General Overview of Fungi

MYCOLOGY

Fungi are eukaryotic microorganisms with absorptive mode of nutrition, which do not contain chlorophyll and reproduce by both asexual and sexual methods. The study of fungi is called Mycology and the scientist who study fungi are called mycologists. About 90,000 fungal species have already been described; however, some estimates of total number suggest that 1.5 million species may exist.

TERMINOLOGY USED IN MYCOLOGY

Some of the terminology used in mycology is as follows.

   A. Fungal element: Fungi include unicellular yeast, multicellular filamentous fungi called molds, macroscopic puffballs, and mushrooms. The body or vegetative structure of a fungus is called thallus. It varies in complexity and size, ranging from the single cell microscopic yeast to multicellular molds, macroscopic puffballs, and mushrooms. Fungi are eukaryotic microorganisms. The fungal cell consists of cell wall made up of chitin, cytoplasmic membrane, and other cell organelles (e.g. nucleus, ribosome, mitochondria etc.

    B. Yeast: Yeast is a unicellular fungus that has a single nucleus and reproduces either asexually by budding and transverse binary fission or sexually through formation of spores (Ascospores). Each bud that separates can grow into new yeast. Generally yeast cells are larger than bacteria, vary considerably in size and are commonly spherical or egg shaped.  They do not have flagella but do possess most of the other eukaryotic organelles. E.g. Saccharomyces spp., Candida spp., Cryptococcus spp.

     C.     Molds: Molds are multicellular filamentous fungi. E.g. Aspergillus, Mucor, Rhizopus

    D.    Hyphae: Hyphae is the unit structure of filamentous fungi. Hyphae are composed of an outer cell wall and inner lumen which contain the cytosol and cell organelles. The hyphae of some fungi have cross walls called septa and called septate hyphae. Hyphae of other fungi do not have septa and are called aseptate or coenocytic hyphae.

      E.     Mycelium: Mycelium is the tangled mass of the fungal hyphae of filamentous fungi.

    F.      Pseudomycelium or pseudohyphae or sprout mycelium: A mycelium like structure consisting of chains of cells formed by sequential budding is called pseudomycelium.

    G.    Germ tube: A short, hyphae like structure which develops from certain types of spore on germination or in yeast (e.g. Candida albicans) undergoing yeast to mycelium transition. A germ tube usually develops into hyphae.

     H.    Rhizoids: Rhizopus (bread molds) is fungi that grow in bread, vegetable, fruits, and other food products and cause food spoilage. These molds produce clusters of root like holdfasts which are called rhizoids. Morphologically, Rhizopus has nonseptate, cottony mycelia with sporangiophores arising at the nodes where the rhizoids form.

     I.       Dimorphic fungi: Many fungi, especially those that cause diseases in humans and animals, are dimorphic, i.e. they have two forms. Dimorphic fungi can change from the yeast (Y) form in the animal to the mold or mycelial form (M) in the external environment. E.g. Histoplasma capsulatum, Blastomyces dermatidis etc.

     J.       Mycoses: Diseases caused by fungi are called mycoses.

   K.    Dermatophytes: Three genera of cutaneous fungi, Epidermophyton, Microsporum, and Trichophyton which are are involved in cutaneous mycoses (skin, nail and hair infections) are called dermatophytes. Dermatophytic infections—also called dermatomycoses, ringworms, or tineas—occur worldwide and represent the most common fungal diseases in humans.

·         Epidermophyton: Smooth thin-walled Macroconidia only present, no microconidia, colonies a green-brown to khaki color.

·         Microsporum: Macroconidia with rough walls present, microconidia may also be present.

·         Trichophyton: Microconidia present, smooth-walled macroconidia may or may not be present.

GENERAL PROPERTIES OF FUNGI

• They are eukaryotic; cells contain membrane bound cell organelles including nuclei, mitochondria, golgi apparatus, endoplasmic reticulum, lysosomes etc. They also exhibit mitosis.
• Have ergosterols in their membranes and possesses 80S ribosomes.
• Have a rigid cell wall and are therefore non-motile, a feature that separates them from animals. All fungi possess cell wall made of chitin.
• Are chemoheterotrophs (require organic compounds for both carbon and energy sources) and fungi lack chlorophyll and are therefore not autotrophic.
• Fungi are osmotrophic; they obtain their nutrients by absorption.
• They obtain nutrients as saprophytes (live off of decaying matter) or as parasites (live off of living matter).
• All fungi require water and oxygen and there are no obligate anaerobes.
• Typically reproduce asexually and/or sexually by producing spores.
• They grow either reproductively by budding or non-reproductively by hyphal tip elongation.
• Food storage is generally in the form of lipids and glycogen.

·         Table: Difference between bacteria and fungi

	FUNGI	BACTERIA
Cell type	Eukaryotic	Prokaryotic
Cell membrane	Sterol present	Sterol absent except in mycobacteria
Cell wall	Consists of chitin, mannans, glucans	Consists of peptidoglycan
Flagella, pili	Absent	Present or absent
Reproduction	Sexual and asexual	Asexual
Spore formation	As part of reproduction	For survival in adverse condition
Metabolism	Heterotrophic	Autotrophic, heterotrophic
Oxygen requirement	Aerobic, facultative anaerobic	Aerobic, facultative anaerobic, microaerophilic, anaerobic

MORPHOLOGY OF FUNGI

Fungi exist in two fundamental forms; the filamentous (hyphal) and single celled budding forms (yeast). But, for the classification purpose they are studied as moulds, yeasts, yeast like and dimorphic fungi.

Fungi exist in three fundamental forms;

• Filamentous and multicellular fungi (molds): The unit structure of filamentous fungi is known as hyphae. Hyphae are composed of an outer cell wall and inner lumen which contain the cytosol and cell organelles. The hyphae of some fungi have cross walls called septa called septate hyphae. Hyphae of other fungi do not have septa and are called aseptate or coenocytic hyphae. The tangled mass of the fungal hyphae of filamentous fungi is called Mycelium.
• Unicellular fungi (yeast): Yeast is a unicellular fungus that has a single nucleus and reproduces either asexually by budding and transverse binary fission or sexually through formation of Ascospores. Each bud that separates can grow into new yeast. Generally yeast cells are larger than bacteria, vary considerably in size and are commonly spherical or egg shaped.  A typical yeast cell possesses most of the other eukaryotic organelles. Some produce pseudohyphae and germ tube (e.g. Candida spp.) and some are capsulated (e.g. Cryptococcus spp.). Yeast are separated on the basis of their size, shape, nature of budding, and capacity to form pseudohyphae, capsule and germ tube.
• Dimorphic fungi (which occur both as mold and yeast): Many fungi, especially those that cause diseases in humans and animals, are dimorphic, i.e. they have two forms. Dimorphic fungi can change from the yeast (Y) form in the animal to the mold or mycelial form (M) in the external environment.

All fungi have typical eukaryotic morphology. They have rigid cell wall composed of chitin, which may be layered with mannans, glucans and other polysaccharides in association with polypeptides. Some lower fungi possess cellulose in their cell wall. Some fungi such as Cryptococcus and yeast form of Histoplasma capsulatum possess polysaccharide capsules that help them to evade phagocytosis.

Inner to the cell wall is the plasma membrane that is a typical bi-layered membrane in addition to the presence of sterols. Fungal membranes possess ergosterol in contrast to cholesterol found in mammalian cells. The cytoplasm consists of various organelles such as mitochondria, golgi apparatus, ribosomes, endoplasmic reticulum, lysosomes, microtubules and a membrane enclosed nucleus. A unique property of nuclear membrane is that it persists throughout the metaphase of mitosis unlike in plant and animal cells where it dissolves and re-forms. The nucleus possesses paired chromosomes.

Reproduction in fungi and fungal spores:

Reproduction in fungi can be either asexual or sexual.

Asexual reproduction is accomplished in several ways.

• Asexual reproduction in yeast takes place by

a.       Fission (a parent cell can divide into two daughter cells by central constriction and formation of a new cell wall) or

b.      Budding (somatic vegetative cells may bud to produce new organisms. 

• The most common method of asexual reproduction in filamentous fungi is spore production. Asexual spore formation occurs in an individual fungus through mitosis and subsequent cell division. There are several types of asexual spores:

a.       Arthroconidia or arthrospores:  A hypha can fragment (by the separation of hyphae through splitting of the cell wall or septum) to form cells that behave as spores. These cells are called arthroconidia or arthrospores.

b.      Chlamydospores: If the cells are surrounded by a thick wall before separation, they are called chlamydospores.

c.       Sporangiospores: If the spores develop within a sac at a hyphal tip, they are called sporangiospores.

d.      Conidiospores: If the spores are not enclosed in a sac but produced at the tips or sides of the hypha, they are termed conidiospores.

e.       Blastospores Spores produced from a vegetative mother cell by budding are called blastospores.

Sexual reproduction in fungi involves the union of compatible nuclei. Some fungal species are self-fertilizing and produce sexually compatible gametes on the same mycelium (homothallic). Other species require out crossing between different but sexually compatible mycelia (heterothallic). Depending on the species, sexual fusion may occur between haploid gametes, gamete-producing bodies called gametangia, or hyphae. This sexual reproduction yields spores. E.g.:

a.       Ascospores: These single celled spores are produced in a sac called ascus. There are usually eight ascospores in each ascus.

b.      Basidiospores: These single cell spores are produced on a club shaped structure called basidium.

c.       Zygospores: Zygospores are large, thick walled spores formed when the tips of two sexually compatible hyphae of certain fungi fuse together.

d.      Oospores: These are formed within a special female structure called oogonium. Fertilization of the eggs by male gamete formed in an anthridium give rise to oospores.

