MYCOLOGY

BRIEF HISTORY OF (MEDICAL) MYCOLOGY

Mycology is an interesting field or branch of microbiology that deals with the study of fungi. Fungi play many important roles in the environment; and their activities affects man, plants, animals, other microbes and even the environment. Fungi is an important group of microorganisms that lack photosynthetic ability but possess tremendous economic, health and scientific value. First, some fungi such as mushroom are a source of protein and food for humans; and other forms of fungi especially yeasts are used in the production of many food products that benefits man and animals. For example, Saccharomyces cerevisiae is a yeast that has immense benefit in bread production where they serve as leavening agent in baking. This yeast, which is a type of fungi also has benefits in brewery where it is used for the production of alcoholic and non-alcoholic beverages.

Fungi are important agents of decay or decomposition because they help in the degradation of dead organic matter in the environment. This way, fungi play important role in maintaining the biosphere and tranquility of the earth. Generally, fungi are saprophytic microorganisms because of their ability to live and feed on dead or decaying organic matter including those of animal and plant origin from where they derive their energy and nutrient source as heterotrophs. As agents of disease and infections, fungi cause a number of clinically important disease and infections in humans, plants and animals. Fungi are also food spoilage organisms that cause the spoilage of food and food products. However, majority of fungi are of immense economic, industrial, medical and pharmaceutical importance because they are sources from which major antibiotics used for treating infectious diseases of man are sourced from. For example, penicillin, an important antibiotic used clinically for treating and managing infectious diseases was first isolated from a fungus known as Penicillium chrysogenum (also known as P. notatum). Fungi have been known since prehistoric times, and their economic value have since been exploited by mankind even till date.

What is mycology? Mycology is simply defined as the study of fungi. Fungi (singular: fungus) are eukaryotic and heterotrophic microorganisms that do not contain chlorophyll but obtains its nutrient through the absorption of food and/or nutrients from dead or decaying organic matter in its environment. The study of fungi actually began in the early 1830’s following the serendipitous discovery of fungi as the causative agent of the white muscardine disease of silkworm. Nonetheless, the benefit and use of fungi by mankind have been noted even before this time period, but the discovery and description of fungi could not be clarified until the discovery of the microscope.

Augustino Deo Bassi in 1835 discovered that a fungus was responsible for the white muscardine disease of silk worm. Lageaibeak in 1839 proved that Candida albicans was responsible for oral thrush (candidiasis). Also in 1839, Schoenileini discovered that favus was caused by Trichophyton schoenleinii. The study of medical mycology was not well developed early enough because the mortality rate of fungal diseases was very low. In 1910, a French dermatologist, Sabouraud studied the disease of the ringworm fungi, Le Teignes (Tinea). The medium he used to cultivate the fungi was named after him as Sabouraud Dextrose agar (SDA) medium. SDA is a selective culture medium for the isolation of fungi. He cultivated the medium in large scale. Emmons, C.W in 1935 classified dermatophytes between 200,000-300,000 of both pathogenic and non-pathogenic fungi existed on earth. And most cause plant diseases. About 100 species of fungi are known to be pathogenic to man. Those fungi that cause disease in animals are called Zoo-pathogenic fungi.

ECONOMIC IMPORTANCE OF FUNGI

  1. Fungi help to breakdown organic matter
  2. They help in the recycling of organic matter in the ecosystem (or in nature).
  3. They contribute to the production of food and spirits
  4. In medicine, fungi are employed in the production of antibiotics (e.g. penicillin) and immunosuppressive drugs (e.g. cyclosporine).
  5. They are also used by scientist to investigate a variety of eukaryotic processes.
  6. They cause diseases in humans and animals
  7. They cause the majority of plant diseases.

Fungi (singular: fungus) are eukaryotes. They generally occur in two forms: yeast and mould. Yeast is round or oval and basically unicellular, but capable of forming long chains called pseudomycelium while mould is filamentous in nature. Some pathogenic fungi can be yeast-like inside animal tissue and filamentous in their natural habitat. Such fungi are known as dimorphic fungi. They exist in two phases; and can be yeast-like or mould-like at different conditions. Also, fungi have a highly developed form of sexual reproduction, but most can also multiply asexually. The natural habitat of a majority of fungi is soil where they perform their primary function in nature. Fungi are saprophytic in nature, and they decompose plant and animal materials and recycle the biomass in the ecosystem. A majority of fungi are harmless to humans and animals. Only a small number of species are known to cause diseases in humans and animals though a majority of plant diseases are caused by fungi. The tissue reaction is usually granulomatous. Fungal infections do not respond to antibacterial antibiotics. Instead, they respond to antifungal agents.

GROWTH AND ISOLATION OF FUNGI

Most fungi occur in nature and grow readily on simple sources of N2 and carbohydrate. The optimal temperature of growth for most fungi (whether pathogenic to man, animals or plants) is between 25-280C. Traditionally, SDA has been used to culture fungi because it does not support bacterial growth. SDA contains cycloheximide and chloramphenicol which inhibit the growth of saprophytic fungi and bacteria respectively. The morphologic appearances of fungi on SDA have been used to identify them in the laboratory. Fungi require low pH (about 5.0-5.5) to grow. Fungi are not easily adapted to growth on the body and because of this, most exhibit dimorphism – in which they exist as both yeast and mould. To culture medical fungi from non-sterile specimens, antibacterial antibiotics and cycloheximide are usually added onto the SDA medium or inhibitory mould agar medium during preparation in order to inhibit bacterial and saprophytic fungi growth respectively as aforementioned.

Classification of Fungi

The classification of fungi is usually based on:

  1. The spores they produce
  2. Morphology (appearance) in the host and culture media.

According to their morphology, we have yeast and yeast-like fungi. Dimorphic fungi are fungi that have 2 growth forms, and these are:

  1. The mycelial form, which grow best at 25-280C.
  2. The yeast form, which grow best at 350C.
  3. capsulatum (American histoplasmosis) and A. duboisii (African histoplasmosis) are examples of dimorphism.

Dimorphism in fungi depends on:

           Tissue requirement

           Temperature requirement

           Nutritional requirement

Taxonomically, fungi are divided into four divisions namely:

Zygomycota (Zygomycetes) or Phycomycota (Phycomycetes)

Ascomycota (Ascomycetes)

Basidiomycota (Basidiomycetes)

Deuteromycetes (Fungi Imperfecti)

These taxonomic groups of fungi are explained in detail in the later part of this section.

But from the perspective of medical mycology, pathogenic fungi are divided into following three groups:

  • Yeast-like fungi
  • Dimorphic fungi
  • Filamentous fungi

Dimorphic Fungi: A unique group of fungi that exhibit properties of both the yeasts and the moulds. A majority of dimorphic fungi are pathogenic. In their natural habitat, these fungi occur in their filamentous or mould form, but in host tissue or at human body temperature (37°C), they exhibit yeast form.

REPRODUCTION IN FUNGI

Fungi reproduce both sexually by meiosis and asexually by mitosis. The reproduction of fungi either asexually or sexually is usually dependent on the type of fungus and on the environmental conditions available for growth. Asexually, fungi reproduce by fragmentation, budding, and by spore formation. The sexual forms of fungi are known as teleomorphs while asexual forms of fungi are known as anamorphs. The major steps of sexual reproduction in fungi include the meiotic development of haploid nuclei, the fusion of the haploid nuclei from different fungal cells, and the emerging diploid nuclei or zygote that emanated from the fusion process. Perfect fungi reproduce both sexually and asexually, while imperfect fungi (Deuteromycetes) reproduce only asexually (by mitosis). Fungi produce spores that disperse from the parent organism to a suitable substrate or surface for further development. And these spores also aid in the dispersal of the organism aside their reproduction potentials. Fungal spores help in the spreading of fungi.

Fungi can also be classified as perfect fungi or imperfect fungi depending on their type or mode of reproduction.  If a fungus reproduce by sexual reproduction alone or sexual and asexual reproduction, the fungus is called perfect fungus. This can be seen in Saccharomyces cerevisiae. However, if a fungus reproduces only by asexual reproduction, such a fungus is called an imperfect fungus or fungi-imperfecti.

Fungi produce two types of spores viz: sexual spores and asexual spores. Spores allow fungi to expand their distribution and colonize new environments. They may be released from the parent thallus (structure), either outside or within a special reproductive sac called a sporangium. The two types of spore-bearing structures of fungi are: conidiophores (which release unicellular or multicellular spores that are released directly from the tip or side of the hypha) and sporangiophores (which release spores from a sporangium). Fungi spores emanate basically from any of these two structures (i.e. the conidiophores and sporangiophores).

