis in hospital settings. The contamination of indwelling catheters in patient pools often initiates systemic candidiasis (Jarvis, 1995). Plastics also have the great capacity to collect biofilms, which promotes the adherence of C. albicans. In studies that focused on the adherence of C. albicans to different medical catheters, it was found that polyvinyl catheters supported the most for biofilm formation and polyurethane supported it the least (Hawser & Douglas, 1994). Of all the species of Candida that were tested, C. albicans contributed to the most biofilm mass (Hawser & Douglas, 1994). This nature is an important one for experimental investigation because it could correlate with organisms that are embedded in biofilms have more resistance to antifungal drugs. Recent studies have shown that there is an increased resistance to amphotericin B for organisms that are in a biofilm (Baillie and Douglas, 1999). Enzyme Hydrolases Gaining entry into the host is an important strategy for C. albicans and many other pathogenic organisms. To aid in doing so, C. albicans produces hydrolytic digestive enzymes such as the secreted aspartyl proteinases (SAPs) and phospholipase B (PLB) (Calderone & Gow, 2002). Hydrolytic enzymes play a central role in the pathogenesis of C. albicans, just as they do in many other pathogenic fungi, bacteria, and protozoa (Naglik et. Al., 2003). In contrast to bacteria, C. albicans tends to produce hydrolytic enzymes that are broad-spectrum rather than highly-substrate specific (Hube and Naglik, 2002). Hydrolytic enzymes serve multiple purposes from the digestion of molecules for nutrient absorption to host tissue invasion by the destruction of cell membranes (Hube and Naglik, 2002). Three major categories of hydrolytic enzymes are known to be involved with C. albicans’ virulence (Hube and Naglik, 2002). These enzymes are proteinases, phospholipases, and lipases (Naglik et. al., 2003). Proteinases hydrolyze peptide bonds, phospholipases hydrolyzes phospholipids, and lipases hydrolyze lipids. More specifically, C. albicans secrete aspartyl proteinases, PLB2, PLB2, and PLD-type phospholipases, and Lip1 through Lip10 lipases (Ghannoum, 2000). It has been purported that the phospholipases enhance virulence by mediating the adhesion and lyssis of host cell membranes during an infection (Ghannoum, 2000). While evidence implicates these findings, more research is needed to determine the actual relationship and mechanism (Ghannoum, 2000). Further study on lipases is also necessary to understand their involvement in the organism’s pathogenesis. However, it has been implicated that the broad lipolytic activity may contribute to its persistence and virulence (Hube and Naglik, 2002). The secreted aspartyl proteinases are encoded by a family of ten SAP genes and are concluded to be key virulence determinants of Candida albicans (Felk et. al., 2000). Studies have shown their involvement in hyphal formation, adhesion, and phenotypic switching (Naglik et. al., 2002). The correlation between SAP and Candida virulence has been determined by isolating C. albicans from various candidal diseases and noting the SAP activity. For instance, increased SAP activity occurred in C. albicans strains that were isolated form HIV-positive patients with oral candidiasis compared with HIV-negative C. albicans strains carriers (De Bernardis et. al., 19992). The same effect was found in isolates from patients with oropharyngeal candidiasis and vaginal candidiasis. In animal models, the strains with more SAP production led to higher levels of tissue colonization in the liver, kidneys, and spleen (Abu-Elteen et. al., 2001). Ten different kinds of SAP have been identified that range in size from 35 to 50 kDA. SAP1 to SAP3 execute high activity at a low pH while SAP4 to SAP6 execute high activity at a high pH (Naglik et. al., 2003). The different optimal pH levels for each SAP may be an evolutionary advantage for proteinases to adapt to varying kinds of environment. For instance, the pH of the oral cavity is different than the pH of the gastrointestinal tract, and C. albicans would have to demonstrate the ability to survive in each one. SAP is also significant for facilitating the adherence of C. albicans onto host cells and tissues followed by the degradation of host proteins (Naglik et. al., 2003). It is also found to play some role in hyphal formation and in the regulation of phenotypic switching, which enhances C. albicans’ virulence (Naglik et. al., 2003). The molecular mechanisms are unclear, however, and more research is necessary. Morphogenesis The characteristic that C. albicans is dimorphic and can convert from yeast buds to filamentous hyphae/pseudohyphae under varying conditions indicates another virulent property. One study of an isolated C. albicans strain that was regulated for suppressed hyphal growth was found to be avirulent in an animal mouse model compared to its wild type (Calderone & Gow, 2002). Moreover, the normal environment of the vagina is a classic example to illustrate the non-pathogenic and pathogenic nature of C. albicans. In normal healthy individuals, C. albicans exists as yeast whose growth is mainly suppressed by beneficial bacteria that reside in the vagina. When C. albicans turns virulent, however, its morphology changes into filamentous hyphae (Brown, 2002). Phenotypic Switching The ability for C. albicans to undergo phenotypic switching is a postulated mechanism of virulence. Switching could changes in the expression of cell-surface antigens, tissue affinities of the organism, enzyme production, and drug sensitivity (Calderone & Gow, 2002). Switching also had profound effects on the susceptiblity to antifungal drugs, producing strains that are resistant (Vargas et. al., 2000).

