Fungal (mycotic) cultures are microbiology laboratory tests to detect or rule out the presence of fungi (plural of fungus) in specimens taken from patients, animals, and the environment. The laboratory uses optimal conditions to grow and identify any fungus present in the specimen while attempting to eliminate or identify contaminants. The specimen is cultured by spreading a small portion of it on various agar media (inoculation). The media are then incubated in a warm, moist environment and examined regularly to detect growth of any organisms. The isolated fungus is identified primarily by its colony morphology and microscopic structures.
Mycology, the study of fungi
Fungi are simple plantlike organisms that do not have roots, stems, or leaves and that live off organic matter such as skin, hair, and vegetation. The group includes mushrooms, yeast, rusts, smuts, molds, and mildews, but only yeast and molds typically cause disease in humans. The basic structural unit of a fungus is either a single yeast cell, or multicellular filamentous hyphae, which are the tubular projections of molds. Molds grow by elongation and branching of hyphae during the vegetative (feeding) stage and produce spores during the reproductive stage. Spores are small reproductive bodies that are capable of sprouting new hyphae. Multiple, loosely intertwined hyphae strands called mycelia are the fluffy, colorful colonies seen as mold growing on rotting fruit. These mycelia are spore producing structures. Yeast primarily reproduce by budding, which is the out-pouching and eventual pinching off of part of the cell. A chain of budding yeast cells adhering together may appear to be like hyphae and are called pseudohyphae. Some fungi, capable of existing in either a yeast or filamentous mold form, depending on the environment, are called dimorphic. The term fungal elements includes any of the structures that may be seen during examination of specimens
Fungi differ from higher plants in that they do not contain chlorophyll and thus cannot manufacture their own carbohydrates. They must use preformed carbon and nitrogen compounds made by other organisms and are therefore either saprophytic (living on dead or decaying organic matter) or parasitic (living on or within other living organisms). Most fungi trace back to a soil origin. All are obligate aerobes (require oxygen to survive), and tend to thrive in a dark, moist, undisturbed atmosphere. They grow well at room temperature, but some of the pathogenic (disease causing) dimorphic fungi also grow well at body temperature.
Of the more than 50,000 species of fungi, only 100 to 150 species of yeast and molds cause disease in humans. The number routinely seen is much lower. Humans are generally resistant to fungi even when they become accidental hosts by inhaling spores or by having a cut or scrape exposed to a fungus. However, inhalation of spores of some of the dimorphic fungi produces illness ranging from mild cough and fever to severe disseminated disease. People with weakened immune systems (immunocompromised) are susceptible to illness from many normally harmless fungi. The characteristics of fungi that make them pathogenic to humans are:
- a small enough spore size to be able to reach the alveoli of the lungs
- the ability to grow at body temperature
- the ability of a dimorphic fungus to convert from a mold to a yeast form within the host
- toxin production
The purpose of the fungal culture is to attempt to grow and identify any fungus originating from a patient's specimen when the medical staff of a hospital, clinic or doctor's office suspects fungal infection. Further, the goal is to determine whether the isolated fungus is clinically significant; that is, the causative agent of the patient's disease. While the physician makes the final decision regarding clinical significance, the laboratory may assist in this process by noting the presence of common contaminants, etc. Since the goal is also to provide information in a timely manner, fungal cultures will usually include smears and stains taken directly from the specimen for microscopic examination. The direct examination attempts to visually detect such fungal elements as hyphae, yeast, or spores.
It should be noted that 90% of fungal infections are due to dermatophytes (fungi that infect skin, hair and nails), which do not normally need a fungal culture for diagnosis. A dermatologist can generally make the diagnosis in the office from a KOH preparation (see section on direct examination of specimens) along with the patient's symptoms and site of infection. However, a culture may be needed to prove that a skin condition is not due to nerves, psoriasis, or metabolic imbalances.
Another contraindication for fungal cultures is if the patient has already been treated with antifungal medication. Many patients who see their doctor for the first time with a possible fungal infection have already been treating themselves with such over-the-counter antifungal medication as imidazoles. A fungus may be causing an infection in the patient, yet antifungal agents present within the specimen suppress the growth of the organism in culture. All topical antifungal therapy should be stopped for at least one week prior to culture.