Asexual reproduction in fungi is characterized by the formation of conidia (asexual spores) by the type of cell division known as mitosis (Figure 3). In asexual reproduction of fungal cells, budding yeast-like structures are formed and hyphae elements undergo fragmentation or they disintegrate into several components. Some of the spores formed in asexual reproduction include arthrospores, blastospores and sporangiospores. Most fungi especially yeast cells such as the Saccharomyces species reproduce asexually by budding, a process in which new daughter cells originate from vegetative (parent) cells as buds or outgrowth. These spores form the basis for the dispersal of fungi in the environment, and their formation can also be used for fungal identification in the laboratory. Fragmentation of fungal hyphae leads to the formation of arthrospores or arthroconidia (which are individual components of broken hyphae that behave like spores) and chlamydospores (which are round-thick walled and resistant hyphae cell components formed before separation of hyphae). When the spores develop within a sac they are known as sporangiospores but when they develop at the side of the hyphae without enclosure in a sac they are known as conidiospores. Conidia formed asexually by fungi are usually borne or encased in sac-like structures known as sporangia (singular: sporangium). The sporangia contain numerous amounts of fungal spores, and they serve as route via which spores are released and dispersed in the natural environment especially when they rupture. The conidia released in this manner are known as sporangiospores.

TYPES OF ASEXUAL SPORES

  • Macroconidium: These are large, multicellular conidium.
  • Microconidium: These are small, single celled conidium.
  • Chlamydospores: These are thick-walled, resistant spores formed by the direct differentiation of the mycelium. This type of spore is formed by Candida albicans.
  • Arthrospores: These are asexual spores formed by the disarticulation of This type of spore is formed by Geotrichum candidum.
  • Blastospores: These are spores produced as a result of budding process of the mycelium or          from a single spore. This type of spore is formed by Saccharomyces
  • Sporangiospore: These are asexual spore produced by closed, often spherical structure called sporangium. This type of spore is formed by Rhizopus.

In sexual reproduction, the reproductive components formed are generally known as sexual spores. Conidia are formed in asexual reproduction. Sexual spores are formed by meiosis (Figure 3).

 TYPES OF SEXUAL SPORES

  • Ascospores: Ascopores are sexual spores characteristic of the true yeasts. They are formed by Ascomycetes. They are produced in a sac-like structure called ascus. Ascospores results from the fusion of two nuclei. This can be seen in Saccharomyces
  • Basidiospores: Basidiospores are sexual spores formed by Basidiomycetes. They are produced on a specialised club-like structure called basidium. Mushrooms and Cryptococcus species are typical examples of fungi that form basidiospores.
  • Zygospores: Zygospores are thick walled sexual spores produced through the fusion of two similar gametangia found in the class Phycomycetes. Gametangium (plural: gametangia) is a structure in which gametes are produced. They are the gamete producing structures of fungi. Zygospores are commonly seen in the bread mould Rhizopus nigricans.

CLASSIFICATION OF PATHOGENIC FUNGI

Fungi are classified into four (4) main groups as follows:

  1. ZYGOMYCETES (ZYGOMYCOTA): Zygomycetes are commonly known as the bread moulds. Fungi in this division or phylum include Rhizopus species and Mucor Fungi in the division Zygomycota undergo both sexual and asexual reproduction. They are usually characterized by the presence of sexual spores, asexual sporangiospores and the presence of septate or aseptate hyphae. Zygomycetes are largely saprophytes, and they live on decaying plant and animal matter in the soil. Fungi in this division form asexual spores or conidia through the formation of sporangia which contain numerous sporangiospores but sexual spores known as zygospores are formed via the mating of two haploid nuclei or fusion of morphologically similar gametangia that divide by the process of meiosis and mitosis. The zygomycetes are parasites of plants, animals and humans. However, fungi in this division are mainly known for their food spoilage activities. 
  1. ASCOMYCETES (ASCOMYCOTA): Ascomycetes are fungi that reproduce sexually via the formation of endogenous ascospores normally enclosed in a sac known as the ascus. Fungi in this division are commonly known as sac fungi, and they contain a large and diverse group of fungi with various economic importance. They also reproduce asexually to produce conidia. Ascomycetes form well developed branching septate hyphae. They are ubiquitously found in the soil as saprophytes, and a handful of them parasitize plants, humans and animals. Some ascomycetes are unicellular cells while others exhibit dimorphism. The division Ascomycota includes moulds, mildews and yeasts; and representative fungi in this group are Microsporum, Trichophyton, Piedraia, Blastomyces, Aspergillus, and Saccharomyces cerevisiae (Baker’s yeast). Majority of the ascomycetes are causative agents of dermatophytosis in humans (e.g. ringworm). Ascomycetes contain economically important fungi, and most cause food spoilage as well as mycoses in humans. Aspergillus flavus produces a potent toxin known as aflatoxins which are cancer-causing in nature. Aflatoxins are common contaminants of food especially grains which they infest to produce the potent toxin.
  1. BASIDIOMYCETES (BASIDIOMYCOTA): Basidiomycetes are fungi that form sexual spores known as basidiospores. The basidiospores are carried on a club-shaped structure known as the basidium (plural: basidia). Fungi in this division are commonly known as club fungi. Basidiomycetes have septate hyphae like the ascomycetes; and their basidiospores are enclosed within fruiting bodies known as basidiocarps. Examples of fungi that are basidiomycetes include toadstools, shelf fungi, mushrooms, smut fungi, Cryptococcus neoformans, rust fungi and puffballs. Fungi in the division basidiomycota possess tremendous economic importance. For example, the mushrooms in the Agaricus species such as campestris and A. bisporus are amongst the important mushrooms used as a source of human food. Mushroom cultivation either for domestic or commercial use is an important aspect of microbiology; and all over the world, mushroom is cultivated and sold because of their perceived medical and health importance. Basidiomycetes are ubiquitously found in the natural environment as saprophytes and parasites of animals and humans as well as plants.

   4.  DEUTEROMYCETES (DEUTEROMYCOTA): Deuteromycetes are generally known as        “imperfect fungi” unlike the ascomycetes, zygomycetes and basidiomycetes which are “true fungi”. Deuteromycetes are characterized by an anamorphic stage in which asexual spores or conidia are produced. Fungi in the division deuteromycetes have no known sexual cycle of reproduction, and they mainly reproduce via the formation of conidia or asexual spores. The division deuteromycota contain many important human pathogens including Candida, Histoplasma, Coccidioides and Paracoccidioides and other dematiaceous fungi. Other imperfect fungi such as Penicillium are important to humans. For example, the antibiotic (antibacterial agent) known as penicillin is naturally soured from Penicillium species such as notatum; and this antimicrobial agent have the ability to kill or inhibit the growth of pathogenic bacteria. The deuteromycetes are ubiquitously found in the environment especially in the soil, and they are important pathogens of plants, animals and humans.

Fungal morphology

Morphology is a branch of biology that deals with the study of the form and structure of organisms and their specific structural features. Morphologically, fungi exist in two basic structural forms. These are the yeast form and the mould form. In order words, fungi can either exist as mould (mycelial/hyphal form) or as yeast. However, there are some fungi that can exist in the two forms. These type of fungi are generally known as dimorphic or dematiaceous fungi because they exist in both the yeast and mycelial forms.

Fungi (singular: fungus) are eukaryotic organisms that are mostly saprophytic in nature. They basically include unicellular microorganisms such as yeasts and multicellular organisms such as moulds. Fungi vary widely in size and shape, and they have both microscopic and macroscopic structures. Individual cells range from 1 µm to 30 µm. Microscopic fungi exists as either moulds or yeasts or both. Fungi have chitin in their cell wall. They are heterotrophic in nature. Fungi are major decomposers of organic materials in the environment. They do not photosynthesize. Some fungi are dimorphic in nature. Dimorphic fungi are those fungi that exist in two morphologic growth forms. These two morphologic growth forms are yeast phase and mould (filamentous) phase. The process by which fungi exist in two different morphologic growth forms is known as dimorphism. Examples of dimorphic fungi are Blastomyces dermatitidis (that causes blastomycosis) and Coccidioides immitis (that causes coccidioidomycosis). Fungi cause diseases known as mycoses in humans. Fungi possess chitinous cell walls, plasma membranes containing ergosterol, and 80S rRNA. Fungi can occur as yeasts, molds, or as a combination of both forms. Fungi have both microscopic morphology and macroscopic morphology (Figure 1).

Figure 1. Illustration of fungal thallus. Fungal thallus means fungal structure.

Microscopically, most fungi grow as hyphae (singular: hypha) in their environment. Hypha is a long, branching filamentous structure of a fungus. They are the main mode of vegetative growth in fungi; and collectively, hyphae grow into a network known as mycelia. Mycelia (singular: mycelium) are a mass of filamentous, branching network of hyphae.  The two types of hyphae are coenocytic hypha and septate hypha. Coenocytic hyphae are those hyphae that lack compartmentalization i.e. they lack compartments separated by cross walls. Coenocytic hyphae can also be called non-septate hyphae. Septate hyphae have compartments separated by cross walls. These compartments found in septate hyphae contain one or more nuclei (Figure 2).

 

Figure 2. Illustration of the two types of hyphae formed by fungi: coenocytic and septate hyphae.

Macroscopically, fungi can become visible to the naked eye especially when they form mycelia on substrates. On such surfaces, the fungi can be called moulds. Fungi can grow on culture media (e.g. Sabouraud dextrose agar, SDA) to form various colonies with different shapes and colours.

Moulds: The moulds form large multicellular aggregates of long branching filaments, called hyphae. There are vegetative hyphae and reproductive hyphae – which help in the propagation of the organism. Spores are borne on the reproductive hyphae. While fungal spores are formed for both reproductive purposes and dispersal purposes, bacterial spores are resistant bodies formed for bacterial survival rather than for reproductive purposes. Spore size, shape and structure are used in the classification and identification of fungi.