Fungal spores are important for several reasons. The size, shape, color, and number of spores are useful in the identification of fungal species. The spores are often small and light; they can remain suspended in air for long periods. Thus they frequently aid in fungal dissemination, a significant factor that explains the wide distribution of many fungi.

DISTRIBUTION OF FUNGI

Fungi are primarily terrestrial organisms, although a few are freshwater or marine. Many are pathogenic and infect plants and animals. Fungi also form association with other organisms. The association of fungi with root of the higher plant is called mycorrhizae and association between fungi and algae is called lichen.

IMPORTANCE OF FUNGI

Fungi are important to humans in both beneficial and harmful ways. They degrade complex organic materials in the environment to simple organic compounds and inorganic molecules. In this way carbon, nitrogen, phosphorus, and other critical constituents of dead organisms are released and made available for living organisms. Besides this, fungi are the major cause of plant disease. Over 5,000 species of fungi attack economically valuable corps and garden plants and also many wild plants. Similarly many diseases of animals and humans are caused by fungi. Some of the fungi have wide industrial importance. Fungi play important role in making of bread, wine, beer, cheese, soy sauce, and commercial production of many organic acids, , certain drugs (e.g. cortisone, cyclosporine) and many antibiotics (e.g. penicillin). In addition, fungi are important research tools in the study of fundamental biological processes. Cytologists, geneticists, biochemists, biophysicists and microbiologists use fungi in research.

Beneficial Effects of Fungi:

·         Decomposition - nutrient and carbon recycling.

·         Biosynthetic factories. The fermentation property is used for the industrial production of alcohols, fats, citric, oxalic and gluconic acids.

·         Important sources of antibiotics, such as Penicillin.

·         Model organisms for biochemical and genetic studies. E.g.: Neurospora crassa

·         Saccharomyces cerviciae is extensively used in recombinant DNA technology, which includes the Hepatitis B Vaccine.

·         Some fungi are edible (mushrooms).

·         Yeasts provide nutritional supplements such as vitamins and cofactors.

·         Penicillium is used to flavour Roquefort and Camembert cheeses.

·         Ergot produced by Claviceps purpurea contains medically important alkaloids that help in inducing uterine contractions, controlling bleeding and treating migraine.

·         Fungi (Leptolegnia caudate and Aphanomyces laevis) are used to trap mosquito larvae in paddy fields and thus help in malaria control.

Harmful Effects of Fungi:

·         Destruction of food, lumber, paper, and cloth.

·         Animal and human diseases, including allergies.

·         Toxins produced by poisonous mushrooms and within food (Mycetism and Mycotoxicosis).

·         Plant diseases.

·         Spoilage of agriculture produces such as vegetables and cereals in the godown.

·         Damage the products like magnetic tapes and disks, glass lenses, marble statues, bones and wax.

Medically important fungi:

Among 90,000 species of fungi that have been described, fewer than 500 have been associated with human disease, and about 100 are capable of causing infection in normal individuals. The remainder of fungi produces disease in imunocompromized hosts. Medical mycology is the discipline that deals with the fungi that causes human disease. It should be noted that the importance of opportunistic fungal pathogens is increasing because of the expansion of the imunocompromized patient population.

Pathogenesis of fungal diseases (Mycoses):

Most fungi are saprophytic or parasitic to plants and are adapted to their natural environment. Infection in humans is a chance event, occurring only when conditions are favorable. Except for few fungi such as the dimorphic fungi that cause systemic mycoses and dermatophytes, which are primary pathogens, the rest are only opportunistic pathogens. Human body is a hostile environment and offers great resistance to fungal invasion. Most fungi are saprophytic and their enzymatic pathways function more efficiently at the redox potential of non-living substrates than at the relatively more reduced state of living metabolizing tissue. Some fungi such as Candida and Malasezzia have adapted to human environment and exist as commensals.

The complex interplay between fungal virulence factors and host defence factors will determine if a fungal infection will cause a disease. Infection depends on inoculum size and the general immunity of the host.

Virulence factors of medically important fungi:

• Ability to adhere to host cells by way of cell wall glycoproteins
• Production capsules allowing them to resist phagocytosis
• Production of a cytokine (e.g. GM-CSF) by Candida albicans that suppress the production of complement.
• Ability to acquire iron from red blood cells as in Candida albicans
• Ability to damage host by secreting enzymes such as keratinase, elastase, collagenase
• Ability to resist killing by phagocytes as in dimorphic fungi
• Ability to secrete mycotoxins
• Having a unique enzymatic capacity
• Exhibiting thermal dimorphism
• Ability to block the cell-mediated immune defences of the host.

Host defence factors:

• Physical barriers, such as skin and mucus membranes
• The fatty acid content of the skin
• The pH of the skin, mucosal surfaces and body fluids
• Epithelial cell turnover
• Normal flora
• Chemical barriers, such as secretions, serum factors
• Most fungi are mesophilic and cannot grow at 37oC.
• Natural Effector Cells (polymorphonuclear leucocytes) and the Professional Phagocytes (monocytes and macrophages)

Factors predisposing to fungal infections:

• Prolonged antibiotic therapy
• Underlying disease (HIV infection, cancer, diabetes, etc.)
• Age
• Surgical procedures
• Immunosuppressive drugs
• Irradiation therapy
• Indwelling catheters
• Obesity
• Drug addiction
• Transplants
• Occupation

CLASSIFICATION OF FUNGAL INFECTIONS (MYCOSES)

Taxonomic classification of fungi is complex and has little value in clinical mycology laboratory. Among the large number of fungi identified, only few may cause disease in human. In medical setting, medically important fungi are categorized on the basis of basic morphology (e.g. yeast, mycelial fungi and dimorphic fungi) and disease they cause (e.g. superficial mycoses, cutaneous mycoses, subcutaneous mycoses and opportunistic mycosis.

Fungal infections are classified on the basis of the site of infections. These fungal diseases, known as mycoses, are divided into five groups according to the type of infected tissue in the host: superficial, cutaneous, subcutaneous, systemic, and opportunistic mycoses.

1) Superficial Mycoses

The fungi responsible are limited to the outer surface of hair and skin and hence are called superficial. The superficial mycoses mostly occur in the tropics. Infections of the hair shaft are collectively called piedras (Spanish for stone because they are associated with the hard nodules formed by mycelia on the hair shaft). For example, black piedra is caused by Piedraia hortae and forms hard black nodules on the hairs of the scalp. White piedra is caused by the yeast Trichosporon beigelii and forms light-colored nodules on the beard and mustache. Some superficial mycoses are called tineas [Latin for grub, larva, worm], the specific type being designated by a modifying term. Tineas are superficial fungal infections involving the outer layers of the skin, nails, and hair. Tinea versicolor is caused by the yeast Malassezia furfur and forms brownish-red scales on the skin of the trunk, neck, face, and arms. Treatment involves removal of the skin scales with a cleansing agent and removal of the infected hairs. Good personal hygiene prevents these infections.

2) Cutaneous Mycoses

Cutaneous mycoses—also called dermatomycoses, ringworms, or tineas—occur worldwide and represent the most common fungal diseases in humans. Three genera of cutaneous fungi, or dermatophytes, are involved in these mycoses: Epidermophyton, Microsporum, and Trichophyton. Diagnosis is by microscopic examination of biopsied areas of the skin cleared with 10% potassium hydroxide and by culture on Sabouraud dextrose agar. Treatment is with topical ointments such as miconazole, tolnaftate, or clotrimaz for 2 to 4 weeks. Griseofulvin and itraconazole are the only oral fungal agents currently approved by the FDA for treating dermatophytoses.

·         Tinea barbae [Latin barba, the beard] is an infection of the beard hair caused by Trichophyton mentagrophytes or T. verrucosum. It is predominantly a disease of men who live in rural areas and acquire the fungus from infected animals.

·         Tinea capitis [Latin capita, the head] is an infection of the scalp hair. It is characterized by loss of hair, inflammation, and scaling. Tinea capitis is primarily a childhood disease caused by Trichophyton or Microsporum species. Person to-person transmission of the fungus occurs frequently when poor hygiene and overcrowded conditions exist. The fungus also occurs in domestic animals, from whom it can be transmitted to humans. A Wood’s light (a UV light) can help with the diagnosis of tinea capitis because fungus-infected hair fluoresces when illuminated by UV radiation.

·         Tinea corporis [Latin corpus, the body] is a dermatophytic infection of the smooth or bare parts of the skin. The disease is characterized by circular, red, well-demarcated, scaly, vesiculopustular lesions accompanied by itching. Tinea corporis is caused by Trichophyton rubrum, T. mentagrophytes, or Microsporum canis. Transmission of the disease is by direct contact with infected animals or humans or by indirect contact through fomites.

·         Tinea cruris [Latin crura, the leg] is a dermatophytic infection of the groin. The pathogenesis and clinical manifestations are similar to those of tinea corporis. The responsible fungi are Epidermophyton floccosum, T. mentagrophytes, or T. rubrum. Factors predisposing one to recurrent disease are moisture, occlusion, and skin trauma.

·         Tinea pedis [Latin pes, the foot], also known as athlete’s foot, and tinea manuum [Latin mannus, the hand] are dermatophytic infections of the feet and hands, respectively. Clinical symptoms vary from a fine scale to a vesiculopustular eruption. Itching is frequently present. Warmth, humidity, trauma, and occlusion increase susceptibility to infection. Most infections are caused by T. rubrum, T. mentagrophytes, or E. floccosum. Tineapedis and tinea manuum occur throughout the world, are most commonly found in adults, and increase in frequency with age.

·         Tinea unguium [Latin unguis, nail] is a dermatophytic infection of the nail bed. In this disease the nail becomes discolored and then thickens. The nail plate rises and separates from the nail bed. Trichophyton rubrum or T. mentagrophytes are the causative fungi.