The medical staff should observe their institution's procedures to protect both themselves and their patients from spread of infectious diseases. The use of such protective barriers as gloves, gowns, aprons, masks or protective eyewear can reduce the risk of exposure of the health care worker to potentially infective materials. All medical staff should take precautions to prevent injuries by needles, scalpels and other sharp instruments during procedures; when cleaning used instruments; and during disposal of used needles. Gloves should be changed after contact with each patient. Hands and other skin surfaces should be washed immediately after gloves are removed. When collecting blood, genital, tissue, cerebrospinal and other body fluid specimens universal cautions for the prevention of transmission of bloodborne pathogens should be followed.
Laboratory personnel need to take several precautions when working with fungal cultures. The cultures should never be sniffed. Fungus colonies may release airborne spores that can be inhaled. Processing of specimens and all work with exposed fungus cultures should be performed in a biological safety hood. Spattering of infectious material by careless flaming of wire needles or loops should be avoided. Cultured petri dishes should be sealed with oxygen-permeable tape. Test tubes should have screw-type caps rather than cotton plugs. All contaminated materials must be autoclaved before discarding, and the work area should be disinfected daily.
The fungus culture is a microbiology laboratory test that is part of a comprehensive attempt to determine if any microorganism is causing an infection in a patient. The physician and nursing staff must first suspect the possibility of infection and order the appropriate cultures. The possibilities include testing not only for fungi, but also for aerobic and anaerobic bacteria; mycobacteria (also called acid-fast bacteria, AFB, a family of bacteria which includes the tuberculosis organism); viruses; or parasites. Many fungal infections are clinically similar to mycobacterial infections and so the same specimen is often cultured for both fungi and mycobacteria. Although fungal cultures are usually covered by insurance, they are costly and time-consuming. Cultures should be ordered only when the medical staff is reasonably suspicious of a fungal infection or when the patient is very ill and the culture is a critical part of assessing the illness. The test is also performed in the offices of dermatologists who frequently encounter fungal skin infections. The office staff may report the presence of dermatophytes or yeast, but a reference laboratory is usually required for specific identification.
Direct examination of specimens
The direct examination of specimens is an important first step in processing the specimen for several reasons. Since fungi take days to weeks to grow in culture on agar media, a direct examination result provides a rapid report to the physician, which may allow early treatment to begin. Sometimes easily identifiable morphological characteristics of a fungus may be seen on a direct exam, giving a clue as to the identification of the organism. This information may be helpful not only to the physician but also to the laboratory personnel who can set up additional specialized media to hasten the identification of the fungus. The information may also indicate the need to harvest specimens from other body sites and order serological tests. Direct examinations may also provide evidence of infection even when the fungus culture is negative.
KOH PREPARATION WITH CALCOFLUOR WHITE. The potassium hydroxide (KOH) preparation is used to detect fungal elements in virtually all types of clinical specimens, especially skin, nails, hair, sputum, concentrated urine, and tissue. A 10% to 20% solution of KOH is mixed with the specimen on a slide, a coverslip is placed on top of the preparation and the slide is gently heated. The preparation is then viewed under the 10x (low power) objective of a light microscope with fairly low illumination. KOH dissolves keratin and other cellular material but leaves the fungal structures intact and refractile. Hyphae, pseudohyphae, yeast, and spores can be seen. Calcofluor white, a fluorescent dye, may be added to the KOH preparation to aid in visualizing the fungal elements. Calcofluor white binds to polysaccharides in the cell wall of fungi, and fluoresces either apple-green or blue-white, depending on the combination of filters used in a fluorescent microscope.
INDIA INK PREPARATION AND CRYPTOCOCCAL ANTIGEN TEST. Traditionally, India ink preparations have been used to examine cerebrospinal fluid (CSF) and other clear body fluids for the presence of the encapsulated yeast Cryptococcus neoformans. A drop of India ink is mixed with a drop of sediment from a centrifuged specimen and the preparation is examined under the light microscope. Budding yeast in CSF surrounded by a large clear capsule against a black background is presumptive evidence of C. neoformans meningitis. Since only 50% of these meningitis patients have at least one positive India ink preparation, many laboratories now use the latex agglutination test for cryptococcal antigen in place of the India ink examination. This test detects the capsular polysaccharide antigen of C. neoformans in the supernatant of centrifuged CSF and serum.