MYCOSIS

Mycosis (plural: mycoses) is a fungal infection of animals and humans. Mycoses are common and a variety of environmental and physiological conditions can contribute to the development of fungal diseases especially the individual’s immune system status and type of occupation. People with debilitating disease such as diabetes and patients on chemotherapy are also prone to fungal infections. The elderly and very young people are also prone to fungal infections than the young adults because the immune status of the former is lower than that of the latter. Fungal infection (mycosis) is usually initiated following the inhalation of fungal spores. The infection can be systemic and disseminated in form – in which form the diseases affect several organs of the body; and in most of the cases fungal infections are usually localized in nature – affecting some parts of the skin. The best way of preventing fungal infection is by keeping the skin clean and dries, as well as maintaining good personal hygiene. Because fungal infections are contagious, it is important to wash after touching other people or animals. Sports clothing should also be washed after use.

Types of mycosis

The diseases caused by fungi are broadly divided into the following categories depending on the type of tissue affected:

Superficial Mycoses: Superficial mycoses are limited to the outermost layers of the skin and hair. The fungus infects mostly keratinized tissues, such as the epidermis, hair, and nails. However, the tissue invasion is usually minimal unlike in cutaneous mycosis. Certain forms of mucous membrane infections may involve some degree of tissue invasion. Most superficial mycosis is acquired through close contact with already infected individuals; and superficial mycosis is common amongst children who have poor personal hygiene and even amongst overcrowded people. An example of such a fungal infection is Tinea versicolor, a fungus infection that commonly affects the skin of young people, especially the chest, back, and upper arms and legs. Tinea versicolor is caused by a fungus that lives in the skin of some adults. It does not usually affect the face. This fungus produces spots that are either lighter than the skin or a reddish-brown.

Cutaneous mycoses: Cutaneous mycoses extend deeper into the epidermis of the skin, and also include invasive hair and nail diseases. These diseases are restricted to the keratinized layers of the skin, hair, and nails. The host immune responses may be evoked resulting in pathologic changes expressed in the deeper layers of the skin unlike the superficial mycoses where the host immune system is rarely evoked. The organisms that cause these diseases are called dermatophytes. The resulting diseases are often called ringworm or tinea. Cutaneous mycoses are caused by Microsporum, Trichophyton, and Epidermophyton fungi – which are all dermatophytes.

Subcutaneous Mycoses: Subcutaneous mycosis involves deeper layers of the skin and may eventually reach to the bones. The infections mostly result from the traumatic implantation of the pathogen into subcutaneous tissue. Most causal agents are soil-borne.

Systemic Mycoses: The etiologic agents of systemic mycoses are generally soil-borne and the respiratory tract is often (but not always) the portal of entry. Infections may involve any part of the body including the lungs, liver, kidneys, and brain. Systemic mycoses are usually caused by opportunistic fungal pathogens that gain entry into the body of individuals on chemotherapy or with weakened immune system. Systemic mycoses due to opportunistic pathogens are infections of patients with immune deficiencies who would otherwise not be infected. Examples of immunocompromised conditions include AIDS, alteration of normal flora by antibiotics, immunosuppressive therapy, and metastatic cancer. Candidiasis (caused by C. albicans), cryptococcosis (caused by C. neoformans) and aspergillosis (caused by Aspergillus species) are typical examples of opportunistic mycoses. Systemic mycosis may also be caused by some primary fungal pathogens that are dimorphic in nature. Systemic mycoses due to primary pathogens originate primarily in the lungs and may spread to several organs of the body. Organisms that cause systemic mycoses are inherently virulent. In general primary pathogens that cause systemic mycoses are dimorphic.

TYPES OF SUPERFICIAL MYCOSES

1. Black Piedra

The infection involves hair of the scalp. The fungus grows as a compact mass of cells forming a black nodule around the hair shaft. It may eventually invade the hair shaft. The size of the fungus mass may range from a few microns to greater than a millimeter in diameter. Piedra hortae is the main causal agent of black piedra. The infections are common in humid and warm countries. The laboratory diagnosis is mostly based on the microscopic examination of the hair shaft. Cultures can be made on Sabouraud agar on which it grows slowly and forms dark brown to black colonies. Multiple lines of treatment are available, but shaving off the hair appears to be the cheapest and most effective therapy.

2. White Piedra

White piedra is caused by Trichosporon beigelii, a yeast-like fungus that is considered dimorphic by some mycologists. The disease is characterized by white to creamy nodular fungal growth on the hair shaft. The fungus grows around and within the hair and mostly involves hairs of scalp. Unlike black piedra which is mostly restricted to scalp hair, white piedra may involve hairs of the beard and pelvic region. Cases of white piedra are seen all over the world, but the disease is more common in tropical regions. The laboratory diagnosis is often based on the microscopic examination of infected hair and demonstration of the mycelium (2–4 µm in diameter) and arthroconidia. Trichosporon beigelii grows well on Sabouraud agar. Heaped, cream-colored, yeast-like colonies can be seen after 4 or 5 days. Hyphae are septate and tend to break into small fragment called arthroconidia. A number of commercial over-the-counter products are available for the treatment. Topical prepa- rations containing azole derivatives are useful. As stated in the case of black piedra, shaving off the hair and observing good personal hygiene is an effective means of controlling white piedra.

3. Tinea Versicolor

Tinea versicolor, also known as pityriasis, is caused by a yeast-like fungus, Malassezia furfur. The fungus is normally present on human skin. Infection mostly occurs on the upper torso and other areas rich in sebaceous glands. The infection is characterized by altered skin pigmentation that may look lighter in color than the normal skin. No tissue invasion or inflammatory reaction is observed. Tinea versicolor occurs worldwide, but more commonly in tropical countries. The fungus is lipophilic and requires olive oil or any other vegetable oil for growth. Diagnosis is mostly based on clinical examination but the pathogen can be cultured on Sabouraud agar supplemented with olive oil and incubated at 35°C. Direct microscopic examination of skin using a 10% sodium hydroxide solution can be helpful. Spherical to elongated yeast- like cells can be easily seen. The condition can be treated using an aqueous solution (20% w/v) of sodium hyposulfite, or topical preparations containing azole deriva- tives. However, the recovery to normal skin is a long, drawn out process.

4. Dermatophytoses

Dermatophytosis (plural: dermatophytoses), also known as tinea or ringworm, is a clinical condition involving keratinized tissues including skin, nail, and hair. The causal agents usually belong to genera Trichophyton, Microsporum, and Epidermophyton, and are collectively called dermatophytes. The term “dermatophyte,” meaning skin-tree, goes back to the era when fungi were considered plants. Most dermatophytes produce keratinase, a proteolytic enzyme that hydrolyzes keratin. Therefore, many mycologists also refer to dermatophytes as keratinophilic fungi. Dermatophytes as aforementioned are fungi that cause various skin diseases (particularly ring worms) in humans and animals. They invade the superficial keratinized areas of the body such as the skin, feathers, nails, horns and hairs. They do not involve deeper tissues. Dermatophytes are usually found in the soil.

With reference to their predilection (or geographical distribution) and natural habitat, the dermatophytes are divided into three groups:

  • Anthropophilic: Anthropophilic fungi are dermatophytes that cause disease in humans. These are primarily isolated from human sources and include species belonging to the genus Trichophyton (T. rubrum, T. tonsurans, T. violaceum and others), Microsporum (M. audouinii), and Epidermophyton (E. floccosum).
  • Zoophilic: Zoophilic fungi are dermatophytes that parasitize animals. Animals are the primary reservoir of zoophilic dermatophytes. Important members include species of Microsporum (M. canis and M. nanum) and Trichophyton (T. mentagrophytes and T. verrucosum).
  • Geophilic: Geophilic fungi are dermatophytes that are found in the soil. Soil is the primary reservoir of geophilic dermatophytes. Notable species include Microsporum gypseum and Trichophyton species.

Infections are acquired by direct contact with infected humans or animals or exposure of the bruised skin to soil. Dermatophytoses occur all over the world but they are more common in developing countries. There is a direct relationship between good personal hygiene and contracting dermatophytoses. A daily shower with generous soap application has dramatically reduced occurrences of dermatophytoses in much of the world. The lesions are initially localized, highly inflamed, and pruritic. The infection may remain localized, forming a round ring-like lesion, or may spread fast, covering a large area of skin. Some of the common clinical conditions along with the dermatologists’ classifications of dermatophytoses are summarized below:

1. Tinea Corporis (Ringworm of the body)

Tinea corporis, also known as ringworm of the glabrous skin, is perhaps the most common form of dermatophytoses. The infection is limited to skin and the lesions are characterized by inflammation, erythema, and vesicle formation. Species most frequently involved include T. rubrum, T. mentagrophytes, and M. canis.

2. Tinea Capitis (ringworm of the scalp)

Tinea capitis refers to scalp infection. The lesions are characterized by inflammation, ulceration, and hair loss. Microsporum canis and T. tonsurans are most frequently involved in this clinical type.

3. Tinea Barbae (ringworm of the beard)

Tinea barbae is characterized by pustular folliculitis mostly affecting the hairs of the beard. Species commonly involved include T. rubrum, T. violaceum, and M. canis.

