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Author: Dr. Shruti Nayak, Dr. Amarnath Shenoy, Dr. U. S. Krishna Nayak.

Abstract:
Fungi are diverse group of algae. They are found in varied environments like tropical forests, oceans, and deserts. Various fungi adapt to Ph extremes and are found in hot and cold climates, although they are not as heat or cold resistant as prokaryotes. A pathogenic fungus belongs to the plant kingdom, possesses no chlorophyll, and so has to depend on organic matter for energy. Opportunistic invasive fungal infections remain an important cause of morbidity and mortality. Fungi have been implicated directly as agents of human or animal disease, and fewer than a dozen of these species cause about 90% of all fungus infections. Early and accurate diagnosis of these infections is important for several reasons, including timely institution of anti fungal therapy and to decrease the unnecessary use of toxic antifungal agents.

Introduction:
The fungi are saprophytic (derives nourishment from dead organic matter) and parasitic eukaryotic organisms. Although formerly considered to be plants, they are now generally assigned their own kingdom, Mycota.1They resemble plants because they have rigid cell walls and are non motile. Unlike plants, the fungi lack chlorophyll and are unable to photosynthesize. The fungi also lack the multicellular complexity and organization of most animals. 2

The biological kingdom of the fungi is composed of approximately 70,000 known species of fungi. Fewer than 300 species of fungi have been implicated directly as agents of human or animal disease, and fewer than a dozen of these species cause about 90% of all fungus infections. 3

Historically, the fungi were regarded as relatively insignificant causes of infection. It is now well documented that the fungi are the common cause of infection, particularly in immunocompromised patients.²

Opportunistic invasive fungal infections remain an important cause of morbidity and mortality. The most common fungi that cause disease in transplant recipients and other immunocompromised patients are Candida and Aspergillus species.4 There are several approaches to manage these infections, including prevention, anti-fungal prophylaxis, and empiric therapy. The first essential component of the treatment is diagnosis. The diagnosis of deep seated Candidiasis is difficult and often depends primarily on the clinical picture, with definitive diagnosis established by histopathology of visceral tissues. 4

Characterstics Of Fungi
General Features Fungi:
Fungi seen in the clinical laboratory can generally be separated into two groups based on the appearance of colonies formed. The yeasts produce moist, creamy opaque or pasty colonies on media, whereas the filamentous fungi or molds produce fluffy, cottony, wooly or powdery colonies. Several pathogenic species of fungi that exhibit either yeast (or yeast like) and filamentous form are referred to as being dimorphic. The dimorphism is temperature dependent; the fungi are designated as thermally dimorphic.5,6

Morphology/Structure Of The Fungi:
With the notable exception of yeasts, fungi consist of masses of intertwined filaments of cells called hyphae. In many species of fungi, individual cells are separated by cross-wall, or septa, such fungi are described as septate. The septa are incomplete, however, and pores allow adjacent cytoplasm to mix. In other fungal species, the cells have no septa, and the cytoplasm and organelles of neighboring cells mingle freely. These fungi are said to be coenocytic. The common bread mould Rhizopus stolonifer is coenocytic, while the blue-green mould that produces penicillin, penicillim notatum, is septate.

The hypha is the morphological unit of fungus and is visible only with the aid of a microscope. Hyphae have broad diversity of forms and many hyphae are highly branched with reproductive structures. A thick mass of hyphae is called a mycelium. This mass is usually large enough to be seen with the unaided eye, and generally it has a rough cottony texture. The study of fungi is called mycology, and a person who studies fungi is called a mycologist. 5,6

Yeasts:
Yeasts are unicellular organisms that are round to oval and range in size from 2 to 60μm.The microscopic morphologic features usually appear similar for different genera and are not particularly helpful in their separation. Fungi grow in two basic morphologic forms, as yeasts or moulds.6

Molds (Moulds):
The mold (or mould) form of growth refers to the production of multicellular, filamentous colonies. These colonies consist basically of branching cylindrical tubules varying in diameter from 2 to 10 am and termed hyphae. Hyphal growth occurs by apical elongation. The mass of intertwined hyphae that accumulates during active growth in the mold form is called a mycelium.