3) Subcutaneous Mycoses

The dermatophytes that cause subcutaneous mycoses are normal saprophytic inhabitants of soil and decaying vegetation. Because they are unable to penetrate the skin, they must be introduced into the subcutaneous tissue by a puncture wound that has been contaminated with soil containing the fungi. Most infections involve barefooted agricultural workers. Once in the subcutaneous tissue, the disease develops slowly—often over a period of years. During this time the fungi produce a nodule that eventually ulcerates and the organisms spread along lymphatic channels producing more subcutaneous nodules. At times such nodules drain to the skin surface. The administration of oral 5-fluorocytosine, iodides, amphotericin B, and surgical excision are the usual treatments. Diagnosis is accomplished by culture of the infected tissue.

• One type of subcutaneous mycosis is chromoblastomycosis. The nodules are pigmented a dark brown. This disease is caused by the black molds Phialophora verrucosa or Fonsecaea pedrosoi. These fungi exist worldwide, especially in tropical and subtropical regions. Most infections involve the legs and feet.
• Another subcutaneous mycosis is maduromycosis, caused by Madurella mycetomatis, which is distributed worldwide and is especially prevalent in the tropics. Because the fungus destroys subcutaneous tissue and produces serious deformities, the resulting infection is often called a eumycotic mycetoma or fungal tumor. One form of mycetoma, known as Madura foot, occurs through skin abrasions acquired while walking barefoot on contaminated soil.
• Sporotrichosis is the subcutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii. The disease occurs throughout the world and is the most common subcutaneous mycotic disease in the United States. The fungus can be found in the soil, on living plants, such as barberry shrubs and roses, or in plant debris. Infection occurs by a puncture wound from a thorn or splinter contaminated with the fungus. The disease is an occupational hazard to florists, gardeners, and forestry workers. After an incubation period of 1 to 12 weeks, a small red papule arises and begins to ulcerate. New lesions appear along lymph channels and can remain localized or spread throughout the body, producing extracutaneous sporotrichosis.

4) Systemic Mycoses

Except for Cryptococcus neoformans, which has only a yeast form, the fungi that cause the systemic or deep mycoses are dimorphic—that is, they exhibit a parasitic yeastlike phase (Y) and a saprophyticmold or mycelial phase (M). Most systemic mycoses are acquired by the inhalation of spores from soil in which the mold-phase of the fungus resides. If a susceptible person inhales enough spores, an infection begins as a lung lesion, becomes chronic, and spreads through the bloodstream to other organs (the target organ varies with the species).

• Blastomycosis is the systemic mycosis caused by Blastomyces dermatitidis, a fungus that grows as budding yeast in humans but as a mold on culture media and in the environment. The disease occurs in three clinical forms: cutaneous, pulmonary, and disseminated. The initial infection begins when blastospores are inhaled into the lungs. The fungus can then spread rapidly, especially to the skin, where cutaneous ulcers and abscess formation occur. B. dermatitidis can be isolated from pus and biopsy sections. Diagnosis requires the demonstration of thick-walled, yeast like cells 8 to 15 μm in diameter. Complement-fixation, immunodiffusion, and skin (blastomycin) tests are also useful. Amphotericin B, itraconazole, or ketoconazole are the drugs of choice for treatment. Surgery may be necessary for the drainage of large abscesses.
• Coccidioidomycosis, also known as valley fever, San Joaquin fever, or desert rheumatism, is caused by Coccidioides immitis. C. immitis exists in the dry, highly alkaline soils of North, Central, and South America. In the soil and on culture media, this fungus grows as a mold that forms arthroconidia at the tips of hyphae. In humans the fungus grows as a yeast-forming, thick-walled spherule filled with endospores. Most cases of coccidioidomycosis are asymptomatic or indistinguishable from ordinary upper respiratory infections. Almost all cases resolve themselves in a few weeks, and a lasting immunity results. A few infections result in a progressive chronic pulmonary disease. The fungus also can spread throughout the body, involving almost any organ or site. Diagnosis is accomplished by aspiration and identification of the large spherules (approximately 80 μm in diameter) in pus, sputum, and aspirates. Culturing clinical samples in the presence of penicillin and streptomycin on Sabouraud agar also is diagnostic. Newer methods of rapid confirmation include the testing of supernatants of liquid media cultures for antigens, serology, and skin testing. Miconazole, itraconazole, ketoconazole, and amphotericin B are the drugs of choice for treatment. Prevention involves reducing exposure to dust (soil) in endemic areas.
• Cryptococcosis is a systemic mycosis caused by Cryptococcus neoformans. This fungus always grows as large budding yeast. In the environment C. neoformans is a saprophyte with a worldwide distribution. Aged, dried pigeon droppings are an apparent source of infection. Cryptococcosis is found in approximately 15% of AIDS patients. The fungus enters the body by the respiratory tract, causing a minor pulmonary infection that is usually transitory. Some pulmonary infections spread to the skin, bones, viscera, and central nervous system. Once the nervous system is involved, cryptococcal meningitis usually results. Diagnosis is accomplished by detection of the thick-walled spherical yeast cells in pus, sputum, or exudate smears using India ink to define the organism. The fungus can be easily cultured on Sabouraud dextrose agar. Identification of the fungus in body fluids is made by immunologic procedures. Treatment includes amphotericin B or itraconazole. There are no preventive or control measures.
• Histoplasmosis is caused by Histoplasma capsulatum var. capsulatum, a facultative parasitic fungus that grows intracellularly. It appears as small budding yeast in humans and on culture media at 37°C. At 25°C it grows as a mold, producing small microconidia (1 to 5 μm in diameter) that are borne singly at the tips of short conidiophores. Large macroconidia or chlamydospores (8 to 16 _m in diameter) are also formed on conidiophores. In humans the yeast like form grows within phagocytic cells. H. capsulatum var. capsulatum is found as the mycelial form in soils throughout the world and is localized in areas that have been contaminated with bird or bat excrement. The chlamydospores, particularly the microconidia, are easily spread by air currents. Histoplasmosis is an occupational disease among spelunkers (people who explore caves) and bat guano miners. Humans acquire histoplasmosis from airborne microconidia that are produced under favorable environmental conditions. Histoplasmosis is a disease of the monocyte-macrophage system; thus many organs of the body can be infected. More than 95% of cases have either no symptoms or mild symptoms such as coughing, fever, and joint pain. Lesions may appear in the lungs and show calcification; most infections resolve on their own. Only rarely does the disease disseminate. Laboratory diagnosis is accomplished by complement fixation tests and isolation of the fungus from tissue specimens. Currently the most effective treatment is with amphotericin B, ketoconazole, or itraconazole. Prevention and control involve wearing protective clothing and masks before entering or working in infested habitats. Soil decontamination with 3 to 5% formalin is effective where economically and physically feasible.

E) Opportunistic Mycoses

An opportunistic microorganism is generally harmless in its normal environment but becomes pathogenic in a compromised host. A compromised host is seriously debilitated and has a lowered resistance to infection. There are many causes of this condition, among them the following: malnutrition; alcoholism; cancer, diabetes, leukemia, or another infectious disease; trauma from surgery or injury; an altered microbiota from the prolonged use of antibiotics (e.g., in vaginal candidiasis); and immunosuppression by drugs, viruses (HIV), hormones, genetic deficiencies, chemotherapy of patients, and old age. The most important opportunistic mycoses include systemic aspergillosis, candidiasis, and Pneumocystis carinii pneumonia.

• Aspergillosis: Of all the fungi that cause disease in compromised hosts, none are as widely distributed as the Aspergillus species. Aspergillus is omnipresent in nature, being found wherever organic debris occurs. Aspergillus fumigatus is the usual cause of aspergillosis. A. flavus is the second most important species, particularly in invasive disease of immunosuppressed patients. The major portal of entry for Aspergillus is the respiratory tract. Inhalation of conidiospores can lead to several types of pulmonary aspergillosis. One type is allergic aspergillosis. Infected individuals may develop an immediate allergic response and suffer typical asthmatic attacks when exposed to fungal antigens on the conidiospores. In bronchopulmonary aspergillosis the major clinical manifestation of the allergic response is bronchitis. Although tissue invasion seldom occurs in bronchopulmonary aspergillosis, Aspergillus often can be cultured from the sputum. A most common manifestation of pulmonary involvement is the occurrence of colonizing aspergillosis, in which Aspergillus forms colonies within the lungs that develop into “fungus balls” called aspergillomas. These consist of a tangled mass of mycelia growing in a circumscribed area. From the pulmonary focus, the fungus may spread, producing disseminated aspergillosis in a variety of tissues and organs. In patients whose resistance has been severely compromised, invasive aspergillosis may occur and fill the lung with fungal mycelia. Laboratory diagnosis of aspergillosis depends on identification, either by direct examination of pathological specimens or by isolation and characterization of the fungus. Successful therapy depends on treatment of the underlying disease so that host resistance increases. Treatment is with itraconazole.
• Candidiasis is the mycosis caused by Candida albicans and other species Candida. In contrast to the other pathogenic fungi, C. albicans is a member of the normal microbiota within the gastrointestinal tract, respiratory tract, vaginal area, and mouth (see figure 31.2). In healthy individuals C. albicans does not produce disease. However, if anything upsets the normal microbiota, Candida may multiply rapidly and produce candidiasis. Recently Candida species have become important nosocomial pathogens. In some hospitals they may represent almost 10% of nosocomial bloodstream infections. No other mycotic pathogen produces as diverse a spectrum of disease in humans as does C. albicans. Most infections involve the skin or mucous membranes (Oral candidiasis; onychomycosis; Intertriginous; candidiasis; Napkin (diaper) candidiasis; Candidal vaginitis; balanitis). This occurs because C. albicans is a strict aerobe and finds such surfaces very suitable for growth. Cutaneous involvement usually occurs when the skin becomes overtly moist or damaged. Besides this, Candida can cause many systemic infections. Diagnosis of candidiasis is difficult because (1) this fungus is a frequent secondary invader in diseased hosts, (2) a mixed microbiota is most often found in the diseased tissue, and (3) no completely specific immunologic procedures for the identification of Candida currently exist. There is no satisfactory treatment for candidiasis. Cutaneous lesions can be treated with topical agents such as sodium caprylate, sodium propionate, gentian violet, nystatin, miconazole, and trichomycin. Ketoconazole, amphotericin B, fluconazole, itraconazole, and flucytosine also can be used for systemic candidiasis.
• Pneumocystis carinii pneumonia: Pneumocystis carinii are eucaryotic protists found in the lungs of a wide variety of mammals. The natural history of P. carinii is poorly understood, in large part because of a lack of a continuous in vitro culture system for its propagation. Although it was once considered a protozoan parasite, recent comparisons of rRNA and DNA sequences from several genes have shown that P. carinii is more closely related to fungi than protozoa. The disease that this protist causes, pneumocystis pneumonia or Pneumocystis carinii pneumonia (PCP), occurs almost exclusively in immunocompromised hosts. Extensive use of immunosuppressive drugs and irradiation for the treatment of cancers and following organ transplants accounts for the formidable prevalence rates noted recently. This pneumonia also occurs in more than 80% of AIDS patients. Both the organism and the disease remain localized in the lungs—even in fatal cases. Within the lungs Pneumocystis causes the alveoli to fill with a frothy exudate. Laboratory diagnosis of pneumocystis pneumonia can be made definitively only by microscopically demonstrating the presence of the microorganisms in infected lung material or by a PCR analysis. Treatment is by means of oxygen therapy and either combination of trimethoprim and sulfamethoxazole, atovaquone, or trimetrexate. Prevention and control is through prophylaxis with drugs in susceptible persons.