4. Tinea Pedis (ringworm of the foot)

Tinea pedis, also known as athlete’s foot, refers to infection involving toe-webs and sole. Infections are frequently associated with persistent moist conditions. The lesions are scaly, erythematous, and inflamed. Species most commonly involved include T. rubrum and T. mentagrophytes.

5. Tinea Unguium (ringworm of the nails)

Tinea unguium, also known as onychomycosis, refers to a clinical condition involving the nails. The infection may involve the nail-plate and spread to the area under the nail, resulting in a total deformation and loss of the infected nails. Species most commonly associated with tinea unguium include T. rubrum and T. mentagrophytes. Species of the yeast-like fungus Candida are also known to cause onychomycosis. Therefore, a differential diagnosis is required because of the differences in the effective therapeutic measures.

Laboratory Diagnosis of mycoses (dermatophytoses)

Dermatologists tend to diagnose dermatophytoses on the basis of affected sites and clinical symptoms. However, a definitive diagnosis requires isolation of the causal agents in culture. For a presumptive diagnosis, samples of skin scrapings are mixed with a 10% solution of sodium hydroxide and examined directly under a bright field microscope. The nail specimens should be cut into finer pieces and a 20% sodium hydroxide solution should be used. Fungal elements, if present, can be easily seen. Dermatophytes grow well on Sabouraud agar fortified with chloram- phenicol (50 mg/L) and cycloheximide (500 mg/L). Chloramphenicol will suppress bacterial growth and cycloheximide is toxic to most of the environmental fungi that are commonly present on the skin and nails as transient microbiota. Inoculated plates should be incubated at 25°C. Visible colonies appear within 1 week but sporulation may require a longer period of incubation, often up to 2–3 weeks. Species are identified on the basis of colony appearance and size and shape of conidia. Microsporum and Trichophyton spp. tend to produce two types of conidia, the macroconidia and the microconidia. Size and shape of macroconidia is most useful in species identification. The macroconidia of Microsporum are fusiform or spindle-shaped (Fig. 13.3), thick-walled, and measure 7–20 × 35–120 µm, and those of Trichophyton are clavate with smooth walls and measure 4–8 × 8–50 µm. The macroconidia of Epidermophyton spp. are widely clavate with smooth walls, rounded distal ends, and measure 6–10 × 8–15 µm. The macroconidia of all the three species are multinucleate. Sexual stage (perfect stage) has been recognized in many species.

MUCOCUTANEOUS MYCOSES

Several pathogenic fungi, especially dimorphic fungi, are known to infect mucous membranes, often as a secondary complication. This type of fungal infection is generally known as mucocutaneous mycoses. Typical examples include thrush and vulvovaginitis or vaginitis due to fungal infection. In this subsection, we will focus only on the mucous membrane infections by Candida spp. Some of the differentiating features of yeast-like fungi associated with mucocutaneous infections are depicted in Table 13.1. The common clinical conditions caused by Candida and related species can be divided into two distinct categories.

1. Thrush

Thrush is characterized by curd-like growth of the yeast on tongue and palate. A confluent growth of yeast-like cells and pseudomycelium can form a biomembrane on the surface. The causal agent is frequently Candida albicans, but other species are also occasionally incriminated. Thrush mostly occurs in newborns, who acquire the disease during passage through the birth canal. It can also be noted in immunecompromised patients, such as those with AIDS, persons on steroids, and diabetics. The condition is often indicative of host’s immune status. Thrush is frequently diagnosed on the basis of clinical symptoms. A definitive diagnosis can be made by direct microscopic examination of swabs or tongue scrapings, which would reveal yeast-like cells and abundant pseudomycelium. Cultures can be made on Sabouraud agar containing chloramphenicol. Most Candida spp., except C. albicans, are sensitive to cycloheximide. Thrush can be also an annoying complication in stomatitis. Topical medications are often sufficient to treat thrush but systemic treatment may be needed for more severe cases, most commonly with azoles.

2. Vulvovaginitis or Vaginitis

Vulvovaginitis or vaginitis is commonly referred to as yeast infection. The disease is characterized by the inflammation of vagina, labia, and surrounding areas. The symptoms may include burning, itching, and curd-like odorless vaginal discharge with painful intercourse. In some cases, as a complication of vaginitis or as a disease by itself, Candida spp. can cause urinary tract infection (UTI), especially urethritis, which can be painful and chronic. Vulvovaginitis is a common complication during the late stage of pregnancy. The primary cause of infection appears to be loss of resident microbiota in vagina, often due to prolonged antibiotic therapy and occa- sionally due to hormonal changes. Vulvovaginitis is also a common infection in postmenopausal women. The disease is sexually transmissible. In males, the infection may result in balanitis characterized by inflammation of the glans of penis, accompanied by a burning and itching sensation. However, in most males the infec- tion is mild and frequently unnoticed. Asymptomatic males can play a role in trans- mitting the disease to females. Candida vulvovaginitis occurs all over the world but the incidence rate seems to be higher in industrialized countries, perhaps due to excessive use of douche and other tools of intimate hygiene, which can disturb or even dislodge the resident microbiota. Though not a mucocutaneous infection, Candida spp. can also cause perianal infection in males and females. No well-defined virulence factor is known, but many strains of Candida albicans are known to produce proteinases and possibly certain extracellular toxins, which probably play important roles in the inflammation and tissue damage.

Laboratory Diagnosis of mucocutaneous mycoses

A vaginal swab can be cultured on Sabouraud agar supplemented with chloramphenicol. Inoculated plates should be incubated aerobically at 35°C. In some chronic cases of vaginitis, Candida spp. tend to form a biofilm which makes it hard to dislodge and isolate the fungus in culture. Species identification may not be considered essential for therapy, but it can be achieved by some simple tests and enforced by physiological tests including carbohydrate assimilation tests (see Table 13.1). Candida albicans forms a germ tube in serum and egg albumin, and produces chlamydospores on cornmeal agar (Fig. 13.4). Candida albicans is the principal causal agent of thrush and vulvovaginitis, responsible for infection in 70%–80% of cases. During the past few decades Torulopsis glabrata (formerly called Candida glabrata) and Candida tropicalis have emerged as important causal agents, especially in vaginitis and UTI.

OPPORTUNISTIC FUNGAL PATHOGENS

Opportunistic fungal pathogens include:

  1. Candida albicans
  2. Cryptococcus neoformans
  3. Aspergillus species
  4. Penicillium species
  5. Species of Mucor, Absidia, Rhizopus and other Zygomycetes

Opportunistic fungal pathogens are pathogens which cause disease in immune compromised individuals. Patients with compromised host defenses are susceptible to ubiquitous fungi to which healthy people are exposed but usually resistant. Candidiasis: Candidiasis is an opportunistic mycosis that is caused by Candida albicans. C. albicans is an endogenous fungus (opportunists) belongs to the normal microflora of the body unlike other fungi that are exogenous and are found in the soil, H2O and in air. C. albicans are deuteromycetes and they cause disease when the immune level of an individual is compromised either by infection or any other means. This makes them opportunistic agents.

Types of Infection caused by Candida

  1. Intertrigenous Candidiasis: It is characterized by lesions in the groin, inflammatory folds and the umbilicus. Obesity, alcoholism and diabetes can lead to this type of Candidiasis.
  2. Paronychia: It can otherwise be called onychomycosis. Paronychia is a candidal invasion of the finger or nails. It is a painful erythematous swelling of the nail fold. It occurs in individuals with occupational exposures.
  3. Generalized Cutaneous Candidiasis: It is characterized by widely disseminated lesions seen occasionally in patients with diabetes. It is found in the angles of the mouth, in the intestine or stomach, and it causes gastrointestinal discomfort if intestine or stomach is affected.
  4. Vulva Vaginal Candidiasis: This is yeast invasion of the vaginal mucosa. It is otherwise called Vulvovaginitis. It is characterized by irritation, pruritus and vaginal discharge. This condition is preceded by diabetes, pregnancy or antimicrobial drugs that alter the microbial flora.
  5. Oral Thrush (Candidiasis): The tongue, soft palate, and buccal mucosa are covered with discrete patches of cream-white to grey pseudomembrane composed of hyphae of C. albicans. It is a disease of the newborn, aged, diabetic patients, and some pregnant women.
  6. Broncho-candidiasis: It causes acute bronchitis characterized by cough caused by Candida.
  7. Endocarditis: This occurs mostly in drug addicts. It causes cough with severe pains in the chest.
  8. Meningitis: This occurs as a result of previous antibacterial or corticosteroid therapy or as a result of a debilitating disease.

OVERVIEW OF ANTIFUNGAL AGENT

Antifungal agents are antibiotics or antimicrobial agents that are used to treat fungal infections. Examples of antifungal agents are amphotericin B, flucytosine, ketoconazole, fluconazole, itraconazole, nystatin, cotrimoxazole, and miconazole. Most fungal agents are topical, and are thus prepared and applied as cream on the affected part of the body. And there are fewer antifungal drugs than there are antibacterial drugs because fungi like humans/animals have prokaryotic cells; and most of the available antifungal drugs especially those used systemically may pose some untoward effects to the host since the fungal cell and that of the host are the same.