Molds are usually identified by observation of their morphology. Macroscopic examination of a mold isolate should include notation of such characteristics as the rate of growth, topography (e.g., glabrous, verrucose), surface texture (e.g., velvety, cottony, powdery) and any pigmentation (surface, reverse, or diffusible into the medium). Microscopically the types of spores or other reproductive structures (pigment, size, shape, mode of attachment) and their ontogeny are characteristic for each species.6

General Considerations For The LAb Daingosis Of fungal Infections

Daignosis Fungal Infections:
There is remarkable change in the pattern of pathogens responsible for serious human infections.7 The frequency of invasive fungal infections has risen dramatically in recent years. Early and accurate diagnosis of these infections is important for several reasons, including timely institution of anti fungal therapy and to decrease the unnecessary use of toxic antifungal agents. In addition the availability of accurate and timely diagnosis could reduce the use of empirical anti fungal therapy, thereby reducing antifungal resistance. 8

Collection And Transport Of the Clinical Specimen
The diagnosis of fungal infections is dependent entirely on the selection and collection of an appropriate clinical specimen for culture. Many fungal infections are similar clinically to mycobacterial infections, and often the same clinical specimen is cultured for both fungi and mycobacteria. It is common for many years, such as Candida species to be recovered in routine bacteriology media and fungal culture media. 9

Specimens Include 9

  • Skin scrapings.
  • Hair and nails.
  • Respiratory tract secretions.
  • Cerebrospinal spinal fluid.
  • Blood.
  • Mouth and vagina.
  • Urine.
  • Pus.
  • Ocular specimen.
  • Tissue.
  • Bone marrow.

Direct Examination of Clinical Specimen 10
Direct microscopic examination of fungal cells within the clinical specimen is a valuable diagnostic procedure for the following reasons:
  • In many instances, a tentative or even a definitive diagnosis can be made before the growth of fungal cells would be apparent in culture.
  • Observing fungal cells in a clinical specimen may be more valuable as a criterion for diagnosis than isolating in a culture.
Preparations for direct examination of clinical specimen include KOH, India ink, and calcoflour white; in addition, a few staining techniques such as Giemsa and periodic acid-Schiff are effective. 10

  • Wet Smears
Preparations With Koh (Unstained):
Patches from the mucous membrane of the mouth, vagina, skin, or nails scrapping, sputum etc are collected in a sterile container. These are examined in a KOH wet mount or gram stain. Yeast cells of 4-8μm with budding mixed pseudohyphae are seen. The presence of pseudohyphae shows colonization and tissue invasion and so their demonstration is significant. For detection of Candida,wet smear microscopy has been positive in the majority, but not in all cases with positive culture. 10

  • Stained Preparations
Preparations With Potassium Hydroxide (Koh):
The specimen should first be examined microscopically for necrotic, purulent, bloody, or caseous areas. Because these are the areas most likely to yield evidence of fungal growth, they are selected for direct examination. Preparation with KOH clears the tissue and cellular debris from all types of clinical specimens without damaging the fungal cells. This clearing process requires only 5 to 10 min, after which one can observe the fungal morphology as well as the pigment of the fungal cell wall.

The disadvantage of using KOH stems from its reaction with pus, sputum, and skin; in these instances, artifacts can be produced that superficially resemble hyphae or budding forms of fungi. The number of these artifacts increases as time after preparation elapses, and experience is required in interpreting the results. KOH can damage the microscope stage if the slide overflows. In addition, crystals can form on standing so that reading of smear becomes difficult.

The slide is examined under a phase-contrast or bright-field microscope, using low-power followed by high-power objectives. With a bright-field microscope, one can detect hyphae and yeast cells more readily by closing the aperture of the iris diaphragm to reduce the intensity of light. Brown-walled hyphal cells can be detected without a reduction in light.

To highlight the fungal cell walls, parker super chrome blue-black ink can be incorporated into the KOH preparation. Adding the dye however will mask the cell-wall pigment of dermatiaceous fungi.

Tentative diagnosis can be derived from the presence of fungal elements compatible to the etiologic agents of aspergillosis, mucormycosis, dermatophytosis, candidiasis, sporotrichosis, or cryptococcosis. To confirm such a diagnosis, however, cultural proof is necessary. 9

Preparations With Calcoflour White And koh :
Calcoflour White is used as a whitening agent in the textile and paper industry. The dye is useful for demonstrating the presence of fungal cells in clinical specimens because it binds to β 1-3, β 1-4 polysaccharides. The dye then fluoresces as it is exposed to the shorter wavelengths of ultraviolet light. A Fluorescence microscope is needed for detecting fungal cells prepared with Calcoflour White.

