The laboratory diagnosis of fungal infection is mainly based on microscopy and cultural techniques. However, molecular biology techniques such as the use of PCR can also be employed in some cases. In addition, serology and antigen detection tests from clinical samples are the non-cultural approaches of laboratory diagnosis of mycoses. The type of laboratory technique to be employed is usually dependent on the severity and type of mycoses being investigated. The early detection of fungal infection is of utmost importance because this strategy is critical to effective treatment and management of the mycoses. Several culture media exist for the selective isolation of pathogenic fungi from clinically important specimens.
The choice of culture media to be used is largely dependent on the type of mycoses and the category of samples to be analyzed. Specimens collected for fungal investigations in the microbiology laboratory include skin scrapings, nail clippings, hair, blood, thrush lesions from oral cavity, cerebrospinal fluid (CSF), urine, vaginal swabs, corneal scrapings, pleural fluid, sputum, bronchial washings, gastric washings, nasopharyngeal specimens, biopsy materials and abscess or pus samples. However, the type of specimen collected from the patient is usually a reflection of the site of entry of the fungal pathogen and the ensuing mycoses in the individual.
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CULTURE MEDIA USED TO RECOVER FUNGI FROM CLINICAL SPECIMENS
Brain heart infusion (BHI) agar: BHI agar is composed of beef heart infusion, glucose, peptone, NaCl, distilled water, agar, calf brain infusion, and disodium phosphate as ingredients. It is used to convert diphasic or dimorphic fungi to yeast forms. BHI agar is also used for the culture of fastidious microorganisms; and it is supplemented with 5 % sheep blood in this instance. BHI agar can be prepared in Petri dishes and test tubes. It is used to recover fungi that cause systemic mycoses such as H. capsulatum and B. dermatitidis.
Sabouraud dextrose agar (SDA): SDA is a general purpose medium used for the recovery of fungal organisms from clinical and environmental samples. It is composed of agar, glucose, distilled water and neopeptone. SDA is usually used to study the colonial morphology of dermatophytes (i.e., fungi that infect the skin). It is widely used for most fungal studies. This type of SDA is not incorporated with any antibiotics. Superficial fungi such as Trichophyton species, Microsporum species and Epidermophyton species are best studied with SDA.
Caffeic Acid Agar (CAA): CAA is a light sensitive medium for fungal isolation. Thus, it should be protected from direct light. CAA is mainly used to recover fungi that cause opportunistic mycoses, such as Cryptococcus neoformans. Fungal growth on CAA appears as black colonies, thus showing the production of melanin.
Corn meal agar: Corn meal agar is composed of cornmeal, agar and distilled water. It is used for the recovery of fungi that causes cutaneous mycoses such as Candida and Trichophyton. When Tween 80 is added; corn meal agar can be used for the cultivation and differentiation of Candida species usually on the basis of the mycelia features of the organism on the growth media. Corn meal agar can also be used to induce the formation of spores or conidia in sporulating fungi. It enhances the production of chlamydospores in Candida species.
Cream of rice agar with Tween 80: Cream of rice agar is composed of cream of rice (obtained as filtrate of rice boiled in water), agar, Tween 80 and distilled water. The cream of rice agar with Tween 80 is used to recover Candida species from environmental and clinical samples.
SDA supplemented with antibiotic: SDA with antibiotic media is composed of cycloheximide, chloramphenicol, agar, peptone, glucose and distilled water. Cycloheximide inhibits the growth of saprophytic fungi and some yeast while chloramphenicol inhibits the growth of bacteria including actinomycetes. SDA supplemented with antibiotic is known as SDA with antibiotic media. This type of SDA is different from the traditional SDA without antibiotics as aforementioned, and it is commercially available as Mycosel agar. Mycosel agar is used for the selective isolation of fungi from a sample suspected of containing mixed population of fungi and bacteria. They are excellent medium for the isolation of fungi from contaminated body sites or samples. SDA with antibiotic is a general purpose media for the recovery of commonly encountered fungi such as Trichophyton species and Aspergillus species.
Potato dextrose agar (PDA): PDA is composed of potato infusion, glucose, agar and distilled water. Like corn meal agar, PDA is used to induce spore formation in sporulating fungi. PDA also increases pigment production in fungi during culture.