Figure 1. skin equivalent before infection Figure 2. Infection with pathogenic clinical isolate of C. albicans. After 48 h the yeast penetrates the skin equivalent and destroys the tissue This slide illustrates the responses to different C. albicans strains using a skin sample. In Figure 1, there is no infection, and the skin sample is healthy. In Figure 2, the skin has been infected with a pathogenic strain of C. albicans. The skin equivalent is penetrated and tissue is destroyed. In Figure 3, non-pathogenic strains of C. albicans have infected the skin equivalent, but not change is observed. (Fraunhofer, 2002) Hence, the virulence factors that were mentioned in the previous slide must be significant in the success of infection of C. albicans. Figure 3. Infection with non-pathogenic C. albicans. This strain is not able to penetrate into the tissue.

MORPHOGENESIS Figure 1. Morphogenesis. Morphogenesis in C. albicans is a pivotal virulence factor that allows rapid multiplication and subsequent dissemination in host tissue. (www.kent.ac.uk) This slide shows the different routes of morphology that C. albicans may take according to the environment it is in. Figure 2. Morphogenic forms of Candida albicans http://cbr-rbc.nrc-cnrc.gc.ca/thomaslab/candida/caindex.html

Tools for Detection & Diagnosis Diagnosis. 1 - This involves microscopic examination of preparations of different materials, both native and Gram-stained. 2-Candida grows on many standard nutrient mediums, particularly well on Sabouraud agar. 3-Typical yeast colonies are identified under the microscope and based on specific metabolic evidence. 2 - Detection of Candida-specific antigens in serum (e.g., free mannan) is possible using an agglutination reaction with latex particles to which monoclonal antibodies are bound. Due to an increasing incidence of infections caused by C. albicans and the rising number of risk factors that can predispose a population to the harms of its pathogenicity, it is necessary to be able to rapidly diagnose these infections to ensure adequate therapy. Lab techniques should be able to accurately and rapidly identify different strains, more so since there is an emergence of non-Candida albicans species as opportunistic pathogens (Sullivan & Coleman, 2002). Unfortunately, some species (C. glabrata and C. krusei) are showing resistance to antifungal agents that have been used to target C. albicans (Sullivan & Coleman, 2002). The use of molecular biological methods gives promise to fulfilling these goals. Old methods of diagnosis included restriction enzyme analysis, which has been replaced with more effective techniques. In restriction enzyme analysis (REA), the genomic DNA of C. albicans is purified and susbsequently digested with restrictive enzymes. Then the resulting DNA fragments are run in a gel electrophoresis producing bands. However, thousands of bands usually appear, which make comparative analysis rather difficult to do objectively. Despite REA’s simple and inexpensive means, it lacks the needed discriminatory ability (Sullivan and Coleman, 2002). Currently, routine diagnostic laboratories depend on culture and serology to detect Candida cells in sample cultures (Sullivan and Coleman, 2002). For candidiasis infections that are not invasive, swabs will be taken from the patient at the affected site (Intelihealth). For instance, wet mounts would be taken from an infected nail and observed under the microscope for growth of hyphae or pseudohyphae (Hidalgo, 2005). Potassium hydroxide smears and gram stains are also used in detecting the fungus (Hidalgo, 2005). Biopsies may be taken from the lungs to determine respiratory candidiasis and endoscopy is often used to diagnose esophageal/gastrointestinal candidiasis (Hidalgo, 2005). To differentiate among Candida species, CHROMagar is typically applied