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Medical mycology is a growing field of interest because an increased number of clinical diseases are associated with pathogenic fungi. From athlete’s.

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Presentation on theme: "Medical mycology is a growing field of interest because an increased number of clinical diseases are associated with pathogenic fungi. From athlete’s."— Presentation transcript:

1 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 Candida albicans

2 Contents Introduction to Mycology Biological Profile
Disease capabilities Pathogenesis Detection Drug therapy Research This discussion starts with a brief overview of mycology to provide the audience with a background that will aid in understanding the biology of Candida albicans. Thereafter, it will focus on the major diseases caused by C. albicans. How these diseases are possible will be clarified in the discussion of its pathogenesis. Detecting C. albicans and confirming that it is a responsible for a disease that has manifested in a patient is rather recondite. However, the advent of new biotechnological methods has made detection and diagnosis much more feasible. Candidal infections are treated with antifungals, and the different kinds will be discussed. Unfortunately, the rise in incidence of candidal infections and overprescription of antifungals have contributed to an increasing resistance against C. albicans. Therefore, on-going research and attention to medical mycology are necessary to control this organism.

3 The Situation Frequency - most common fungal pathogen worldwide
- 4th leading causes of nosocomial infections, 40% mortality - significant mortality and morbidity in low birth-weight infants - affects 75% women, 45% experience recurrenceA > 10 million visits/year - classified as a STD by CDC Immunocompromised - cancer and HIV-AIDs patientsC - most commonly manifested in patients with leukemia or HIV-AIDs infections. Oral candidiasis is often a clue to acute primary infectionC 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.

4 Mycology Basics Kingdom: Fungi
More than 10 million species, but only ~400 human disease (*) Sexual Groups Ascomycota* Basidiomycota* Zygomycota* Chytridia Fungi Imperfecti* Very few species are in a commensal relationship with humans - includes Candida albicans and Malasezia furfur Diseases caused by fungi are usually accidental Endogenous and Exogenous Sources Increasing problem due to antibacterial & immunosuppressive agents Molecular mechanisms of pathogenesis not well-defined Candida albicans belongs in the kingdom Fungi. Over 10 million species exist in this kingdom, of which only approximately 400 species are known to cause disease. Fungi are separated into four major groups that are based on their reproductive structures (Baron, 1996). These groups are Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota, and the Fungi Imperfecti. The classification depends on whether the fungus has the ability to reproduce sexually, asexually, or by a combination of both (Baron, 1996). See Figure 1 on slide 5. The 400 species that are known to cause human diseases belong mainly in the Ascomycota, Basidiomycota, Zygomycota, and Fungi Imperfecti phyla (Kwong-Chung et. Al., 1992). Most of these species are pathogenic, but very few exist as commensal organisms along with their pathogenic potential. Such organisms are Malassezia furfur and Candida albicans (Baron, 1996). The diseases that are caused by fungi are usually accidental, but fungal infection may arise endogenously or exogenously. Endogenously from the internal environment of a human and exogenously from an external environment that has gained internal access by the penetration of the host (Baron, 1996). It is worthwhile to mention that fungal diseases are a problem in today’s world as the number of disease associated with fungal infections rises, in large part, due to a increased episode of antibacterial and immunosuppressive agent therapies (Baron, 1996). In addition, while the molecular mechanisms of pathogenesis in bacteria are extensively known, the mechanisms in fungi are limited and require much more research (Baron, 1996).

5 The 5 main groups Fungi are classified into five main groups based on their sexual/asexual reproductive abilities and structures. What should be noted here is that C. albicans exists as an asexual (imperfect) yeast. The phylum Fungi Imperfecti is unique in that species under this group do not have a sexual stage (Baron, 1996). Budding yeasts is the most common form of asexual reproduction, and is key to the growth of c. albicans. C. albicans is also recognized by its ability to form chlamydospores by the conversion of its hyphal elements (Baron, 1996). Figure 1. Classification of Fungi. Fungi are classified based on their ability to reproduce sexually, asexually, by a combination of both. The different reproductive structures places them in the appropriate category. (Baron, 1996)