Yeast cells, pseudohyphae and hyphae display a chalk-white or brilliant apple-green fluorescence, depending on filters used, that readily differentiates them from background material. The disadvantages of using Calcoflour are the need for a Fluorescence microscope, the inability of the dye to detect the endospores within a spherule of Coccidioides immitis, and the difficulty in interpreting vaginal secretions. 9

Preparation With INdia Ink:
India ink is useful for indicating the presence or absence of extra cellular polysaccharide capsules of fungal cells. The technique is particularly helpful for detecting Cryptococcus neoformans in CSF. Because India ink serves as a negative stain, the encapsulated yeast cells can readily be detected against the dark back ground. The ink should be free from artifacts or granular carbon particles to ensure a good preparation.

The presence of encapsulated yeast cells in CSF in almost always an indicator of Cryptococcal meningitis. Making the distinction between encapsulated yeasts and artifacts requires considerable experience. Because tests for antigen also have false-positive results, the tests can be used to complement one another. Every spinal fluid that is India ink positive is also antigen positive, although the converse is not true. Antigen may be detected in absence visible Cryptococci on India ink smear. There are reports of Cryptococcal species other then neoformans in CSF. 9

Preparations With Periodic Acid-schif(PAS) Stain:
The PAS stain one of the most widely used stains for fungal histopathology. In a direct examination of clinical specimen, PAS stain is sometimes used when a KOH preparation do not reveal fungi that are suspected to be present.

PAS preparation requires 20 to 25 minutes depending on the weather counterstaining is employed. Light green is preferred as a counter stain because the fungus appears deep purplish red against the contrasting background color. For the counter staining process the slides should be placed in light green stain for 5 sec and washed for 5 to 10 sec between steps.

The PAS reaction stains certain polysaccharides found in the fungal cell wall. For good results, both the periodic acid solution and the sodium Meta bisulphate solution should be fresh and protected from light. For laboratories with a fluorescent microscope, calcoflour white is preferred over PAS. 9

Acid-Fast Stain Procedure For Nocardia (modified Kinyoun Method):
Acid-fast staining is useful for detecting Nocardia species and for differentiating them from other aerobic actinomycetes. Some of the filaments stain red with carbol- fuschin staining, while others may appear blue because of counterstaining effect.
The slides are then examined under a bright field microscope with oil immersion objective. Viewing tissue homogenate specimens with this method is difficult because of background staining. 9

Grams's Stain:
It is effective for some pathogens but not for others. In general, the procedure is more suited to sections than smears. 11

Gormori's Methenamine Silver Stain (Grocott's Modification):
It is based on the liberation of aldehyde groups and their subsequent identification by the reduced silver method. It is used for demonstration of polysaccharide content on the fungus in tissue sections. The aldehydes reduce the methenemine silver nitrate complex, resulting in brown black staining fungal cell wall due to the deposition of reduced silver wherever aldehydes are located. 11

Inference:
The fungi and bacteria are stained black, mucopolysaccaride dark grey, cytoplasm old rose and the tissue pale green. 11

The GMS is better than other fungal stains as:
  • It stains both live and dead fungi in contrast to PAS which stains only live fungi.
  • It stains the higher bacteria (Nocardia and Actinomycosis) also which are not stained by other fungal stains.

Culture Medias:
All fungi require several specific elements for growth and reproduction. The requirements for growth are generally less stringent than for sporulation, so it is often necessary to try several types of media when attempting to identify a fungus in culture. 12

Common media for primary fungal isolation include Sabouraud dextrose agar and brain-heart infusion agar, either in petri dishes or screwtop tubes. The media may be enriched with 5% to 10% sheep blood to support the growth of certain fungi. Specimens that are likely to be contaminated with other microorganisms, such as urine or sputum, are set up on agar media containing antimicrobials. Chloramphenicol, streptomycin, or penicillin are incorporated into the agar to inhibit the growth of bacteria, and cycloheximide is used to inhibit the growth of contaminant. 13