ANALYSIS OF CLINICAL SAMPLES FOR THE ISOLATION OF PATHOGENIC FUNGI
Samples suspected of containing pathogenic fungi are analyzed in the mycology laboratory. The clinical samples collected from patients suspected of having fungal infections include skin scrapings, nails, hairs and body fluids such as blood, CSF, urine, abscess. It is important to always ensure aseptic technique such as using face mask when collecting fungi samples. Face mask can help prevent the inhalation of particles (containing fungal spores) from the samples especially during skin scrapings.
- Skin scrapings: For skin scrapings, the site of sample collection should be cleaned and wiped with cotton wool soaked in 70 % ethanol to remove contaminants, dirt’s or dust and oil from the site of collection prior to scrapping. The disinfected site on the skin should be allowed to dry before collecting the skin scraping. Skin scrapings should be collected aseptically using sterile surgical blades; and it should be done in such a manner that excludes bleeding. The scrapings should be collected in a clean white paper or sample collection vessel/container.
- Nails/ nail clippings: For nails, the site should be cleaned with 70 % alcohol/ethanol and allowed to dry. Nails should be aseptically clipped into clean containers and minced or crushed before inoculation onto solid culture media.
- Hair samples: For hair samples, the hairs for mycological investigation should be obtained directly from the site of infection by plucking or brushing into a clean container or white paper. Woods lamp can be used to identify infected areas of the scalp and other skin parts where hairs should be obtained from for mycological investigation.
- Body fluids (blood, CSF, urine, abscess): For body fluids such as blood, CSF (cerebrospinal fluid), urine and abscess, normal aseptic techniques of collecting clinical samples should be used for optimum result. Specimens for mycological investigations obtained outside the laboratory should be properly collected, packaged, labeled and transported to the laboratory without delay for further processing to avoid the likelihood of spreading the fungal spores in the environment.
Clinical specimens for mycological investigations should be aseptically collected based on the prevailing hospital sample collection procedures in order to get optimum result. Proper sample collection and processing is important in order to avoid the contamination of the sample during collection, transport and processing in the laboratory. Antibiotics such as chloramphenicol are usually added in fungal culture media in order to make it selective in nature.
The laboratory methods involved in the diagnosis of mycoses include serological tests (for example, ELISA), molecular techniques (such as PCR), antigen detection tests (for example, latex agglutination test) and skin tests which detect an individual’s delayed hypersensitivity reactions to fungal antigens in vivo. Fungal culture unlike bacterial culture (which is usually carried out at 37oC for 18-24 hours or overnight) is carried at room temperature (i.e., 25-28oC) and even for a longer period of days (usually 48 hrs and more).
The reason for this is because fungi have a longer generation time than bacteria; and thus fungal cultures are usually cultured for 48 hours or more depending on the culture medium and the organism being sought for. Some fungal culture can also last up to a week before the suspected fungi can start growing on the culture media; and this is because fungi is a eukaryote cell with a longer generation time. And the type of culture media to use for the isolation and identification of fungi (especially those that are of clinical significance) is dependent on the type of specimen/sample to be processed and the nutritional requirement of the suspected fungus.
Most fungal culture media are better prepared in stoppered test tubes or slant form than in Petri dish plates (as is mostly applicable in bacteria cultures). This is because tube media last longer than plate media upon usage, storage and incubation than plated culture media. And tube media is easy to store and there is a lesser chance of fungal spores escaping into the environment when tube culture media is used than in the plate media where fungal spores escape is likely. The tube media used for fungal culture are stoppered with cotton wool; and this prevents the escape of fungal spores into the surrounding environment during culture.
More so, tube media is less amenable to dehydration and possible environmental contamination than plate media; and their small surface area ensure maximum safety of the cultured specimen and/or organism. It is also advisable to conduct fungal experiments or culture in a biological safety cabinet or hood in order to prevent the aerosolization of fungal spores and contamination of the worker and the working environment in general. All culture media for fungal investigations are commercially available, and they should be prepared according to the manufacturer’s instruction for optimum result. However, some fungi culture media can be locally prepared and compounded in the laboratory once the required raw materials are available and used in the correct proportion or amount.