6 Fungal Characteristics
Plant-like lacking chlorophyll Cell wall chitinous matrix Free-living saprobes and heterotrophs needs Carbon source and Nitrogen source Yeasts or Molds or both Success of an infection Accidental Overcoming host barriers Presiding in host with immunological defects Fungi are eukaryotic cells that look like plant cells, but lack cholorophyll. They possess a cell wall that is made up of chitinous myofibrils that are embedded into a matrix with polysaccharides, proeins, lipids, inorganic salts, and pigments that provide structural rigidity. The major polysaccharides are glucans, mannans, chitosan, and galactans (Baron, 1996). Glucans are a large group of D-glucose polymers that are linked by glycosidic bonds. Mannans are mannose polyers with -D-mannan backbones. Chitosan are polymers of glucosamine and galactans are polymers of galactose. The proportions of these cell wall polymers differ from fungus to fungus. The cell wall of C. albicans, for instance, contains 30-60% glucan, 25-50% mannan, 1-2% chitin, 2-14% lipid, and 5-15% protein (Baron, 1996). Studies have indiciated that the mannans and galactomannans are key players in the immunological response to many yeasts and moulds. Morever, identifying serum concentrations of mannan from clinical samples has been a useful diagnostic technique for determining patients with disseminated candiddiasis (Baron, 1996). The plasma membrane of a fungus is similar to mammalian plasma membranes. Its main difference lies in the presence of nonpolar sterol ergosterol rather than cholesterol (Baron, 1996). Ergosterol is significant in its role of drugs that target fungal diseases and a role in making one who is on steroid treatment more susceptible to yeast infections. These relationships will become clearer throughout the discussion. Fungi are free-living saprobes that nourishes from non-living or decaying organic matter. They cannot synthesize their own food and are therefore heterophic, depending on complex organic substances for nutrition (Kwong-Chung et. Al., 2992). Carbon is needed for the synthesis of carbohydrates, lipids, nucleic acids, and proteins. Nitrogen is also a much needed molecule for the synthesis of amino acids and nucleic acids. Fungi may exist as yeast, mould, or an interchanging phase of both. Yeasts are unicellular, solitary rounded cells while moulds are filamentous. See Figure 1 and Figure 2 on slide 6. Fungi that are yeasts reproduce by budding while moulds reproduce by hyphae formation. See Figure 1 on slide 7 and Figure 2 on slide 8. The budding cells of yeasts are known as blastoconidia. This formation involves three stages: bud emergence, bud growth, and conidium separation (Baron, 1996). See Figure 1 on slide 7. Mould spores, on the other hand, grow hyphae into masses called mycelium by elongation. Although C. albicans oftentimes referred to as a yeast, it has a dual morphology (dimorphic) that allows it to be a yeast but alternate to a mould form, as well (Kwong-Chung et. Al.,1992). As mentioned before, fungi do not usually cause disease, and when they do, disease is a result of an accidental penetration of host barriers or the condition of an immunological defect in the host. When a fungus gains entry into the host from an exogenous source or is capable of growing from an endogenous source, it often develops virulence mechanisms that help it adapt and grow in its new environment. Some fungi are quite comfortable at 37 C and can assume many morphological forms like yeasts, hyphae, and pseudohyphae to facilitate their rapid multiplication and dissemination in the host (Baron, 1996). Figure 1. Penicillium chrysogenum

7 Yeast Characteristics solitary, unicellular Mould Characteristics
reproduction via budding rounded shape moist & mucoid colonies Mould Characteristics filamentous hyphae hyphal formation tips may be rounded (conidia/spores) 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 1. Typical Yeast Figure 2. Typical mould

8 Yeast Bud Formation Figure 1. Stages of bud growth and
This slide shows the formation of a yeast bud. Bud cell emergence is regulated by turgor pressure in the parent cell and by the synthesis of cellular materials that have been activated by a polysaccharide synthetase zymogen. As the bud emerges, the cell of wall the parent things, microtubules elongate into spindle microtubules, and a pinching at the end of the cell begins. This emerging bud is the blastoconidium, and a ring of chitin forms between the blastoconidium and the parent cell to encourage a separation when the ring forms a septal wall. A bud scar might be produced. However, if the blastoconidia continue to develop without separating from the parent, structures called pseudohyphae form. This can be visualized as a filament of attached blastoconidia. C. albicans has the ability to form budding yeast cells, pseudohyphae, and true hyphae (Baron, 1996). Figure 1. Stages of bud growth and yeast cell cycle (Baron et. Al., 1996)

9 Hyphal Formation Hyphal formation is characteristic of developing moulds. During a process called apical elongation, hyphae are formed. Apical elongation requires a careful balance between cell wall lysis and cell wall synthesis. Part of the growth is regulated by a series of vesicles that carry and push wall precursors and wall synthetases towards the apical end of the cell at the site of exocytosis (Baron, 1996). These vesicles are derived from the golgi bodies (Baron, 1996). Figure 1. Polarized hyphal formation (Baron et. Al., 1996)

10 Biology of Candida albicans
Commensal Pathogen A thin-walled dimorphic fungus Morphogenesis Unicellular yeast (harmeless) Filamentous (pathogenic) Principal Cell Wall Polymers Gluccan 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) % 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

11 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).