LABORATORY DIAGNOSIS OF FUNGAL INFECTION

• Introduction
• Selection of specimens
• Collection of specimens (Skins, nails and hairs; Mucous membrane; Ear; Eye; Blood; CSF; Urine; Exudates/pus; Stool; Other body fluids (peritoneal, pericardial, synovial); Lower respiratory tract specimens; Bone marrow; Tissue)
• Specimen transport and storage
• Specimen processing
• Laboratory analysis and interpretation (Direct microscopic examination; Culture; Histopathological examination; Mycoserology; Molecular diagnosis)

1.      Introduction

General approach: Diagnosis is based on the combination of clinical observation and laboratory investigation. Diagnosis solely based on clinical basis has limited value;

·         It is not unusual to find that the appearance of lesion has been modified and rendered atypical by previous treatment in case of superficial infection

·         The clinical presentation of deep infection is non specific and can be caused by a wide range of infections, underlying illness or complication of treatment

·         Radiological or other imaging methods are relied upon to distinguish fungal infection from other cause of infection.

Role of laboratory analysis                                                                                

·         Confirming the clinical diagnosis of fungal infection

·         Antifungal susceptibility testing

·         Providing objective assessment of response to treatment

·         Monitoring resolution of the treatment

The successful diagnosis of fungal infection depends on the collection of appropriate specimens and selection of appropriate microbiological test procedures. The laboratory findings can sometimes be unhelpful or even misleading

·         Close liaison between the clinician and the laboratory is particularly important.

·         The clear demarcation between classical pathogen and saprophytic fungi no longer exist in imunocompromized patients.

2. Selection of specimens

·         Selection of specimen differs from mycoses to mycoses as well as the presenting symptoms and clinical signs.

·         The specimen must contain viable etiological agent if it is to be recovered and identified.

·         Must be collected in such a manner that it will allow the fungus to remain viable in its natural state, with no contamination.

3. Collection of specimens

Inappropriately collected/ transported or processed specimen leads to

·         Misdiagnosis/ under diagnosis

·         Delay in appropriate therapy

·         Increased cost of the patients

General guidelines for specimen collection

·         Appropriate site

·         The specimen should be collected from active lesions, old lesions often do not contain viable organisms

·         Appropriate volume

·         Appropriate time

·         Collect the specimen under aseptic condition

·         Collect sufficient specimens

·         Collect specimen before initiating therapy

·         Use sterile collection devices/ container (not required for skin, nail and hairs)

·         Label the specimen appropriately

·         Collect sufficient background information from patients

Collection procedures for some specimens are as follows

3.1 Skins, nails and hairs

• Should be collected in folded square of black paper (10 x10cm)
• Prior cleaning of superficial lesions with 70% alcohol prior to sampling (especially if ointments, creams, or powder have been applied to lesions).
• Fungal agents: Dermatophytes, Candida spp, Trichosporon spp.

Collection of skin:

·         Should be collected from cutaneous lesions by scraping outward from the margin of the lesion with scalpel.

·         Use of adhesive disc (if there is scaling of lesion)

Collection of hairs:

·         Use of wood’s light/ or collect lusterous hairs or stumps/ or hairs broken off al follicle mouth

·         Pluck hais with forceps (cut hairs without root is unsuitable).

·         Brushing of scalp with plastic massage pad (for patient’s with incospicious scalp lesions)

Nail specimens:

·         Should be taken from any discoloured, distrophic or brittle part of nails

·         Specimen should be cut far back as possible from the edge and should be of full thickness of nail.

·         Scrapping can be taken if nail is thickened.

3.2   Mucous membrane

• Scrappings from oral lesions are better than swabs.
• In case of veginal infections, swab should be taken from discharge in the vegina and from the lateral veginal wall.
• Swabs should be moistened with saline or sterile water prior to collection and sent in transport media.
• Fungal agents: Candida spp, C. immitis

3.3   Ear

• Scrappings of material from the ear canal are to be preferred than swab.
• Fungal agents: filamentous fungi

3.4   Eye

• For corneal ulcer: scrap the ulcer with sterile platinum spatula/ swabs are not suitable.
• Endoopthalmitis: collect vitreous humour
• Fungal agents: filamentous fungi, C. albicans, C. neofarmans

3.5   Blood

• Preferred in all cases of suspected deep fungal infections.
• Specialized technique or media are used for isolation of fungi other than Cryptococcus neoformans, Trichosporon spp. and Candida spp.
• Isolation of fungi from blood depends on number of factors:

ü  Amount of blood

ü  Number of sample collected

ü   Method of processing

·         Collection of arterial blood is considered if venous blood culture is unsuccessful.

·         Isolation rates are high when the medium is vented and aerated, use of biphasic media, use of lysis centrifugation system, automated blood culture system.

• Fungal agents: H capsulatum, C. neoformans, Candida spp.

3.6. Cerebrospinal fluid

• Ideal volume: 3-5 ml
• Centrifugation and use of sediment for culture/ microscopy is effective, supernatant can be used for serology and biochemistry.
• Fungal agents:  C. neoformans

3.6   Urine

• Fresh MSU specimes (50- 100 ml) are used for non catheterized patients
• Suprapubic aspiration for children
• Collect urine specimen after prostatic massage for blastomycosis and cryptococcosis.
• Urine specimens are processed for direct microscopy, culture and fungal antigen.
• Fungal agents: yeast, C. immitis, H. capsulatum, B. dermatidis

3.7   Stool

• Requred for profound immunosupressed bone marrow transplant recipients.
• Collect rectal swab or stool in sterile container.

3.8   Pus/ exudates

• Swabs are not suitable to collect material draining abscess or ulcer (if used should be taken from as deep as possible).
• Pus from draining abscess or sinus should be aspirated
• Grains if visible should be collected.
• Fungal agents: yeast and filamentous fungi, actinomycetes

3.9   Other body fluids

·         Peritoneal, pericardial, synovial fluids can be collected from drainage or by aspiration with heparin (heparin is not used for patients with continuous peritoneal dialysis).

·         Fungal agents: H capsulatum, C. neoformans

3.10 Lower respiratory tract specimens

·         Early morning sputum samples, at least three samples

·         Should be processed within two hours of collection (if delay store at 4˚C)

·         Induced sputum (using nubelized serum)

·         Bronchoalveolar lavage (BAL) or bronchial washing

·         Percutaneous needle biopsies for patients with focal lung disease.

·         Fungal agents: yeast and filamentous fungi, actinomycetes

3.11 Bone marrow

• Used for histoplasmosis, cryptococcosis, paracoccidiomycosis
• About 3-5 ml, collect in container with heparin.
• Fungal agents: H capsulatum, C. neoformans

3.12 Tissue

• Collect specimen form both edge and middle of the lesions
• Should be placed in sterile saline and not in formalin.
• Fungal agents: yeast and filamentous fungi, actinomycetes

3.13 Specimens for serological tests/ drug level determination

• Paired or sequential samples are more appropriate
• Blood, CSF, urine or other fluid are used
• Collect without anticoagulants

4.   Specimen transport and storage

·         All clinical specimens should be analyzed promptly as far as practicable (except for suspected dermatophytosis).

·         If processing is going to be delayed, incubate normally sterile specimens (e.g. blood, CSF) at 37˚C and specimens that are potentially contaminated with bacterial contamination (e.g.. Aspirates, pus, urine, sputum) at 4˚C but do not froze.

·         H. capsulatum doesn’t survive for long periods under refrigeration. Limited studies have shown significantly decreased viability of H. capsulatum, C immitis, B. dermatidis and A. fumigatus stored at room temperature or on dry ice.

·         Transport media are not needed except for blood and corneal scrapings.

·         Specimens should not be frozen before culture.

·         Transported specimen should never be allowed to dry.