WOOD LAMP EXAMINATION, MICROSCOPY AND FUNGAL STAINING
Woods lamp examination is a microbiological laboratory and/or clinical diagnosis technique in which a light or transillumination from a specialized type of lamp (known as wood lamp) is used to detect fungal infections on the skin surface or hair. This type of examination is carried out in a dark room, and every other source of light is removed or blocked in order to get the optimal use of the procedure. The wood lamp examination can also be used in the laboratory diagnosis of some bacterial infections such as Propionibacterium acne and Pseudomonas infections.
Wood lamp examination is usually used to detect mycoses of the superficial regions of the skin (i.e., skin infections) and scalp including Tinea capitis, Pityriasis versicolor and vitiligo (patches around the skin especially around the genitals, hands, feet, nostrils and inside the mouth). Vitiligo is a clinical condition in which the cells (melanocytes) around some regions of the skin as aforementioned are destroyed. Such areas of the skin lack the ability to produce the skin pigment known as melanin. It is noteworthy that dead skin cells lack the ability to produce melanin; and they lose their colour and turn white.
KOH mount is a commonly used technique for mycological investigation in the microbiology laboratory.Most fungal specimens (for example, skin scrapings, nail clippings and hair) are initially processed in the mycology laboratory by direct microscopy using 10 % potassium hydroxide (KOH). This is known as the KOH mount of samples meant for fungal investigations; and the technique is a routine in most microbiology laboratory across the world. KOH (10 %) is used to dissolve the proteinaceous material of skin samples (for example, skin scrapings) because it facilitates the detection of fungal elements from clinical specimens.
The KOH dissolves or degrades the keratinized portion of the skin specimen so that fungal elements (for example, spores) can be released and seen microscopically. Other fungal elements looked for in microscopical examination of fungal samples include fungal hyphae, budding yeast cells, conidia and mycelia. Wet mount preparation of the samples can also be carried out to lookout for yeast cells and other fungal structures. Another important procedure in fungal examination is the use of lactophenol cotton blue stain which stains the chitinized component of fungal cell walls so that fungal elements and/or structures can be seen microscopically. Lactophenol cotton blue is usually used as a staining dye to increase the contrast between fungal elements and background during microscopy.
Other important stains used for mycological investigations include:
- Acid fast stain: Acid fast stain helps to differentiates Nocardia species from other aerobic Actinomyces.
- Indian ink stain: Indian ink stain reveals the capsules of C. neoformans in CSF specimens.
- Fluorescent antibody stain: Fluorescent antibody stain is used to detectfungi in tissues or fluid specimens.
- Periodic Acid-Schiff (PAS) stain: PAS stain is used to stain the polysaccharide layer in the fungal cell wall:
- Giemsa Stain: Giemsa stain is used for staining blood and bone marrow samples.
Anaissie E.J, McGinnis M.R, Pfaller M.A (2009). Clinical Mycology. 2nd ed. Philadelphia, PA: Churchill Livingstone Elsevier. London.
Baumgardner D.J (2012). Soil-related bacterial and fungal infections. J Am Board Fam Med, 25:734-744.
Calderone R.A and Cihlar R.L (eds). Fungal Pathogenesis: Principles and Clinical Applications. New York: Marcel Dekker; 2002.
Champoux J.J, Neidhardt F.C, Drew W.L and Plorde J.J (2004). Sherris Medical Microbiology: An Introduction to Infectious Diseases. 4th edition. McGraw Hill Companies Inc, USA.
Gladwin M and Trattler B (2006). Clinical Microbiology Made Ridiculously Simple. 3rd edition. MedMaster, Inc., Miami, USA.
Larone D.H (2011). Medically Important Fungi: A Guide to Identification. Fifth edition. American Society of Microbiology Press, USA.
Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.
Stephenson S.L (2010). The Kingdom Fungi: The Biology of Mushrooms, Molds and Lichens. First edition. Timber Press.
Sullivan D.J and Moran G.P (2014). Human Pathogenic Fungi: Molecular Biology and Pathogenic Mechanisms. Second edition. American Society of Microbiology Press, USA.