AMPHOTERICIN B

This is a complex antibiotic polyene produced by a Streptomyces spp. Amphotericin B has little antibacterial properties. It strongly inhibits the growth of several pathogenic fungi in vitro and in vivo – where it binds to the steroid on the fungal membrane and thus disrupts their function. The cell membrane of fungi is made up of a steroid called ergosterol while that of humans is made up of cholesterol. This takes care of the selective toxicity of the drug when used to treat pathogenic fungal infection.

 

Structure of amphotericin B

Mechanism of action: Amphotericin B is given intravenously as micelles with sodium deoxycholate dissolved in dextrose solution. Though the drug is widely distributed in tissues, it penetrates poorly to the cerebrospinal fluid (CSF). When it comes in contact with a fungal cell, it will strongly bind to ergosterol in the cell wall/membrane of the target pathogenic fungus – resulting in changes in membrane fluidity and perhaps introduction of amphotericin pore. The death of the cell occurs as small molecules and ions are lost from the fungal cell. The lack of ergosterol in mammalian cell makes them resistant to the action of amphotericin B.

Although amphotericin B binds weakly to the cholesterol in mammalian cell membrane and this interaction probably accounts for its toxic side effects. The combination of amphotericin B with flucytosine is beneficial in fungal infections caused by Candida species and Cryptococcus species. Amphotericin B is a broad spectrum antifungal agent with demonstrated efficacy against most of the major systemic mycosis including but not limited to coccidioidomycosis, blastomycosis, histoplasmosis, sporotrichosis, cryptococcosis, mucormycosis, and candidiasis.

Side effects: Intravenous administration of amphotericin B causes some acute reactions like chills, dyspnea and hypotension. The effect can usually be alleviated by prior or concomitant administration of hydrocortisone or acetaminophene and tolerance to the acute side effect develops during therapy. Hypocalemia, anemia, renal tubular acidosis, headache, ulcer and vomiting are frequently observed.

FLUCYTOSINE

Flucytosine is a fluorinated derivative of cytosine (5-fluorocytosine). It is an oral antifungal compound that is primarily used in conjunction with amphotericin B for infections caused by Candida species and Cryptococcus species. When administered, it penetrates well into all tissues including CSF.

Structure of flucytosine 

Mechanism of action: Fungi that are susceptible to flucytosine are capable of accumulating this drug via a membrane bound permease enzyme. Flucytosine is then converted to fluorouracil by cytosine deaminase. Fluorouracil is a potent inhibitor of thymidylate synthetase and the inhibition of thymidylate synthetase results to cell death. The lack of cytosine deaminase in mammalian cell protects it from toxic effects of fluorouracil. Unfortunately, resistant mutants rapidly emerge, limiting the utility of flucytosine for the treatment of fungal infection. The combination of flucytosine and amphotericin B is beneficial particularly in cryptococcal meningitis and this combination has shown to delay or eliminate the appearance of flucytosine resistant mutant.

Side effects: Prolonged administration of flucytosine results to bone marrow depression, hair loss and liver dysfunction. The conversion of flucytosine to fluorouracil by enteric bacteria may cause colitis. Patients with AIDS may be more susceptible to bone marrow suppression by flucytosine and serum level should be closely monitored too.

AZOLES

Azoles are a class of five-membered nitrogen heterocyclic ring compounds containing at least one other non-carbon atom of either nitrogen, sulfur, or oxygen in its structure. Many azoles are used as antifungal drugs – where they inhibit the fungal enzyme 14-α-demethylase which produces ergosterol (an important component of the fungal cell membrane). Some examples of azole antifungal drugs include ketoconazole, fluconazole, itraconazole, posaconazole and voriconazole. Azole antifungals are a group of fungistatic agents with broad-spectrum activity. They are classified into two groups: the triazoles and the imidazoles. The azole antifungals inhibit the cytochrome P450 dependent enzyme lanosterol 14-alpha-demethylase, which coverts lanosterol to ergosterol, the main sterol in fungal cell membrane. Depletion of ergosterol damages the cell membrane resulting in cell death. Azole antifungal agents can be used to treat systemic and topical (athletes foot, ringworm) fungal infections.

 

Structures of some azoles

The antifungal imidazole (ketoconazole) and triconazole (fluconazole and itraconazole) is and orally active agent useful for the treatment of a wide range of systemic and localized fungal infection. Its oral administration with less toxicity makes it a supplement with amphotericin B in many fungal infections. Other imidazoles (iconazole and clotrimazole) which are too toxic for systemic administration are useful as topical agents.

Mechanism of action: All antifungal azoles act by inhibition of fungal ergosterol biosynthesis. This inhibition is achieved by drug binding and interference with the function of the heme group in cytochrome p450 oxidases. The fungi P450 oxidase that is most sensitive to inhibition is 14-lanosteroldimethylase. Other p450 enzymes (including mammalian enzymes involved in steroidogenesis – i.e. involved in sterol synthesis) can be inhibited at higher concentration. The blockage of ergosterol formation leads to alteration in fungal cell membrane function and structure.

Usage of the azoles

Ketoconazole is useful in the treatment of chronic mucocutaneous candidiasis and in extra-meningochronic form of blastomycosis, coccidioidomycosis, paracoccidioidomycosis and histoplasmosis.

Fluconazole’s ability to penetrate the CSF makes it an attractive alternative for amphotericin B in the treatment of coccidiodo meningitis. Life-long fluconazole maintenance therapy can prevent relapse in AIDS patients with cryptococcal meningitis.

Itraconazole may be useful in treating invasive aspergillosis which generally responds poorly to amphotericin B. It is effective in maintaining therapy in AIDS patients with histoplasmosis, in immune competent patients, sporotrichosis, blastomycosis and extra meningial coccidiomycosis.

Side effects: The side effect of antifungal azoles is related to their ability to inhibit mammalian cytochrome P450 enzyme. Ketoconazole is the most toxic in this regard and will at therapeutic doses inhibit the synthesis of testosterone and cortisol which could lead to gynecomastia, decreased libido, impotence, menstrual irregularities and occasionally adrenal insufficiency. Fluconazole at increased dosage in therapeutic level may affect mammalian steroidogenesis. All the antifungal azoles can cause both asymptomatic elevations in liver function test and rare cases of hepatitis. Antifungal azole therapy can also reach to higher than expected serum level of cyclosporine, phenyltoin, oral hypoglycemic, anticoagulants, and diagozine.

GRISEOFULVIN

Griseofulvin is obtained from certain Penicillium species and the drug is administered orally. Griseofulvin suppresses certain dermatophytes; and thus it can be used to treat fungal infections caused by dermatophytes.

 

Structure of griseofulvin

Mechanism of Action: Griseofulvin when taken orally is distributed throughout the body. The drug accumulates in the epidermis or other keratinized tissues (hair and nails). Keratin being the major nutritional source for dermatophytes when degraded by Griseofulvin impregnated keratin by this fungi result in ingestion of the drug within the organism. Griseofulvin is thought to interact with microtubules and disrupts mitotic spindle function resulting in growth inhibition. It is clinically useful for the treatment of dermatophytosis, skin infection, hair and nail infections, and fungal infections caused by Trichophyton, Epidermophyton and Microsporum spp.

Side effects: The most common side effect is headache which usually results without discontinuation of the drug. Less frequently observed side effects are gastrointestinal upset, drowsiness and hepatotoxicty.

TOPICAL AND OTHER ANTIFUNGAL AGENTS

1. NYSTATIN

Nystatin is a polyene antibiotic that is structurally related to Amphotericin B and probably shares a common mode of action. It is useful in treatment of local Candida infection of the mouth and vagina. It may also suppress sub-clinical oesophageal candidiasis and gastrointestinal overgrowth of Candida. There is no systemic absorption of nystatin and it has no noticeable side effect.

2. TOLNAFTATE AND NAFTITINE

Tolnaftate and naftitine are topical antifungal agents used for the treatment of fungal infections caused by Tinea pedis, T. cruris, T. corporis and T. versicolor.

Infections due to candida are relatively resistant to these agents. Tolnaftate and naftitine are available as creams, powders and spray.

3. CLOTRIMAZOLE, MICONAZOLE & OTHER AZOLES

These antifungal agents are usually available in over-the counter formulations. They include ecoconazole, glutoconazole, thioconazole and teconazole.

Topical antifungal azole have a broad spectrum of activity. Fungal infections caused by I. pepis, T. cruris, T. corporis, T. versicolor and cutaneous candidiasis responds well to local application of creams and powders.

Vulvovaginal candidiasis can be treated with vaginal suppositories or creams. Clotrimazole is also available at oral thrush for treatment of oral and oespohageal thrush in immune competent patients.

GENERAL LABORATORY ANALYSIS OF SPECIMEN SUSPECTED TO CONTAIN YEAST LIKE FUNGI OR INFECTIOUS FUNGI

The clinical specimen to be collected includes Sputum, Scrapings, CSF, Urine or Swab samples. After collection of the specimen, the first step in the laboratory diagnosis of fungal infection is usually direct microscopy in which the test sample is mounted on a clean glass slide using potassium hydroxide (KOH) or lactophenol cotton blue stains as the case may be. The slide is usually observed under the microscope using 40x objective lens for the presence of some yeast and presence or absence of arthrospores or hyphae of the suspected yeast-like cell.