and compared for color colonies. Biochemical assays such as API 20C and API 32C provide more precision for the identification of specific species by evaluating the assimilation of carbon substrates (Hidalgo, 2005). For deep candidiasis, blood is drawn for a blood culture. Imaging studies are also commonly used in concurrence with other tests for better verification of the species (Hidalgo, 2005). Polymerase Chain Reaction methods have paved the way for rapid developments in the identification of pathogens by using universal primers and sequencing to determine species of bacteria and fungi in a culture (Raoult et. al., 2004). Molecular assays such as this, however, are still under development. Targets for the PCR technology have been the family of secretory aspartyl proteinases in the C. albicans genome (Sullivan & Coleman, 2004). PCR methods are sensitive enough to detect microbial nucleic acid in blood or tissue cultures (Sullivan & Coleman, 2002). They can also diagnose individuals who are at risk early on. In one study, nine patients who were culture-negative for Candida were assessed using PCR. Four of these patients yielded positive PCR results. One of these four patients became culture-positive seven days later. Hence, the speed of the PCR results is highly recognized for the early detection and essentially early treatment of Candida infections. While the sensitivity and speed of PCR methods are a valuable gain for the detection of C. albicans, this method nonetheless has some disadvantages. False-positives arise easily due to environmental contaminants or cross-contamination from previously amplified products (Sullivan & Coleman, 2004). Furthermore, PCR may generate false-negatives due to the presence of inhibitors of Taq DNA polymerase (Sullivan & Coleman, 2004). To circumvent these problems, care and caution must be used in designing the primer and in preparing the PCR for diagnosis. Nonetheless, the cost for this technology is quite expensive. Thus, most clinics depend on the conventional procedures such as culture and serology for the microbial diagnosis (Sullivan & Coleman, 2004). Current research is thus emphasizing discovering more practical and user-friendly means of implementing PCR methods into diagnostic clinical laboratories. Moreover, the potential incorporation of fluorescent probes and molecular beacons (Taqman and Light Cycler) in PCR technologies would lead to even more rapid identification, diagnosis, and quantification of the microbial load (Sullivan & Coleman, 2002). Another molecular technique that holds great promise and potential is a non-PCR-based design such as ‘fluorescent in situ hybridization’ (Sullivan & Coleman, 2002). Here fluorescently labeled DNA probes are used to detect C. albicans in situ in histological samples of deep tissue biopsies and blood. Several studies showed that this method yielded a close sensitivity result to that of the PCR in detecting Candida at 3 cells per 0.5 ml blood (Sullivan & Coleman, 2002). The main disadvantage with this method is that it is not appropriate for the immediate and direct analysis of infected primary tissue or blood samples (Sullivan & Coleman, 2002). The equipment is also too expensive and highly specialized for a routine diagnostic laboratory (Sullivan & Coleman, 2002).

Tools for Detection & Diagnosis Old Methods: Restriction Enzyme Analysis Current methods Culture; biochemical tests; and Serology PCR Based Molecular Techniques Non-PCR Based: Fluorescent in situ hybridization

Therapy. Nystatin and azoles can be used in topical therapy. In cases of deep candidiasis, amphotericin B is the agent of choice, often administered together with 5-fluorocytosine. Echinocandins (e.g., caspofungin) can be used in severe oropharyngeal and esophageal candidiasis.

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