·         Specimen mailed to laboratories should follow transport guidelines.

5.      Specimen processing

• Concentration by centrifugation
• Highly viscous specimen such as sputum should be liquified before culture
• Homogenization in tissue grinder

6.      Laboratory analysis and interpretation

• The choice of the appropriate tests differs from one disease to another and depends on the site of infection as well as the presenting symptoms and clinical signs. The investigation used for fungal diagnosis includes:

6.1 Direct microscopic examination

• Simpler and must helpful procedure for fungal diagnosis.
• Most useful in diagnosis of cutaneous and subcutaneous mycosis; can stabilize the diagnosis of deep mycoses.
• Helps to determine whether an organism recovered later in culture is a contaminant or pathogenic and to assist the laboratory in selecting the most appropriate culture conditions to recover organism visualized on direct smear.
• Unstained wet mount preparation can be examined by bright field, dark field or pahase contrast microscopy.
• Various methods can be used:

ü  Calcofluor white for detecting fungi, requires fluorescence microscope.

ü  Giemsa stain for examining bone marrow and peritoneal blood smears to detect intracellular H. capsulatum

ü  Grams stains for detecting yeast cells/ bacteria

ü  India ink for detecting Cryptococcus neoformans in CSF and other body fluids

ü  Methylene blue for detecting fungi in skin scrapping

ü  Potassium hydroxide for clearing specimen to make fungi more visible

ü  Methanamine silver stain for detecting fungi in histological specimens

ü  Papanicolaou stain for examining secretion for presence of malignant cell to detect fungal elements

ü  PAS stain for detecting fungi

ü  Wright stains for examining bone marrow and peripheral blood smear for detection of intracellular H. capsulatum.

·         The sensitivity of direct microscopy depends on the quality of specimens, specimen type, quality of microscope, and quality of microscopist in recognizing fungal elements and their diagnostic morphology. Characteristics of fungal elements in clinical specimens are as follows.

Morphology	Organism	Size(µm)	Characteristics
Yeast	Blastomyces dermatidis           Cryptococcus neoformans Histoplasm capsulatum var. capsulate Paracoccidioides brasiliensis Sporothrix schenckii	8-18                         2-15 2-5 5-60 2-6	Usually large and spherical double retractile, buds usually single and connected by broad base, small forms (2-5µm) may be seen. Cells vary in size, usually spherical but may be football shaped, buds usually single and pinched off, capsule may or may not be evident, pseudohyphal forms with or without capsule are rarely seen. Small, oval to round budding cells, often found clustered within histocytes,, difficult to detect when present in small number Cells usually large and surrounded by smaller buds around periphery (mariner’s wheel appearance), smaller cell that resembles Histoplasma may be present, buds have pinched off appearance. Small, oval to round to cigar shaped, single or multiple buds present, often not seen in clinical specimens.
Yeast form or pseudohyphae or true hyphae	Candida spp.	3-4 (5-10)	Cells usually exhibit single budding, pseudohyphae when present are constricted at ends and remain attached like links of sausage, true hyphae when present have parallel walls and are septate
Yeast forms and hyphae	Malassezia furfur	3-8 (2.5-4)	Short, curved hyphal elements, usually present along with round yeast cells that retain their spherical shapes in compacted clusters when found in skin
Spherules	Coccidioides immitis	10-200	Vary in size, some contains endospores, other are empty. Adjacent spherules may resemble B. dermatidis and endospore may resemble H. capsulatum. Hyphae may be found.
Hyaline, septate hyphae	Dermato-phytes Aspergillus spp.  Fusarium spp.	3-15 3-12 3-12	Presence of chain of arthroconidia Hyphae withdichotomous branching (45˚) Septate hyphae, impossible to distinguish from other hyaline molds
Phaeoid septate hyphae	Phialophora spp.	2-6	Budding cells with single septa and chains of swollen rounded cells may be present. Occasionally aggregates may be present.
Muriform bodies	Phialophora verrucosa	5-20	Round to pleomorphic thick walled cells with transverse septa, Cells commonly contain two fission plates that form a tetrad of cell, occasionally bhanched septate hyphae
Granules	Fusarium spp.;  and Aspergillus nidulans	200-500	White soft granules with cement like matrix
Sprongia	Rhinisporidium seeberi	6-300	Large thick walled sporangia containing sporangiophore
Wide, non septate hyphae	Zygomycetes	10-30	Hyphae are large, ribbon like, often fractured or twisted. Occasionally septa may present, branching usually at right angle. Smaller hyphae overlap with those of Aspergillus, particularly A. flavus.

6.2 Culture

• Isolation in culture permit must pathogenic fungi to be identified
• The laboratory must be aware of the particular fungal agents that are suspected in a given sample so that the most appropriate media can be included

6.2.1. Culture media:

Specimens are generally inoculated onto primary isolation media, like Sabouraud's dextrose agar and Brain heart infusion agar supplemented with 5% sheep blood. Several media are available.

·         Dixon's Agar for Malassezia furfur

·         Czapek Dox Agar for For the routine cultivation of fungi, especially Aspergillus, Penicillium, and non-sporulating moulds.

·         Bird Seed Agar For the selective isolation of Cryptococcus neoformans.

·         Cornmeal Glucose Sucrose Yeast Extract Agar for Zygomycetes To stimulate sporulation in some zygomycetes, especially Saksenaea and Apophysomyces.

·         Sabouraud's Dextrose Agar with Cycloheximide, Chloramphenicol, Gentamicin and Yeast Extract  for Dermatophytes

·         Potato Dextrose Agar For the routine cultivation and identification of fungi.

·         Brain Heart Infusion Agar (BHIA) with 5% Sheep Blood For the primary isolation and cultivation of yeasts and moulds.

·         Cornmeal Agar  For the routine cultivation and identification of fungi.

·         Malt Extract Agar For the routine cultivation and identification of fungi.

·         Sabouraud's Dextrose Agar with Chloramphenicol and Gentamicin For the primary isolation and cultivation of yeasts and moulds.

·         Lactrimel Agar For the production of pigment by Trichophyton rubrum

·         Hair Perforation Test for dermatophytes

·         Urease Agar with 0.5% Glucose

·         Trichophyton Agars Nos 2-7For the differentiation of Trichophyton species.

·         Littman Oxgall Agar For routine inoculation of specimens from skin, nails, hair etc.

·         Sabouraud's Dextrose Agar with 5% NACL For the cultivation and differentiation of dermatophytes especially T. rubrum from T. mentagrophytes.

·         CGB (L-Canavanine, glycine, 2 bromthymol blue) Agar For distinction between Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii.

6.2.2 Inoculation, Culture condition                       

6.2.2.1 Cerebrospinal Fluid (CSF):

·         The specimen should be centrifuged. Keep the supernatant for cryptococcal antigen testing and process the sediment as follows.

·         For direct microscopy use 1 drops of the sediment to make an India ink mount.

·         Resuspend the remaining sediment in 1-2 ml of CSF and inoculate onto; (a) SDA with chloramphenicol and gentamicin and incubate duplicate cultures at 26˚C and 35˚C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35˚C. Maintain cultures for at least 4 weeks.

6.2.2.2 Sputum, Bronchial Washings and Throat Swabs

·         Unless it is already sufficiently fluid, sputa may need to be emulsified by shaking with about 3-5ml of sterile distilled water, depending on the volume of the original specimen. Any bits of blood, pus or necrotic material should be plated directly onto media.

·         Make wet mount preparations in KOH (l drop) and Gram stained smears (l drop) of all suspicious areas. The PAS stain may be necessary if the KOH preparation is unsatisfactory.

·         Inoculate sample onto: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26˚C and 35˚C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35˚C. Maintain cultures for 4 weeks.

6.2.2.3 Skin Scrapings and Swabs

Skin scrapings, nail scrapings and epilated hairs where tinea is the provisional diagnosis:

·         Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.

·         Inoculate specimen onto two slopes containing cycloheximide i.e. one DERMASEL agar slope and one LACTRITMEL agar slope also containing chloramphenicol, gentamicin and incubate cultures at 26˚C. Maintain cultures for 4 weeks.

·         Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26˚C. Maintain cultures for 4 weeks.

Skin scrapings and swabs where candidiasis is the provisional diagnosis:

A. Skin scrapings:

·         Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.

·         Inoculate specimens onto Sabouraud's dextrose agar slopes containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35˚C. Maintain cultures for 4 weeks.

B. Skin swabs:

·         Smear swab onto heat sterilized glass slide for Gram stain.

·         Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35˚C. Maintain cultures for 4 weeks.

·         Where secondary bacterial infection is suspected, and separate swabs for routine bacteriology were not collected, the swab should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures should be incubated at 35˚C. Maintain cultures for 4 weeks.

Scrapings from the groin, feet or nails where either a dermatophyte or Candida species may be isolated. This includes the possibility of a non-dermatophyte onychomycosis.

·         Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.

·         Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide (as for Candida) and incubate at 26C. Maintain cultures for 4 weeks.

·         Inoculate specimen onto a DERMASEL agar slope containing cycloheximide, chloramphenicol and gentamicin and incubate cultures at 26˚C. Maintain cultures for 4 weeks.

·         Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.

Skin scrapings from patients with suspected Pityriasis versicolor:

·         Direct Microscopy: Wet mount preparation in KOH for direct microscopy along with the cellotape stripping taken at the time of collection.

·         Inoculate scrapings onto a DIXON'S agar slope for isolation of Malassezia furfur and incubate cultures at 26˚C. Maintain cultures for 4 weeks.

·         Inoculate specimen onto Sabouraud's dextrose agar with chloramphenicol and gentainicin but NO cycloheximide and incubate cultures at 26˚C. Maintain cultures for 4 weeks.