Most fungi also thrive on Potato Dextrose Agar (PDA), but this can be too rich for many fungi, so that excessive mycelial growth is obtained at the expense of sporulation. 12

Constituents of Media:
Media generally contain a source of carbon, nitrogen and vitamins. Glucose (dextrose) is the most widely utilizable carbon source, and hence is the most commonly used in growth media. Fructose and mannose are the next most commonly utilized sugars by fungi and are found in media from natural sources. Sucrose (table sugar) may be used in some media. Nitrogen sources include peptone, yeast extract, malt extract, amino acids, ammonium and nitrate compounds. Casamino Acids, a Difco product, is acid-hydrolyzed casein, a mixture of amino acids. It is a good general source of nitrogen but is vitamin-free. Bacto-Peptone, another Difco product, contains nitrogen and a high peptone and amino acid content. Salts, including Fe, Zn and Mn, are often added to ‘defined’ media, but are usually not added to the common media used for routine culture. Fungi have natural deficiencies for vitamins that are satisfied at mM to nM concentrations. The most common naturally occurring vitamin deficiencies are thiamin and biotin. Deficiency of both is quite common among the Ascomycota. Other organic nutrients such as glucose are often contaminated with vitamins sufficient to supply the growth requirements of fungi. 12

Culture media used for fungal growth: 14,15
The most commonly used cultures media used for fungal growth are as follows:
  • Sabouraud agar
  • Hay infusion agar
  • Potato dextrose agar
  • Potato Dextrose Broth
  • Yeast Agar
  • Yeast Broth
  • Mycological Agar
  • Malt extract agar
  • Malt Extract Broth
  • Mycological Agar
  • Soy Peptone Yeast Extract Agar
  • Water Agar (WA)
  • Antibiotic Agar (AA)
  • Acidified Cornmeal Agar (ACMA)
  • Cornmeal Agar (CMA)
  • Potato Carrot Agar (PCA)

Special Tests:

  • Chlamydospore Formation For Candida
  • Inoculate suspected Candida on plates or slides with Chlamydospore agar. Inoculate by cutting angles into medium. Known culture of Candida is used as culture, and multiple cultures can be tested on the plate.
  • Incubate at room temperature for 48 to 72 hrs. Examine slides, plates or slide mounts from plates, under microscope for presence of Chlamydospore and characteristic growth of mycelia. C albicans produces abundant chlamydospores in the lateral terminal.
  • Inoculate as cut streaks on Eosin Methylene Blue Agar (EMBA) plates and incubate at room temperature for 24 to 48 hrs 10
  • GERM TUBE FORMATION (REYNAULD-BRAUDE PHENOMENON))
Germ Tube Formation (Reynauld-Braude Phenomenon)
The serum germ tube test is a rapid “presumptive test” for C. albicans. A light inoculum of cultured yeast is incubated in bovine serum for 2-3 hours at 37oC. The test is positive if there are short hyphae without a constriction where the hypha joins the parent cell. Not all C. albicans isolates are germ tube positive and false-positive results are a possibility, especially with C. tropicalis. Carbohydrate assimilation tests are commercially available. Rapid identification tests based on preformed enzymes are particularly good for confirmation of a germ-tube positive organism as C. albicans, but C. dubliniensis is positive too. 16

Principle:
Strains of C. albicans produce germ tubes from their yeast cells when placed in a liquid nutrient environment and incubated at 35° C for 3 hours 16 Characteristics
  • It is one of the most common phenomenon in all the kingdom fungi. However, this occurs over the course of 1 to 3 hrs for Candida albicans, so used as rapid presumptive identification test.17
  • It is accepted method for rapid identification for C. albicans but it is recognized that sensitivity and specificity is not absolute.18
  • It is a simple, efficient and economic test for screening and identification of C. albicans.19
  • True germ tube is defined as hyphal projections from the germinating yeast cell, lacking any constriction at the point of origin.20
  • Inoculate lightly the test strain of Candida and culture (control) into 2 separate tubes containing 0.5 ml each of same batch of human serum. Incubate at 37°C. Examine first the control C. albicans for satisfactory formation of germ tubes, this is seen as an outgrowth from the cell with no constriction at the base resembling a ladies hand mirror. The width of the outgrowth is less than half the width of the yeast and length of the outgrowth 2-3 times that of the yeasts cell.
  • Old cultures, heavy inoculum and cultures from SAB can give negative results.
  • It is important to read the test within 3 hrs as other Candidal species will also form germ tubes in serum after this time period
  • In conclusion germ tube appears to be the cheapest test, but it is time consuming and laborious.17