  1. If specimen is a scraping sample, digest the sample with 10 % KOH and observe under the microscope.
  2. If specimen is a feacal sample, perform Gram staining on it.
  3. If the specimen is CSF or urine sample, centrifuge it or pass it via a membrane filter. This is done in order to sieve the sediment and to increase the possibility of harvesting the fungal cells. This is followed by microscopy using Gram staining or Giemsa stain.
  4. If the specimen is sputum, flood with normal saline, in order to reduce the viscosity of the mucous, and then view under the microscope.
  5. If the specimen is biopsy or autopsy, cut and examine under the microscope.

Fungal culture is also important for the isolation and detection of fungal organisms from clinical or environmental samples. The specimen is usually cultured on a selective such as Sabouraud dextrose agar (SDA) or medium containing cycloheximide and chloramphenicol. The reason of introducing antibiotics into culture medium meant for the isolation of fungi is because the antibiotics will help to inhibit the growth of bacteria and other non-fungal organisms present in the sample. Yeast broth can also be used for culturing.

SYSTEMIC MYCOSES (FUNGI INFECTIONS) AND THEIR FEATURES

SPOROTRICHOSIS

Sporotrichosis is a chronic granulomatous subcutaneous infection caused by Sporothrix schenckii. Sporotrichosis is also known as Rose Gardner’s disease because of the granulomatous lesions produced by the organism and/disease on the infected patient or individual. The etiologic agent of sporotrichosis is Sporothrix schenckii. Sporotrichosis results following traumatic introduction of S. schenckii into the skin.

 Aetiologic Agent/Causative agent: Sporotrichosis is caused by Sporothrix schenckii. S. schenckii is a dimorphic fungus (i.e. it exists in the yeast form and mycelia form) that lives on vegetations, and the organism can also be found in soil or humus. It also lives in temperate and moist tropical areas.

Morphology and Identification: S. schenckii is a thermally dimorphic fungus that lives on vegetations. It grows well on routine culture media for fungal isolation including the Sabouraud dextrose agar (SDA). At ambient temperature, S. schenckii grows as a mould, producing branching, septate, hyphae and conidia in tissue or in vitro at 35-370C producing a small budding yeast. The conidium (plural: conidia) of S. schenckii is about 3-5 µm diameter.

Pathogenesis and Clinical Findings: The conidia (hyphal fragments) of S. schenckii are introduced into the skin by trauma (via nails, pins and sharp objects). The initial lesion develops as a granulomatous nodule that may progress to form a necrotic (dead) or ulcerative lesions. The drainage lymphatics become thickened and cord-like and multiple subcutaneous nodules also develop. Abscess also occurs along the drainage lymphatics. There is usually little systemic illness associated with these lesions, and dissemination may occur in debilitated patients. Fixed sporotrichosis is localized and it is more common in endemic areas, where there is a high level of exposure and immunity in the population. It is limited to the local spread of the infection and can give rise to systemic form of fungal infection in complicated cases. Infection with S. schenckii is acquired via cutaneous inoculation of the organism (i.e. S. schenckii). A reddish necrotic nodular papule appears 1-10 weeks after trauma. The lesion is a sporulating granuloma that consists of histiocytes and giant cells with neutrophils that accumulate in the centre. The fungus spreads from the initial lesion along lymphatic channels and from these sites; other tissues are involved by direct extension and less often by haematogenous dissemination. This occurs mostly in immunocompromised hosts resulting in widely disseminated subcutaneous and visceral infection including meningitis. Sporotrichosis is likely more common in males than in females presumably due to a higher exposure rate in males. In terms of prognosis, the early administration of the appropriate drugs remedies the situation of the disorder, despite its prolonged need of therapy.

Forms of the disease: The different forms of sporotrichosis are as follows:

  1. Cutaneous Sporotrichosis

(a)        Lymphocutaneous sporotrichosis – which affects the skin, subcutaneous tissues and regional lymphatics.

(b)       Fixed sporotrichosis – which is a localized skin lesion that has no spread.

  1. Osteoarticular Sporotrichosis

(a)        Arthritis and tenosynovitis – which may affect the joints of the ankles, knees, spine, and hip.

(b)       Sinus tracts and local spread to adjacent bones.

  1. Pulmonary Sporotrichosis

(a)        Classical – which produces enlarging cavitary lesions usually with pulmonary infiltrates

(b)       Pleural, effusion and lymphoadenopathy

(c)        Alcoholism, diabetes mellitus, and tuberculosis (TB).

  1. Disseminated Sporotrichosis

(a)        It affects all other parts of the body.

(b)       Gonorrhea

Signs and symptoms: Sporotrichosis manifests as small painless bump that looks like an insect bite. This bump can be red, pink and purple and it appears in the ear, and fingers of infected individuals. This is usually followed by one or more additional bumps that open. These bumps are usually very slow to heal. Most fungal infections caused by S. schenckii are limited to the skin but cases of joint, lung and central nervous system (CNS) infections are rare.

Laboratory Diagnosis of sporotrichosis

  1. Specimen: Biopsy materials or exudates from granules or ulcerating lesion.
  2. Microscopic examination: Specimens are examined with 10 % KOH but S. schenckii are rarely seen. Gomoris methenamine silver which stains the cell wall black and periodic acid-schiff stain which stain the cell wall read can be used for the microscopical analysis of clinical samples suspected to contain S. schenckii. They can also be alternatively identified by fluorescent antibody staining. The yeast are 3-5 µm in diameter structurally, spherical to ellipsoidal. Asteroid body can also be identified in the microscopical analysis.
  3. Culture: This is the most reliable method of diagnosis. Specimens are streaked on inhibitory-agar mould media or saborauds dextrose agar (SDA) containing antibiotics which inhibit bacterial growth. Identification is confirmed by growth at 350C and conversion to the yeast form. Note: S. schenckii is a dimorphic organism/fungi; and thus the organism exist in two forms: the mould form and the yeast form; and this attribute is vital and considered in the cultivation of S. schenckii in the microbiology laboratory.
  4. Serology: The agglutination of yeast cell suspension of latex particles coated with antigens occurs in higher titer with sera of the infected patients. Serology is not always a preferred diagnostic tool for the identification of S. schenckii in the laboratory.

Treatment

  1. Local application of extreme heat (hyperthermia) can be used to treat cutaneous lesions.
  2. Itraconazole is effective for treating systemic infections of sporotrichosis.
  3. Potassium iodide is also used for treatment, but it leaves behind some side effects.
  4. Amphotericin B is used during serious infections like pulmonary disseminated sporotrichosis.

Epidemiology and Control: S. schenckii occurs worldwide in close association with plants and vegetations from where likely human infections occur. It has a global occurrence and occurs predominantly in U.S.A, South America and some European countries. Most human episode of sporotrichosis follows contact with thorn, syphagnum moss, pine bark and other vegetations that harbour the pathogen. In the U.S.A, sporotrichosis is estimated at 1-2 cases per million people and approximately 200-250 cases occurring in a year. In Peru, it is estimated at one heads per thousands. About 75 % of cases occur in males either because of increased exposure to S. schenckii. Sporotrichosis occurs more among agricultural workers including horticulturists, farmers, basket weavers and gardeners than in people who do not participate in any of this occupations.

Prevention/Control: Preventive measures for sporotrichosis include:

  1. Measures to minimize accidental inoculation of S. schenckii into the body.
  2. Use of fungicides where appropriate to treat woods and vegetation.

COCCIDIOIDOMYCOSIS

Coccidioidomycosis is a systemic (deep) mycosis. It affects the lungs and spreads to other parts of the body. Coccidioidomycosis is also known as San Joaquin valley fever or desert rheumatism. This is because of the geographical location of the fungus causing the disease. Coccidioidomycosis is acquired by inhalation of dust containing spores (arthroconidia) of the pathogenic fungus that causes the disease. Coccidioidomycosis is systemic because it involves the general or entire body of the infected host. It is mycoses that spread in the body.

Etiology: The etiological agent of coccidioidomycosis is called Coccidioides immitis. C. immitis is a soil mould. Humans encounter with their habitat (i.e. the soil) leads to the acquisition of its spores (arthroconidia). C. immitis can be the most virulent of all the other mycotic pathogens.

Biology/Morphology: At 25oC, C. immitis forms a moist white or brown colony with abundant branching septate hyphae. The hyphae fragment into arthroconidia at maturity. When inoculated on a special culture media and incubated at 37-40oC, an arthrosphore germinates into the parasitic phase (spherule) of the organism. The spherule swells into a giant sporangium that cleaves internally to form numerous endospores that resembles bacterial endospores (but the spores of C. immitis do not posses bacterial resistance trait as is applicable to spores produced by bacterial pathogens that are spore formers).

Mode of Transmission: Spores (arthroconidia) of C. immitis are acquired through the inhalation of dust particles that contains these spores. The lungs are usually their first point of attack after inhalation. They are converted to spherules, which swell, sporulate, burst and releases more spores (conidia) that continue the life cycle of the fungal pathogen. Coccidioidomycosis is not transmitted from person to person. This primary pulmonary infection is apparent in 60 % of patient.