·         If dermatophytes are to be excluded also inoculate onto DERMASEL agar slope and incubate cultures at 26˚C. Maintain cultures for 4 weeks.

Skin scrapings from patients where a systemic pathogen is suspected:

·         Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.

·         Inoculate specimens onto: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin but NO cycloheximide and incubate duplicate cultures at 26˚C and 35˚C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35˚C. Maintain cultures for 4 weeks.

 

6.2.2.4 Blood and Bone Marrow

·         Special media are necessary for the optimum recovery of fungi.

·         Numerous blood culture systems are available; however all systems must be vented to atmospheric air and incubated at 30˚C to maximize the rate and time of recovery of fungal organisms.

·         Prepare several smears for Giemsa, Gram and PAS staining. Culture the remaining specimen by one or more of the following methods. With bone marrow aspirates the initial material is generally used for making smears for Giemsa staining, the remaining 3-5 ml of marrow and blood may be cultured on the media listed below.

·         Cultures should be maintained for 4 weeks.

·         Direct culture method: Inoculate 0.5-1.0 ml of buffy coat, prepared by centrifuging 5-10 ml of blood, onto the surface of the media. This inoculum can then be spread over the surface of the agar with a sterile inoculating loop and the plate incubated aerobically at 30˚C.

·         Biphasic culture bottle: A ratio of 1:10 to 1:20 (blood to broth) is recommended, a minimum of 5.0 ml of blood is required for each culture bottle. The biphasic culture bottle is kept vented and is tilted daily to allow broth to flow over the agar surface. These cultures must be carefully checked daily for growth. Because fungi will not turn the broth very cloudy it is imperative to frequently Gram stain the bottle contents to detect fungal elements.

·         Membrane filter technique: Briefly, specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane. This membrane is then placed onto the media.

·         Lysis centrifugation isolator system: The Isolator utilizes a tube that contains components that lyse leukocytes and erythrocytes and also inactivate plasma complement and certain antibiotics. Once lysed, the cells release the microorganisms contained within them, and the centrifugation step in the procedure serves to concentrate the organisms in the blood sample. This concentrate is then inoculated onto the surface of appropriate culture media.

·         Bactec: Bactec have produced a special fungal media (BACTEC Fungal Medium) for enhanced fungal blood culture using their non-radiometric (NR) instruments. Once again, blood cells are lysed by the medium to enhance recovery of fungi. Note antimicrobials have also been added to limit the growth of bacteria.

·         Primary isolation media for blood and bone marrow culture: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26˚C and 35˚C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35˚C. Maintain cultures for 4 weeks.

6.2.2.5 Tissue Biopsies from Visceral Organs

·         If areas of pus and necrosis are present, inoculate directly onto the isolation media. Also perform a smear for direct microscopic examination.

·         If there are no areas of pus or necrosis then process the specimen by mincing it into pieces as small as possible with a sterile scalpel blade, or for soft tissues by grinding in a sterile glass tissue grinder and inoculate onto the media.

·         Warning: zygomycetous fungi will not survive the chopping up or tissue grinding process.

·         For direct microscopy examination of tissue sections stained with H&E, GMS & PAS is essential. Direct smears may also be made by smearing a small amount of tissue onto slides for Gram stain.

·         Inoculate onto: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26˚C and 35˚C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35˚C. Maintain cultures for 4 weeks.

·         Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26˚C for 4 weeks to exclude the presence of a slow growing fungus.

 

6.2.2.6 Urine

·         Yeasts recovered from routine urine bacteriology cultures of catheterized urine or urine obtained by sterile procedure should be identified and reported regardless of colony count. However, the isolation of yeasts from clean catch specimens must be interpreted with caution and is not significant without additional support from other clinical and laboratory investigations.

·         Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26˚C for 4 weeks to exclude the presence of a slow growing fungus.

·         Centrifuge the urine for 10-15 minutes at 2000 rpm. Decant the supernatant and pool the sediment if necessary.

·         Prepare a direct smear of the sediment in KOH for direct microscopy. Note PAS, Gram or India ink preparations may also be helpful.

·         Inoculate 0.05-0.1 ml of the sediment onto Sabouraud's agar with gentamicin and chloramphenicol and incubate duplicate cultures at 26˚C & 35˚C. Maintain cultures for 4 weeks.

6.3 Identification of culture: Key Features include Microscopic Morphology and Culture Characteristics:

Blastomyces dermatitidis

·         On Sabouraud's dextrose agar at 25˚C, colonies are variable in both morphology and rate of growth. They may grow rapidly, producing a fluffy white mycelium, or slowly as glabrous, tan, non-sporulating colonies.

·         Microscopically, hyaline, ovoid to pyriform, one-celled, smooth-walled conidia (2-10 um in diameter) are borne on short lateral or terminal hyphal branches.

·         On blood agar at 37˚C, colonies are wrinkled and folded, glabrous and yeast-like. Microscopically, the organism produces the characteristic yeast phase as seen in tissue pathology. B. dermatitidis can be described as a dimorphic fungus.

Coccidioides immitis/posadasii complex

·         The two species are morphologically identical and can be distinguished only by genetic analysis and different rates of growth in the presence of high salt concentrations (C. posadasii grows more slowly).

·         C. immitis is geographically limited to California’s San Joaquin Valley region, whereas C. posadasii is found in the desert regions of the USA southwest, Mexico and South America. The two species appear to coexist in the desert regions of the USA southwest and Mexico.

·         Colonies of C. immitis/posadasii on Sabouraud’s dextrose agar at 25˚C are initially moist and glabrous, but rapidly become suede-like to downy, greyish white with a tan to brown reverse, however considerable variation in growth rate and culture morphology has been noted.  

·         Microscopy shows typical single-celled, hyaline, rectangular to barrel-shaped, alternate arthroconidia, 2.5-4 x 3-6 µm in size, separated from each other by a disjunctor cell.  

 

Histoplasma capsulatum

·         H. capsulatum exhibits thermal dimorphism by growing in living tissue or in culture at 37˚C as a budding yeast-like fungus or in soil or culture at temperatures below 30˚C as a mould.

·         On Sabouraud's dextrose agar at 25˚C, colonies are slow growing, white or buff-brown, suede-like to cottony with a pale yellow-brown reverse. Other colony types are glabrous or verrucose, and a red pigmented strain has been noted.

·         Microscopic morphology shows the presence of characteristic large (8-14 um in diameter), rounded, single-celled, tuberculate macroconidia formed on short, hyaline, undifferentiated conidiophores. Microconidia, if present, are small (2-4 um in diameter), round to pyriform and borne on short branches or directly on the sides of the hyphae.

·         On brain heart infusion (BHI) blood agar incubated at 37˚C, colonies are smooth, moist, white and yeast-like. Microscopically, numerous small round to oval budding yeast-like cells, 3-4 x 2-3 um in size are observed.

·         Three varieties of H. capsulatum are recognized, depending on the clinical disease: var. capsulatum is the common histoplasmosis, var. duboisii is the African type and var. farciminosum causes lymphangitis in horses. Histoplasma isolates may also resemble species of Sepedonium and Chrysosporium.

Paracoccidioides brasiliensis

·         On Sabouraud's dextrose agar at 25˚C, colonies are slow growing and variable in morphology. Colonies may be flat, wrinkled and folded, glabrous, suede-like or downy in texture, white to brownish with a tan or brown reverse.

·         Microscopically, a variety of conidia may be seen, including pyriform microconidia, chlamydoconidia and arthroconidia. However, none of these are characteristic of the species, and most strains may grow for long periods of time without the production of conidia.

·         On BHI blood agar at 37˚C, the mycelium converts to the yeast phase and colonies are white to tan, moist and glabrous and become wrinkled, folded and heaped. Microscopically, numerous large, 20-60 um, round, narrow base budding yeast cells are present. Single and multiple budding occurs, the latter are thick-walled cells that form the classical "steering wheel" or "Mickey mouse" structures that are diagnostic for this fungus, especially in methenamine silver stained tissue sections.

Hyaline Hyphomycetes

·         Hyaline Hyphomycetes include those conidial fungi which are not darkly pigmented; colonies may be colorless or brightly colored.  These include the agents of hyalohyphomycosis, Aspergillosis, dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum.

·         Identification of the hyphomycetes is primarily based on microscopic morphology including; (a) conidial morphology, especially septation, shape, size, color and cell wall texture; (b) the arrangement of conidia as they are borne on the conidiogenous cells (c) the type conidiogenous cell

·         Culture characteristics, although less reliable may also be useful.  These include surface texture, topography and pigmentation, reverse pigmentation and growth at 37˚C.  For identification, potato dextrose agar and cornmeal agar are two of the most suitable media to use and exposure to daylight is recommended to maximize culture color characteristics.

·         Mandatory to see conidial characteristics to make an identification therefore must have a good slide preparation [needle mounts, tape mounts, slide cultures].  May also need to stimulate sporulation by using different media, such as potato dextrose agar or cornmeal agar.  

 

Aspergillus spp.

·         Aspergillus colonies are usually fast growing, white, yellow, yellow-brown, brown to black or shades of green, and they mostly consist of a dense felt of erect conidiophores.

·         Conidiophores terminate in a vesicle covered with either a single palisade-like layer of phialides (uniseriate) or a layer of subtending cells (metulae) which bear small whorls of phialides (the so-called biseriate structure).  The vesicle, phialides, metulae (if present) and conidia form the conidial head.  Conidia are one-celled, smooth- or rough-walled, hyaline or pigmented and are basocatenate, forming long dry chains which may be divergent (radiate) or aggregated in compact columns (columnar). 

·         For identification, isolates are usually inoculated at three points on Czapek dox agar and potato dextrose agar and incubated at 25˚C.  Most species sporulate within 7 days.