Adhesive (scotch) Tape Preparation)
METHOD
  • Touch the adhesive side of a small length of transparent tape to the surface of the colony.
  • Adhere the length of tape to the surface of a microscope slide to which a drop of lactophenol cotton or aniline blue has been added
  • Observe microscopically for the characteristic shape and arrange­ment of the spores. 17
The transparent adhesive tape prepa­ration allows one to observe the or­ganism microscopically approximately the way it sporulates in culture. The Spores are usually intact, and the mi­croscopic identification of an organism can be made easily. Some laboratories prefer to use the microslide culture for making the microscopic identification of an organism. 17

Biochemical Tests
  • Carbohydrate Fermentation
Yeasts are able to metabolize some foods, but not others. In order for an organism to make use of a potential source of food, it must be capable of transporting the food into its cells. It must also have the proper enzymes capable of breaking the food’s chemical bonds in a useful way. Sugars are vital to all living organisms. Yeast is capable of using some, but not all sugars as a food source. Yeast can metabolize sugar in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen. 21

The fermentation of glucose can be described by the following equation: 21

C6Hl2O6 2 CH3CH2OH + 2 CO2 + energy

glucose ethyl alcohol carbon dioxide.
  • Carbohydrate Assmilation
This test is used for definite speciation of Candida and few other fungal micro organosms.10
Carbohydrate assimilation test (Modified Wickerham Method):- The carbohydrates used are Glucose, Maltose, Lactose, Sucrose, Galactose, Xylose, Trehalose, Cellibiose. 22

Rapid Urease Test:
This test is used to detect presence of urease enzyme produced by different Candida species. Christensens urea agar slants are used. Conversion of the yellow slope to pink or red is considered positive. A negative test is reported when there is no colour change observed. 22

Other Techniques: 23,224,25,26
  • Microslide Culture
  • Hair Preformation Test
  • In Vitro COnversion Of Dimorphic Molds
  • Exo-Antigen Test
  • Nucleic Acid Probe Testing

Newer Diagnostic Techniques: 27,28,30,31,32
  • CT Scanning MRI Techniques
  • Nuclear Magnetic Resonance (NMR)
  • Immundiagnostic Techniques
  • PCR Techniques
  • Serologic Test
  • Latex Agglutination Test
  • Immunfluoresce

References:
  1. William A, Strohl, Pamela C, Champe, Richard A, Pamela C, Harriet Rouse. Lippincott`s illustrated reviews; microbiology. 1st edition, page 265-268.
  2. Michael T, John M, Paul V, David P Clark Brock Biology of microorganisms, part 4 survey of microorganisms, page 357 to 368.
  3. George W Burnette, Henry W Scherp, (1996). Mycotic Diseases With Oral Manifestations, 3rd edition; The Williams and Wilkins Publications, page 558 to 585
  4. Erjavec Z, Verweij P. E. (2002). Recent progress in the diagnosis of fungal infections in the immunocompromised host, Drug resistance updates, 5; 3-10.
  5. Lansing M Prescott, Augustana C, John Hailey, Don Klene. Microbiology 5th edition, page 440 to 446
  6. Micro organisms – fungi, algae, protozoa and viruses, fungi - molds and yeasts page 333 to 348
  7. Michael Ellis; Invasive fungal infections, (2001). evolving challenges for diagnosis and therapeutics. Molecular immunology 38; 947 - 957.
  8. Siew Fah Yeo, Brain Wong; July (2002). Current status of nomenclature methods for diagnosis of invasive fungal infections, Clinical Microbiology Reviews; page 465 – 484.
  9. Bennett and Chang, (1995). Part 1; Laboratory Aspects of Medical Mycology; Laboratory diagnosis, page 45 to 78.
  10. Diagnostic manual, procedures in mycology Chapter XIV page 109 to 132.
  11. More References are available on request

    Comments   

     
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