12 Oropharyngeal Thrush * Pseudomembranous * Atrophic * Angular chelitis
Symptoms Risk Factors HIV Treatment: topical antifungals 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)

13 Genital Yeast Candidiasis
Symptoms Risk Factors - disruption of normal microbiota Treatment - direct genital administration - tablets, suppositories, creams Figure 1. Vaginal Yeast Culture (www.euromeds.co.uk) 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). Most genital yeast infections are caused by Candida albicans. Vaginal yeast infections are most common and affect nearly 75% of women during a lifetime. 45% of women have recurrence rates of two or more yeast infections. Symptoms include intense vaginal itch and/or soreness, secretion of a thick cheeslike discharge, burning, pain, and severe discomfort. Conditions that predispose a woman to an infection are pregnancy, diabetes, frequent douching, the use of birth control pills and antibiotics, and HIV-infection (Owen et. Al., 2004). These are factors that may tempt the virulence capabilities of C. albicans alter the normal vaginal flora (Ruhnke, 2002). For instance, members of Lactobacilli, are beneficial, normal habitants of the vagina that suppress the growth of C. albicans (Mayo Clinic. 2003). These bacteria secrete lactic acid that helps keep the pH of the vagina low, which prevents the growth of many microbes that could only survive at a neutral pH (Prescott, 2002). When these members are eliminated in cases of antibiotic treatment, however, the environment of the vagina becomes favorable for candidal growth. This explains some studies that suggest the consumption of yogurt, which contains lactobacilli cultures, is helpful during yeast infections or urinary tract infections to replenish the environment with the beneficial microbiota (Mayo Clinic, 2003). Figure 1 shows an image of C. albicans in a vaginal yeast sample. Growth is noted by the presence of hyphae. C. albicans also affects men in a manifestation called balanitis. The glans of the penis is inflamed and the symptoms include discharge, redness, and sometimes itchiness. Balanitis is often clinically diagnosed in men who have not had their foreskin circumcised. The domain under the foreskin is warm and moist and thus, ideal for the growth of C. albicans. Figure 2 shows an infiltrate of plasma cells lining in the dermis of a male’s glans that has affected by balanitis. The inflammation that arises from balanitis is a reactive immunological process, which explains the presence of the plasma cells. Also present are numerous capillaries, and extravasated red cells and macrophages may be seen (Ramani, webpathology.com). Figure 2. Plasma cell balanitis. A band-like infiltrate of plasma cells is in the dermis of the male penis. (www.webpathology.com)

14 Deep Candidiasis According to a survey conducted by the CDC, 8 in every 100,000 persons have candidemia each year (CDC). In deep candidiasis, C. albicans infects the bloodstream and spreads throughout the circulatory system and causes severe infection. Newborns, especially those who have low birth weights, severely immunocompromised patients, or those who have severe medical problems are vulnerable to this type of an infections. The fungi may gain access to the bloodstream through catheters, tracheotomies, ventilation, and deep surgical wounds (Intelihealth). It is for this reason that many hospitals need to reevaluate their protocol in treating patients to prevent high rates of nosocomial infections. It is also possible to get infected by C. albicans through intravenous drug abuse, severe burns, and traumatic wounds (Intelihealth). The symptoms include fevers and chills that are not usually alleviated after antibiotic treatment and symptoms associated with different affected organ sites. Death due to organ failure is inevitable if the sepsis is not treated. The treatment for deep candidiasis usually calls for the intravenous administration of amphotericin B. Invasive candiadis is involved in four over-lapping forms that typically begins as an episode of candidemia and leads to various clinical subtypes. These subtypes are catheter-related, acute disseminated, chronic disseminated, and deep organ candidiasis. Catheter-related candidemia is noted as the most common form of candidiasis, which puts C. albicans as the 4th most commonly isolated nosocomial pathogen. Infections of this type are mainly local. A direct contamination of the catheter makes it potentially potent to the patient as the organism can gain entry into the bloodstream and spread to other places in the body. Figure 1. Four forms of invasive candidiasis (www.doctorfungus.org)

15 Pathogenesis Host Recognition Adhesins Enzymes
Hydrolases: Phosphoplipases, Lipases, Proteinases Morphogenesis Yeast form to Filamentous hyphae/pseudohyphae Phenotypic Switching 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).

16 Virulence assay of different C
Virulence assay of different C. albicans strains using the skin equivalent (AST 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 and thus behaves as avirulent as shown in the mouse model of systemic infection. (Fraunhofer, 2002)

17 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

18 Tools for Detection & Diagnosis
Old Methods Restriction Enzyme Analysis Current methods Culture and Serology PCR Based Molecular Techniques targets SAPs Advantages Disadvantages Future 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). Fig. 1. Throat Swab (www.nlm.nih.gov) Non-PCR Based Fluorescent in situ hybridization