·         Descriptions are primarily based on colony pigmentation and morphology of the conidial head.  Microscopic mounts are best made using a cello tape flag or slide culture preparation mounted in lactophenol cotton blue.  A drop of alcohol is usually needed to detach the cellotape flag from the stick, and to act as a wetting agent.

·         Key Features: Hyaline hyphomycete showing distinctive conidial heads with flask-shaped phialides arranged in whorls on a vesicle.

 

Fusarium spp.

·         Colonies are usually fast growing, pale or brightly colored (depending on the species) and may or may not have a cottony aerial mycelium. The color of the thallus varies from whitish to yellow, brownish, pink, reddish or lilac shades.

·         Species of Fusarium typically produce both macro- and microconidia from slender phialides. Macroconidia are hyaline, two- to several-celled, fusiform to sickle-shaped, mostly with an elongated apical cell and pedicellate basal cell. Microconidia are 1- to 2-celled, hyaline, pyriform, fusiform to ovoid, straight or curved. Chlamydoconidia may be present or absent.

·         Identification of Fusarium species is often difficult due to the variability between isolates (e.g. in shape and size of conidia and colony color) and because features that are required are not always well developed (e.g. the absence of macroconidia in some isolates after subculture). .

Dematiaceous Hyphomycetes

ü  Dematiaceous Hyphomycetes are those conidial fungi that produce dark brown, green-black, or black colonies and are the causative agents of phaeohyphomycosis.  These includes: Acrophialophora, Alternaria, Aureobasidium, Bipolaris, Cladophialophora, Cladosporium, Curvularia, Drechslera, Epicoccum, Exophiala, Exserohilum, Fonsecaea, Hortaea, Lecythophora, Ochroconis, Phaeoacremonium, Phialophora, Pithomyces, Ramichloridium, Rhinocladiella, Scedosporium, Sporothrix, Stemphylium, Ulocladium, Veronaea

Dermatophytes

·         Microscopic morphology of the micro and/or macroconidia is the most reliable identification character, but needs good slide preparation and to stimulate sporulation in some strains.

·         Culture characteristics such as surface texture, topography and pigmentation are variable and are therefore the least reliable criteria for identification.

·         Clinical information such as the site, appearance of the lesion, geographic location, travel history, animal contacts and race is important, especially in identifying rare non-sporulation species like M. audouini, T. concentricum and T schoenleinii etc.

·         Epidermophyton:
Smooth thin-walled Macroconidia only present, no microconidia, colonies a green-brown to khaki colour.

·         Microsporum:
Macroconidia with rough walls present, microconidia may also be present.

·         Trichophyton:
Microconidia present, smooth-walled macroconidia may or may not be present.

 

Sporothrix schenckii

·         At 25˚C, colonies are slow growing, moist and glabrous, with a wrinkled and folded surface. Some strains may produce short aerial hyphae and pigmentation may vary from white to cream to black. 

·         Conidiophores arise at right angles from the thin septate hyphae and are usually solitary, erect and tapered towards the apex.  Conidia are formed in clusters on tiny denticles by sympodial proliferation of the conidiophore, their arrangement often suggestive of a flower. 

·         As the culture ages, conidia are subsequently formed singly along the sides of both conidiophores and undifferentiated hyphae.  Conidia are ovoid or elongated, 3-6 x 2-3 um in size, hyaline, one-celled and smooth-walled.

·         In some isolates, solitary, darkly pigmented, thick-walled, one-celled, obovate to angular conidia may also be observed along the hyphae.

·         On BHI blood agar at 37˚C, colonies are glabrous, white to greyish yellow and yeast-like, consisting of spherical or oval budding yeast cells.

 

Yeasts (Blastoschizomyces, Candida, Cryptococcus, Loboa loboa, Malassezia, Rhodotorula, Saccharomyces, Trichosporon)Identification.

·         Yeast-like fungi may be basidiomycetes, such as Cryptococcus neoformans or ascomycetes such as Candida albicans.

ü  Germ Tube Test: lightly inoculated 5 ml of serum, containing 0.5% glucose and incubated at 35˚C for 2-3 hours. 

ü  Positive = Candida albicans or Candida dubliniensis.

• Negative or from HIV positive patient = perform assimilation tests.

·         For the identification of germ tube negative yeasts, morphological (Dalmau plate culture), physiological and biochemical tests are essential.

 

Candida spp.

·         The genus Candida is characterized by globose to elongate yeast-like cells or blastoconidia that reproduce by multilateral budding.

·         Most Candida species are also characterized by the presence of well developed pseudohyphae, however this characteristic may be absent, especially in those species formally included in the genus Torulopsis.

·         Within the genus Candida, fermentation, nitrate assimilation and inositol assimilation may be present or absent, however, all inositol positive strains produce pseudohyphae.

·         CHROMagar Candida plate showing chromogenic colour change for C. albicans (green), C. tropicalis (blue), C. parapsilosis (white) and C. glabrata (pink).

• Candida albicans, Candida colliculosa, Candida dubliniensis, Candida fabianii, Candida famata, Candida glabrata, Candida guilliermondii, Candida haemulonii, Candida inconspicua, Candida keyfyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida norvegensis, Candida parapsilosis, Candida pelliculos, Candida rugosa, Candida tropicalis, Candida viswanathii

 

Cryptococcus

·         The genus Cryptococcus is characterized by globose to elongate yeast-like cells or blastoconidia that reproduce by multilateral budding. Pseudohyphae are absent or rudimentary.

·         On solid media the cultures are generally mucoid or slimy in appearance.  Red, orange or yellow carotenoid pigments may be produced, but young colonies of most species are usually non-pigmented, and are cream in color.

·         Most strains have encapsulated cells with the extent of capsule formation depending on the medium.  Under certain conditions of growth the capsule may contain starch-like compounds which are released into the medium by many strains.

·         Within the genus Cryptococcus, fermentation of sugars is negative, assimilation of nitrate is variable and assimilation of inositol is positive. The genus Cryptococcus is similar to the genus Rhodotorula. The distinctive difference between the two is the assimilation of inositol, which is positive in Cryptococcus.

• Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus neoformans, Cryptococcus gattii

 6.3 Mycoserology

• Useful in early diagnosis of systemic fungal infection.
• Agglutination, precipitation, complement fixation and enzyme immunoassay are widely used.
• Detection of antibodies

ü  Includes most of the currently available standardized procedures

ü  Limited value in diagnosing imunocompromized patients.

ü  Useful in diagnosing fungal disease, such as histoplasmosis and coccidioidomycosis.

• Detection of antigens is now becoming more widely available. Stablished for diagnosis of cryptococcosis and histoplasmosis and being evaluated for other fungi.
• Antigen detection methods are complicated

ü  Often released in minute amount necessitating the use of highly sensitive test procedure

ü  Often cleared very rapidly necessitating frequent collection of samples

ü  Often bound to circulating IgG

• The result of the serological testing are seldom more than suggestive or supportive of a fungal diagnosis and must be interpreted with clinical and laboratory finding.

Disease	Test antigens	Interpration
Aspergillosis	CF (Aspergillin) A. fumigatus, A. niger and A. flavus ID, aspergillin	Titre ≥1:32 is suggestive of infection Higher titer have a stronger correlation with disease Multiple precipitation bands increase the suspicion of active disease. Positive in 95% of cases with fungus ball, 50% with bronchopulmonarry disease. May be positive in invasive disease rarely.
Blastomycosis	Cf, yeast form ID, yeast culture filtrate EIA, extract of B. dermatidis	Titre of ≥1:8 are significant. Higher titer are more indicative of disease. Cross reaction occur with histoplasmosis and coccidioidomycosis. High percentages of cases are negative. Specific band for A antigen. Many culture proven cases are negative. A positive relative value suggests disease. Sensitive than ID
Candidiasis	ID, C albicans sonicate LA, yeast cytosol or mannan	Most cases have bands, however high percentage of normal populations have antibody to C. albicans ≥1:4 or ≥1:8 are considered positive, difficult to interpret, most have clinical correlation
Cryptococcosis	LA, Ab to capsular polysaccharide EIA, Ab to capsular polysaccharide	Detects cryptococcal capsular polysaccharide. Any titer significant. RF causes false positive.  More sensitive than LA
Histoplasmosis	CF,histoplasmin (mycelia) CF, yeast cell ID histoplasmin EIA, H. capsulatum	≥1:8 is significant. Higher titer paralles more severity. ≥1:8 is significant. Higher titer paralles more severe disease. May cross react with blastomyces and coccidioidomycosis Specific band on H and M antigen. M bands usually appear first in disease and persist. H band is associated with active disease & often dissociates with active therapy. A positive relative EIA value indicates disease. Tests for IgG. More sensitive than ID.
Sporotrichosis	LA yeast	Titre ≥1:80 usually indicate active disease. Usually negative with systemic sporotricosis.

6.3 Histopathological examination of tissue section

• Most reliable procedure for the diagnosis of subcutaneous and deep seated fungal infection.
• In addition to culture and serology histopathology is the important means of diagnosis of fungal infection.
• When the mycosis is not suspected, whole specimen is often fixed for histopathology, in such cases histopathology is the only means of fungal diagnosis.
• Recognition of fungal pathogen in tissue specimen depends on its abundance and distinctiveness in appearance.
• Stains

ü  Hematoxylin and eosin-may be insufficient to reveal fungal elements.         

ü  Methenamine silver (Gomori/ Grocott) and Periodic acid Schiff are specific

·         Fungal genus may not be identified in all cases only by staining.

·         Histopathological studies can sometimes confirms the possibility of multiple infections or exclude a fungal disease from clinical differential diagnosis.

·         Immunoperoxidase and immunoflourescence staining are available for some fungi.

·         DNA probe based methods are under investigation.