Clinical Features: Infection with C. immitis may be asymptomatic, resulting in an acute self limiting respiratory disease – that cannot be transmitted from the infected individual to non-infected persons in the community. Coccidioidomycosis resembles a common cold or influenza infection caused by a bacterial pathogen. This is usually accompanied with severe chest pain and mycetoma (fungus balls) may develop in the lungs. Constitutional upsets like headache, fever (pyrexia), and malaise may also result. Less common symptoms of coccidioidomycosis may include joint pains (desert rheumatism) and skin lesions. Patients usually experience dry cough which latter leads to productive cough.

Geographical Distribution: C. immitis exist in dry highly alkaline soil of hot North Central and South America. In the U.S.A, it has been estimated that 1,000 people are infected annually with coccidioidomycosis with about 50-80 deaths occurring. Endemic areas have been defined with massive skin testing for the antigen (coccidioidin) of C. immitis .C. immitis grows as a mould that forms arthroconidia at the tips of hyphae in the soil and in culture. In these endemic areas, visitors can easily inhale the spores of the agent (C. immitis) in dust particles and acquire the disease due to the abundance of its spores in such areas.

Pathogenesis: The primary infection occasionally leads to coccidioidal granuloma (a progressive often fatal disseminated disease) with lesions in the skin, joints and meninges of infected individuals. Secondary infections associated with C. immitis infection includes: chronic and progressive pulmonary disease, and single or multiple extra-pulmonary dissemination (generalized systemic infection). Extra-pulmonary lesions involve damages outside the lungs (skin bones). Most cases of coccidioidomycosis are asymptomatic. They usually present with dry cough which late leads to productive cough, chest pain and other light symptoms.

Epidemiology: C. immitis occurs endemically in various natural reservoirs and casually in areas where the spores of the fungal pathogen have been carried by winds and animals. High carbon and salt content, as well as a semi-arid relatively hot climate favours its settlement in a given habitat. C. immitis has been isolated from soil, plants and a large number of invertebrates. It undergoes a period of dormancy in winter and spring followed by growth in summer. Wind storms, psychos of draught and heavy rains increases its growth in the environment. Earthquake, like that of the 1994 earthquake in North Ridge California can lead to dispersion of C. immitis spores (arthroconidia) in endemic regions. Local winds can easily carry the dust containing these spores into the residential areas; and this phenomenon increases the chances of more people becoming infected with C. immitis.

Laboratory Diagnosis of coccidioidomycosis

In the laboratory investigation for C. immitis, the following tests are carried out:

  1. Specimens: Specimens include sputum, exudates from cutaneous lesions, spinal fluid, blood, urine and tissue biopsies.
  2. Microscopic examination: Materials should be examined fresh for typical spherules. KOH mount or calcofluor white stain will facilitate finding of endospores and spherules.
  3. Culture: Culture of samples on inhibitory mould agar or SDA containing antibiotics and cycloheximide to inhibit bacterial and saprophytic mould growth respectively are incubated at 37oC. Suspicious cultures are examined in a biosafety laminar flow cabinet because the arthroconidia of C. immitis are highly infectious. Identification of the organism is done by detection of a C. immitis antigen (spherulin or coccidioidin); via animal inoculation or by the use of a specific DNA probe.
  4. Skin test: In skin test, the antigens spherulin or coccidioidin is used for the identification and/or detection of fungal antigens in the patient’s sample. This testing reaches maximum in duration between 24 and 48 hrs after cutaneous infection of 0.1 mL of a standardized dilution. If patients with disseminated diseases become anergic, the skin test will be positive and this implies a very poor prognosis. Spherulin is more sensitive than coccidioidin in detecting reactors.
  5. Serological test: This is the key test for diagnosis of C. immitis infection. Immunodiffusion (ID) testing and complement fixation (CF) testing are usually used in serological tests for laboratory diagnosis of coccidioidomycosis.

Treatment/Prevention: The drug of choice for treating coccidioidomycosis is basically ketoconazole, amphotericin B (which is given intravenously during severe diseases), itraconazole and miconazole. Some measure of control can be achieved by reducing dust in endemic areas, paving roads and airfield, planting grass or crops and using oil spills to control dust aerosolization.

HISTOPLASMOSIS 

Histoplasmosis is an infectious disease which is caused by inhaling spores of the fungus, Histoplasma capsulatum. It is also known as Darling’s disease. Histoplasmosis is both a disease of man and animal and it is found worldwide. Histoplasmosis is non-contagious. It occurs when spores of H. capsulatum are inhaled. Because histoplasmosis is a systemic infection, it can be seen in some organs of the body like spleen, kidney, lymph node, heart, and intestine.

Clinical Features: The lung is the primary point of infection and because of this, its symptoms vary greatly. A vast majority of infected people are asymptomatic and symptoms may be so mild that medical attention is not sought.

Incubation period: The incubation period of H. capsulatum infection is usually 3-17 days; and the disease is usually characterized as a mild flu-like respiratory illness that has a combination of symptoms which are usually constitutional upset (e.g. fever, headache). The most severe and rare form of this disease is disseminated histoplasmosis.

People and Occupation at Risk: Anyone working on a job or activities where materials contaminated with H. capsulatum become airborne can contact the disease if enough spores are inhaled. People that are likely at risk are:

–           Infants, children, the elderly (especially those with chronic diseases), and people with weakened immune systems, AIDS and cancer patients.

Occupations at risks include:

–           Demolition workers and those that roof houses

–           Construction workers and pest control workers

–           Chimney workers and farmers,

–           Gardeners and horticulturists

–           Microbiology laboratory workers and heating air conditioners

–           The spelunkers (cave explorers) installer

Geographical Distribution: H. capsulatum grows in the soil throughout the world. In USA, the fungus is endemic with higher infections in the centrals & eastern states of America especially along the valleys of ohid. H. capsulatum grow well in N2-rich soils and those enriched with bat droppings. Spores become aerosolized or airborne when the soil or contaminated materials are disturbed. And once airborne, they can easily be carried by wind currents over long distances.

LABORATORY DIAGNOSIS

  1. Specimens: They include sputum, Buffy coat of blood, urine, bone marrow aspirates, spleen and lymph node exudates containing spores.
  2. Microscopic examination: Fungal stains such as Gomoris are methenamine silver, calcofluor white and periodic acid Schiff stains are used. Under the microscope, using x10 or x40 objective lens, the appearance of the hyphae will be branching and septate. Macroconidia and microconidia are the two spores to be seen. After incubation at 37oC, yeast cells are seen microscopically. They are small, oval and budding between 2-3 µm.
  3. Culture: Culture is the major way of diagnosis of histoplasmosis. SDA containing antibiotics is used for culturing. After this you incubate for about 3 days at 25-28oC. Fine, dense cottony are likely to be seen. After cultivation, the colony morphology looks white-green and it is smooth and moist.

PREVENTION

–           Avoiding aerosolization of contaminated materials and subsequent inhalation of dust containing the spores.

–           Dust suppression technique: This is done by water spraying in order to reduce the amount of dust that is aerosolized during some activities such as road construction and farming.

–           Disinfectants like formaldehyde can also be used to treat contaminated soil

TREATMENT

Ketoconazole and Amphotericin B are the drugs of choice for treating histoplasmosis. For acute pulmonary histoplasmosis, supportive therapy and rest is recommended. Prolonged therapy is required for disseminated disease and in AIDS patients.

BLASTOMYCOSIS

Blastomycosis is a common infection of dogs in endemic areas. Blastomycosis is a systemic-pyogranulomatous mycosis caused by the thermally dimorphic fungus, Blastomyces dermatitidis. Blastomycosis which is also known as Northern America disease or Gilchrist’s disease was described by Gilchrist and Stokes in 1894 and 1896 respectively. Blastomycosis is uncommon in children.

ANTIGENIC STRUCTURE (ANTIGENICITY): The 2 serotypes of B. dermatitidis are the perfect serotype (sexual form) and the mycelial form (second serotype). These 2 serotypes are based on hexo-antigen analysis. The sexual form (perfect serotype) is heterothallic and requires two compatible mating types for spore formation. The mycelial form bears conidiophores and produce single terminal conidia (2-10 µm in diameter). They are round or oval in shape.

PATHOGENESIS: The spores of B. dermatitidis are usually inhaled into the lung tissue following exposure especially in areas where dust particles containing the organism’s spores become aerosolized. Human infection is initiated in the lungs after spore inhalation. The primary infection may be sub-clinical (i.e. asymptomatic). After deposition of the fungal conidia in the distal air ways (e.g. alveolus of the lung tissue), there is transition of the fungi from the mould form (second serotype) to the yeast form (first serotype). This phase shift occurs as a result of heat-related stress allowed by uncoupling of the oxidative phosphorylation in the organism. The cells increase in number in the lung parenchyma in the absence of non-specific host defense mechanism to ward-off the pathogenic/virulent activities of the organism in vivo. The incubation time of B. dermatitidis infection averages 4-6 weeks. However the incubation time of the disease may vary widely based on several environmental or host factors.