19 Current Drug Therapies
Major Drug Categories Polyenes Problems: Azoles Problems: Enhanced drug efflux Catalase activity, ergosterol production FDA approved antifungal drugs Amphotericin B (Fungizone) Clotrimazole (Mycelex) Fluconazole (Diflucan) Itraconazole (Sporanox) Ketoconazole (Nizoral) Nystatin (Mycostatin) The rise of incidences of candidal infections is not the only problem. Antifungal treatments against Candida are strained in the success due to the emergence of refractory fungal species and the development of drug resistance (Sanglard and Balk, 2002). Because the ergosterol biosynthesis pathway is quite specific to fungi, most antifungal drugs target it. These agents include polyenes and the azoles. Polyenes were discovered in the early 1950s and include one of its most successful derivatives in treating C. albicans, amphotericin B (AmB). AmB binds to ergosterol of the plasma membrane and causes the leakage of the cell’s electrolytes by puncturing it. AmB forms salts in acidic and basic environments and is insoluble in water. Its intense potency, however, is associated with severe side effects such as systemic and renal problems. To reduce these problems, AmB has been suspended with lipid complexes to lower its toxicity in mammalian cells (Sanglard and Balk, 2002). A decrease of ergosterol in the cell, however, gives resistance to this agent, which is another drawback. Resistant yeast strains may develop with lowered levels of ergosterol, which would render AmB useless as an antifungal drug. AmB is also known to cause oxidative damage to the cell, but resistant strains may develop with decreased catalase activity, which would diminish AmB’s ability as a powerful antifungal weapon. AmB is usually administered to patients with invasive candidiasis, although its treatment is also necessary for patients with oropharyngeal candidiasis who have had no success under azole therapy (Sanglard and Balk, 2002). The other major category of antifungal agents is the azoles. The group of azoles was discovered in the 1960s and contains the largest group of antifungal agents. Their mode of action is to inhibit the 14-lanosterol demethylase. It binds to the heme iron of the cytochrome P450 in yeast, which inhibits the enzymatic reaction that is involved in demethylating lanosterol into ergosterol (Sanglard and Balk, 2002). Ergosterol, which is needed for the production of Vitamin D in yeast cells, is not synthesized. Thus Vitamin D is not produced either, and the vitality of the yeast cell is supposedly lost. The azole antifungal agents have a broad-spectrum of activity. Perhaps because it is widely used, resistance to these agents is most frequently reported (Sanglard and Balk, 2002). Clinical studies have shown that resistance is related to the cells’ enhanced effluxes that are mediated by multidrug transporters that were upregulated (Sanglard and Balk, 2002). This upregulation explains for the failure resistant yeasts to accumulate the azoles. Fig. 1. Fungizone (www.bms.se)

20 Research Biotechnological methods for rapid identification and detection of Candida strains New antifungal agents Molecular pathogenesis Emerging opportunistic strains Public Health Measures in limiting nosocomial-related infections In conclusion, the epidemics of diseases caused by Candida albicans is a growing concern and must be publicly acknowledged before it is too late. Future research should include investigations for improved biotechonological methods in detecting Candida cells, as well as making the equipment and techniques accessible for routine laboratory diagnostics. With the increasing problem of resistance to antifungals, research into new antifungal agents is necessary. For instance, antibodies against the fungal antigen heat shock protein hsp90 may play a huge role in antifungal therapy (Burnie and Matthews, 2003). Hsp90s are responsible for yeast survival as a virulence factor at higher temperatures. It also serves as a molecular chaperone for various fungal proteins. It is typically releases as a result of cell lysis in sepsis, and is part of the consequence of septic shock (Burnie and Matthews, 2003). Moreover, hsp90 is upregulated in the presence of increased estradiol (Burnie and Matthews, 2003), which references back to why the use of steroids and birth control pills are a few of the predisposing factors for candidal infection. Antibodies to hsp90s are naturally occurring in the host’s endogenous immune response to an infection (Burnie and Matthews, 2003). Commercially known as Mycograb, these antibodies hold the purpose of serving as targets for for C. albicans in combating the spread of resistance, and has so far not been associated with side effects in the patient (Burnie and Matthews, 2003). Exploration of more antifungal therapies that are based on using antibodies against fungal molecules is an ongoing exciting field of research in medical mycology. A better approach to the points listed on the slide is supported by the need for a greater understanding of the molecular pathogenesis of C. albicans. Morever, evidence indicates that other isolates from infections that are non-C. albicans, but related fungal strains, are emerging and may contribute to wider variety of opportunistic infections. These emerging strains should also be studied. And finally, public health measure must be taken especially in hospitalized settings where nosocomial infections are notorious for the spread and cause of disease. Perhaps better guidelines and work protocol will assist in alleviating the problem. It is in great hopes that fungal infections will remain at a controlled level and not become a part of a greater human problem as emerging infectious diseases.

21 References 23. Burnie J. & R. Matthews The role of antibodies against hsp90 in the treatment of fungal infections Drug News Perspect 16(4):


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