6.5 Molecular diagnosis

• Numerous methods: for the detection of fungal nucleic acid in clinical specimens (esp. Blood, CSF, Respiratory specimens etc).
• Polymerase chain reaction based methods are common and widely used.

ü  Conventional PCR, Multiplex PCR, Nested PCR,  Real time PCR

• Several regions within the genome have been evaluated as potential targets: mostly ribosomal DNA gene complex (18S, 5.8 S and 28S).

SOME PRACTICAL TECHNIQUE

Preparation of 20% KOH – glycerol solution:

Potassium hydroxide is a strong alkali used as a clearing agent to observe fungi in the specimen in a wet mount preparation for specimen such as sputum, pus, urine sediment, homogenate from biopsy tissue, nail, hair, etc., to clear cell debris. The formula consists of

• KOH – 20 g
• Glycerol – 20 ml
• Distilled water – 80 ml

(1)   The relative amounts of KOH and distilled water must be adjusted according to the percentage of KOH used.

(2)   Addition of glycerol to KOH solution will prevent crystallization occurring in the solution, thus enhancing the shelf life of this reagent. It will also permit keeping the KOH preparation for a couple of days before it drys.

Preparation of Sabouraud dextrose agar (SDA):

This modification differs from the original formula in that it has an approximately neutral pH and contains only 2% dextrose. The original medium (with high sugar, up to 40% and low pH of 5.4) devised by Raymond Sabouraud is more suitable for isolation and study of dermatophytes.

• Dextrose – 20 g
• Neopeptone – 10 g
• Agar – 15 g
• Distilled water-1000 ml.

Preparation: The ingredients are mixed and dissolved by boiling and then autoclaved. After autoclaving dispence in sterile petriplates of fungul cultute bottle (make slant). This formulation is available in prepared or dehydrated form. The final pH is around 6.8 (near neutral).

Storage: The prepared medium can be stored at 4˚C.

KOH mounts preparation:

Potassium hydroxide is a strong alkali used as a clearing agent to observe fungi in the specimen in a wet mount preparation. 10%-20% KOH is usually used depending on the specimen; occasionally 40% is also used when not cleared by 10%-20% KOH. Used for specimen such as sputum, pus, urine sediment, homogenate from biopsy tissue, nail, hair, etc., to clear cell debris.

Preparation of the mount

(1)   Take a clean grease-free glass slide.

(2)   Place a large drop of KOH solution with a Pasteur pipette.

(3)   Transfer small quantity of the specimen with a loop or the tip of a scalpel into the KOH drop.

(4)   Put a clean cover slip over the drop gently so that no air bubble is trapped.

(5)   Place the slide in a moist chamber, and keep at room temperature.

(6)   Tissue usually takes 20-30 minutes; sometimes overnight contact with KOH is useful for getting a positive result.

(7)   Clearing can be hastened by gentle heating of the slide, but it is best avoided.

Observation

1.      Examine the clear specimen under low power (10X or 20X objective). Scan the entire cover slip from end to end in a zigzag fashion.

2.      If any fungal elements are suspected, examine under high power (40X objective).

3.      Reduce the light coming into the condenser while examining at high power.

4.      Look for budding yeast cells, branching hyphae, type of branching, the colour, septation and thickness of hyphae.

Modification

For more distinction, stains like methylene blue or Parker blue-black fountain ink may be used along with KOH. This will impart a colored background and fungal elements, if present, will show as prominent refractile objects.

Advantages

·         Simple, economical and rapid

Disadvantages

·         Pus and sputum may contain artifacts, which may superficially resemble hyphal and budding forms of fungi. These artifacts may be produced by cotton or wool fibres, starch grains (in pleuritis) or cholesterol crystals.

·         It gives an idea about the presence of hyphal element, but cannot distinguish different fungi.

·         Preparation cannot be kept for too long; but drying can be prevented/prolonged by keeping the slides in a moist chamber (covered Petri dish with a wet filter paper on which a triangular glass rod is placed).

Precautions

(1)   The drop of KOH should not be so large that the cover slip floats.

(2)   If kept outside a moist chamber, the KOH dries and crystals form that restrict the visibility of the fungus.

(3)   After clearing, pressure is to be gently applied on the top of the cover slip with a fold of filter paper or the handle of a teasing needle. This ensures even spreading of the material onto the slide.

(4)   KOH should be kept in a closed container in small aliquots ready to use on the workbench.

Quality assurance

Fungal spores or hyphae may contaminate the KOH solution kept in the laboratory and may give false positive results. A negative control should therefore be put up every day.

India ink or nigrosin preparation for C. neoformans

It is the simple methods to identify the capsulated varieties of Cryptococcus neoformans.

Procedure:

(1)   The preparation should be made in the centre of a clean, grease free glass slide.

(2)   Put 1 drop of India ink or Nigrosin on the centre of the slide. Too much stain makes the background too dark. (Upon examination, if the staining appears too dark, a small amount of water may be applied on the edge of the cover slip and the cover slip gently tapped: this will dilute the stain to some extent.)

(3)   Put 1 loopful of the specimen or preferably centrifuge sediment from the fluid specimen to be tested (e.g. CSF, spinal fluid, urine, and other body fluids) close to the drop of the stain.

(4)   Mix the two well with the loop, or preferably a sterile needle. The loop should be cooled before use; otherwise the stain tends to precipitate.

(5)   Hold the cover slip vertically such that one edge just touches the fluid on the slide. The fluid will spread along the edge by surface tension.

(6)   Keeping that edge in contact with the fluid surface, drop the cover slip gently on the fluid, so that no air bubble is trapped inside. If there are air bubbles, the surface of the cover slip may be gently tapped by the needlepoint, so as to move the bubbles towards the edge. But this should be avoided as far as possible.

(7)   Examine slide immediately under the microscope. Since the stain tends to dry fast in air, if immediate examination is not feasible, the slide should be kept in a moist chamber.

Observation:

Scan the entire cover slip from end to end in a zigzag fashion. Encapsulated yeast cells (Cryptococcus neoformans) are seen under low power as luminous dots in an otherwise dark background. Under high power, the cells can also be seen, containing refractile bodies, and surrounded by the unstained thickness of the capsule. Characteristic pinched-off budding, when observed, is confirmatory for diagnosis.

Please note

§  Besides the classical budding-yeast form, various unusual forms can also be seen, including elongated forms that look like pseudohyphae; this is mainly due to a very high multiplication rate of the organism in HIV/AIDS patients.

§  In the very late stage in progressive AIDS, it may be difficult to differentiate the capsules of individual cells; the organism may remain enmeshed in a matting of the capsular material.

§  Micro- or non-capsulated strains of the organism are also reported on rare occasions. In such cases, gram stain of the sample is helpful in identification.

§  The edges of the cover slip should be specially examined. When placing the cover slip, the yeast cells tend to move towards the periphery along with the fluid. For this reason the common practice of draining the extra fluid from the sides should be avoided.

§  If the protein content of the CSF is too high, India ink sometimes may form floccules, which make it difficult to demonstrate the capsule.

§  In the case of Nigrosin stain, the preparation dries up quite quickly, which is a problem in hot climatic conditions, so quick examination is essential.

§  In case of HIV-positive patients, > 90% of cases may be positive by the India ink/Nigrosin test, whereas in non-HIV cases, < 60 % positivity is seen.

Precautions

• The India ink or Nigrosin should be shaken well before every wet mount preparation.
• The stain should be regularly checked for contamination by examining just the stain under a microscope.
• False positive readings may occur with air bubbles or monocytes or neutrophils. Air bubbles, under high power, will be hollow and will not show the typical cell with characteristic nuclei. Monocytes and neutrophils have a crenated margin (and not the entire margin seen in cryptococcal cell) and will not show the characteristic refractive cell inclusions, and the luminous halo around the cell is not well demarcated.

Rapid identification by germ tube (GT) test

The germ tube test is used for presumptive identification of Candida albicans. It is a rapid screening test wherein the production of germ tubes within 2 hours in contact with the serum is considered as indicative of Candida albicans. This test must be validated with a CMA (corn meal agar) test.

Procedure

(1)   Ensure that the test starts with a fresh growth from a pure culture.

(2)   Make a very light suspension of the test organism in 0.5 ml of sterile serum (pooled human serum or fetal calf serum). The optimum inoculum is 10⁵-10⁶ cells per ml.

(3)   Incubate at 37˚C for exactly 2 hours.

(4)   Place 1 drop from the incubated serum on a slide with a cover slip. Observe under the microscope for production of GTs.

Observation: Germ tubes represent initiation of hyphal growth, arising directly from the yeast cell. They have parallel walls at their point of origin and are not constricted. To record a positive, about 30% of the cells should show GT production.

Quality control: Suitable controls should be kept with each test; a known strain of Candida albicans should be tested with each new batch of serum. C. albicans and C. tropicalis are run with each group of GT determinations to serve as positive and negative controls respectively.

Precautions:

(1)    The medium, inoculum size, temperature of incubation, concentration of simple carbohydrates and microaerobic conditions influence GT formation.

(2)    Increased concentration of inoculum causes a significant decrease in the percentage of cells forming GTs. Maximum percentage of GT formation occurs when 10⁵-10⁶ cells/ml are used as inoculum. As the concentration of cells increases, the percentage of GT formation decreases. A faintly turbid serum suspension is ideal for maximum GT development.

(3)    Approximately 95% of clinical isolates of C. albicans produce GTs when incubated in serum at 37˚C for 2-3 hours.

(4)    A neutral pH (7.4) facilitates maximum development of germ tubes. Bacterial contamination may interfere with production of germ tubes.

(5)    Antimicrobial substances in the isolation medium may interfere with this test; human serum may contain inhibitory substances (such as ferritin) that suppress GT development.