CLINICAL FEATURES: The clinical spectrum of blastomycosis is highly variable. It may be asymptomatic, chronic or pneumonia-like. Extra-pulmonary cases like infection of the bone marrow and skin may occur. Its constitutional symptoms includes: fever, chills, weight loss, malaise and anorexia. In children, it presents as acute pulmonary blastomycosis and its symptoms include: dyspnea, productive cough and whizzing respiration.

IMMUNITY: Some patients have natural immunity/resistance to blastomycosis since 50 % of the infections are sub-clinical in nature. One attack of blastomycosis does not confer life-long immunity. Cellular immune response mediated by antigen T lymphocytes and lymphokines-derived macrophages cell-mediated immunity plays a critical role in stopping this fungal pathogen.

EPIDEMIOLOGY: Blastomycosis is a common infection of dogs in endemic areas. Its natural habitat is unknown and it cannot be transmitted by animals or humans. Blastomycosis is often referred to as Northern American blastomycosis because of its endemicity in that part of the world. Most clinical cases of blastomycosis are reported in states surrounding the Mississippi and Ohio rivers and the great lake regions. It can also be found in parts of Africa, Canada, Israel and India. B. dermatitidis requires wet sandy acidic soil rich in organic matter and close to water for its propagation or spread.

MODE OF TRANSMISSION: Infection occurs through inhalation of fungal spores. Other roots of infections include skin lesions or penetrating injuries that introduce the organism into the body. Dissemination can occur in patients that are immunocompromised. Dogs (especially male dogs and large breed dogs) are ten times more likely to be infected with B. dermatitidis than humans. Thus, the ratio of B. dermatitidis infection in dog and man is 10:1 respectively.  

LABORATORY DIAGNOSIS

  1. Specimens: This consists of sputum, pus, exudates, urine, and biopsies from lesions of infected individuals. Skin and lymph node samples may also be used. Pulmonary blastomycosis may be diagnosed by trans-tracheal wash (TTW), Broncho-alveolar lavage (BAL) and fine needle aspirate of the lung parenchyma.
  2. Microscopic examination: B. dermatitidis usually seen in form of round yeast ranging from 5-20 µm in diameter. It stains blue with Wright’s or Diff stains. It has a thick cell wall and broad-base budding.
  3. Culture: Identification of B. dermatitidis is the only surest way of diagnosis. Colonies usually develop within two weeks on SDA or enriched blood agar at 30oC. Identification is confirmed by its conversion to yeast form after cultivation on a rich culture medium at 37oC.

Note: Culture of cytologic specimens is not recommended because of the potential danger to laboratory personnel.

  1. Serology: This is used for if identification of the organism is not possible. The Agar Gel Immunodiffusion (AGID) testing is the most common serologic test used.

TREATMENT: Amphotericin B and 2-hydroxystilbamidine are usually used for the treatment of blastomycosis. Other azole drugs like itraconazole can also be used in place of Amphotericin B for the treatment of blastomycosis.

CHROMOBLASTOMYCOSIS

Chromoblastomycosis is a chronic subcutaneous mycosis. It is a long-term fungal infection of the skin and subcutaneous tissue. Chromoblastomycosis is caused by different type of fungi which become implanted under the skin, often by thorns or splinters following exposure to the causative agent of the disease. Chromoblastomycosis spreads very slowly, and it is rarely fatal and usually has a good prognosis, but it can be very difficult to cure.

Etiologic agents: Chromoblastomycosis is caused by the traumatic inoculation of any of any of the following five recognized fungal agents that resides in the soil and vegetation. These fungi are mainly identified by their different modes of conidiation or spore (conidia) formation. And they are all dematiaceous fungi that colonize the melanized skin of the body.

The aetiologic agents that causes chromoblastomycosis include:

           Phialophora verrucosa

           Fonsecaea pedrosoi

           Rhinocladiella aquaspersa

           Fonsecaea compacta

           Cladophialophora carrioni

Morphology and Identification (Biology): The agents of chromoblastomycosis are identified by their modes of conidiation. The colonies are compact, deep brown-back and develop a velvety, often wrinkled surface. They appear the same in tissue producing spherical brown cells (measuring about 4-12 µm). These cells are termed muriform (sclerotic) bodies and they divide by transverse septation.

Pathogenesis and Clinical Findings: The initial trauma causing the infection is often not noticed or forgotten. The infection builds at the site of the fungus inoculation over a period of years and a small red papule appears. The lesion is usually not painful and there are a few, if any symptoms associated with the disease. Patients rarely seek medical care at this point of the disease development. And several complications may occur. The infection remains localized at the area around the wound as it gradually spreads to other areas or parts of the body. Secondary infection with bacteria may occur, and this may lead to lymphstasis (i.e. the obstruction of the lymph nodes) and even elephantiasis in some cases.

LABORATORY DIAGNOSIS

  1. Specimen: Specimens consist of skin scrapings and biopsies from lesions.
  2. Microscopic examination: Scrapings are placed in 10 % potassium hydroxide (KOH) and examined microscopically for dark, spherical cells (medlar or sclerotic bodies). Detection of sclerotic bodies is diagnostic of chromoblastomycosis regardless of its etiologic agent.
  3. Culture: Specimens are cultured on SDA with antibiotics in order to identify the organisms involved. They are identified by their different conidial structures.

Treatment: Chromoblastomycosis is very difficult to care. Chemotherapy with flucytosin (5-FC) or itraconazole may be efficacious for larger lesions. Surgical excison with wide margins is the therapy of choice for small lesions. Local application of heat is also reliable. Relapse is common.

Epidemiology: Chromoblastomycosis occurs most commonly in tropical or subtropical climates, often in rural areas. Madagascar and Japan have the highest incidence of the disease. Over two thirds of patients are male and usually between the ages of 30 and 50 years of age. Chromoblastomycosis occurs chiefly on the legs of barefoot agrarian workers following the traumatic inoculation of the fungus into the host. Chromoblastomycosis is not a communicable disease and the prevention of the diseases is usually by wearing shoes and protecting the legs when working in the farm or in areas where the foot come in constant contact with the soil.

PARACOCCIDIOIDOMYCOSIS

Paracoccidioidomycosis is caused by the thermally dimorphic fungus, Paracoccidioides brasiliensis. Paracoccidioidomycosis (South American blastomycosis) is geographically restricted to the endemic regions of Central and South America.

Morphology and Identification (Biology): Cultures of the mould form of P. brasiliensis grow very slowly and produce chlamydospores and conidia. It forms large multiple budding yeast cells at 36oC on rich medium. The buds are attached by a narrow connection.

Pathogenesis and Clinical Findings (Manifestation): Initial lesions of the disease occur in the lungs. It is caused by inhalation of spores of P. brasiliensis. Clinical manifestations vary from subclinical infections that are detected only by skin test reactivity to chronic unifocal infection where only a single organ is involved and to chronic multifocal infection where more than one organ is involved. The different forms of this mycosis are:

  1. Pulmonary form: Here, patients present with chronic symptoms such as cough, fever, night sweats, malaise and weight loss. Chest x-ray are characteristics but not diagnostic. It must be distinguished from histoplasmosis and tuberculosis (TB).
  2. Mucocutaneous form: The mouth and nose are the most usual mucosal sites of infection. Painful ulcerated lesions develop on the gums, tongue, lips or palate and can progress over weeks or months.
  3. Lymphonodular form: Lymphadenitis (inflammation of the lymph nodes) is common in younger patients. The inflammation of the lymph nodes may progress to form abscesses with draining sinuses.
  4. Disseminated form: Haematogenous spread of P. brasiliensis can result in widespread disseminated disease that affect several parts or organs of the body, including lesions of the small or large intestine, hepatic lesions and osteomyelitis arthritis

LABORATORY DIAGNOSIS

  1. Specimen: Skin scrapings, sputum, bronchial washings, CSF, plural fluid and blood, bone marrow, and tissue biopsies from various visceral organs.
  2. Direct Microscopy: Skin scrapings should be examined using 10 % KOH. Exudates and body fluids should be centrifuged and the sediment examined using 10 % KOH. Tissue sections should be examined using PAS digest, Grocott’s metherramine silver (GMS) or Gram stained. A positive direct microscopy demonstrating the presence of large, 20-60 µm round, narrow base budding yeast cells with multiple budding steering wheels” from any specimen should be considered significant.
  3. Culture: Specimens should be inoculated onto primary isolation media like SDA and Brain heart infusion agar supplemented with 5 % sheep blood. Cultures are incubated at room temperature and confirmed by conversion to yeast form in vitro at 36oC.
  4. Serology: This is most useful for diagnosis. Antibodies to paracoccidioidin (P. brasiliensis antigens) can be measured by the immunoflourescence (IF) or immunodiffusion (ID) test. Healthy persons in endemic areas do not have antibodies to this antigen (paracoccidioidin).

Treatment: Itraconazole is used for treatment but ketoconazole and trimethoprim are also efficacious against the causative organism of the disease.

Epidemiology: Paracoccidioidomycosis occurs mainly in rural areas of Latin America, particularly among farmers. The disease manifestations are much more frequent in males than in females, but the infection and skin test reactivity occur equally in both sexes. The natural habitat of P. brasiliensis has not been well defined. And paracoccidioidomycosis is not a communicable disease as is applicable to other endemic mycosis.

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