OTHER DIRECT PREPARATIONS. Stains used in other parts of the laboratory will detect fungal elements. The gram stain, which is performed on specimens sent for bacterial culture, will detect most fungi, if present. The acid-fast stain used for the detection of mycobacteria will also detect Blastomyces dermatitidis. The Papanicolaou stain, which is used to examine secretions for the presence of malignant cells, also stains fungal elements well. The Wright stain, which is used routinely on peripheral blood smears and bone marrow aspirates, will also detect Histoplasma capsulatum and Cryptococcus neoformans. Common tissue stains that are excellent for the detection of fungal elements are the periodic acid-Schiff (PAS) stain, the Gomori methenamine-silver nitrate (GMS) stain, and the Mayer mucicarmine stain. Fluorescent antibody stains can be used to detect fungi directly in tissue or fluids and stains for specific organisms are available at the national Centers for Disease Control (CDC) in Atlanta, Georgia. Molecular probes capable of detecting fungi in clinical specimens are promising but not yet commercially available.
As the direct examination of the specimen is proceeding, the specimen is set up as a culture without delay. The viability of fungi decreases with time, and contaminant overgrowth may hinder the recovery of the pathogen. When delay cannot be avoided, the specimens, with the exception of blood, CSF, and dermatological (skin, hair, nail) specimens, can be refrigerated for a short time. It is not necessary to concentrate most mycology
For optimal recovery of fungi, a battery of agar media should be inoculated. 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 fungi.
Fungus cultures are incubated at either at 30°C. (86°F.) or at room temperature (22-25°C or 72-77°F). A temperature of 30°C is recommended because nearly all pathogenic fungi grow better and more rapidly at this temperature. A relative humidity of 40% to 50% is desired to prevent the agar from drying out over time and can be achieved by placing an open pan of water in the incubator. Cultures should be incubated for 30 days and examined at least three times weekly before reporting as negative. Dermatophyte cultures should be incubated six weeks at room temperature.
Identification of positive cultures
The fungi grown in positive cultures are identified by noting their growth rate, colonial morphology, and microscopic structures. Rapid-growing fungi appear in one to three days, intermediate growers take five to nine days to colonize and slow growers take up to four weeks to appear. Colonial morphology traits include color, size, texture, and topography of the colony. Yeast colonies resemble bacterial colonies (moist, rounded, opaque and raised), whereas mold colonies are described variously as wrinkled, heaped, folded, etc. The pigments of both the underside and top of the colony are noted.
The examination of microscopic structures of fungi usually provides definitive identification for molds. Microscopic morphological features that are looked for are the type, size, shape and arrangement of spores and the size and color of hyphae. It is also important to note whether the hyphae have cross walls (septations). Yeast identification may require biochemical tests for fermentation and assimilation of carbohydrates.
Microscopic observation of molds is done by using several techniques, always prepared in a biological safety hood. Transparent tape preparations are a rapid method for observing the arrangement of spores. The tape is pressed, sticky side down, on the surface of a mold colony and then onto a slide containing a drop of dyemounting fluid such as lactophenol cotton blue (LPCB). The preparation is then examined under low and high magnification on the microscope for spore type and arrangement. In a wet mount preparation, portions of colonies are teased apart with a dissecting needle, transferred to a drop of LPCB and observed under a coverslip on the microscope. Occasionally, a slide culture is needed to observe spore production. In this procedure, a small cube of agar media is inoculated on four sides with the fungus, placed between two coverslips and incubated five to ten days. When spores are evident, the coverslips are removed and mounted in LPCB and examined under the microscope.
Susceptibility testing of fungal isolates to antifungal agents is generally done only after treatment failure. Testing is done by a reference microbiology laboratory using the minimal inhibitory concentration (MIC) method. Common antifungal agents include amphotericin B, griseofulvin, fluconazole, itraconazole, ketoconazole, miconazole, clotrimazole, flucytosine and nystatin.
The proper collection of appropriate specimens is critical to successful fungus cultures and diagnosis of
Direct microscopic examination of specimens for fungal elements should always be requested along with fungus culture. Specimens or cultures that need to be sent to a reference laboratory over a long distance should be sent by air-express service. Taped screw-top tubes must be placed in a container, then placed in a second container. The outside label must include a biohazard label.
Respiratory tract secretions including sputum, bronchial washings, and tracheal aspirates are the most common specimen types submitted for fungus cultures, as many fungal infections have a primary focus in the lungs. Patients ideally should provide sputum from a deep cough after rising in the morning. All respiratory specimens should be collected into sterile, leak-proof, screw-top containers.
Hair, skin, and nail scrapings submitted for dermatophyte culture are generally contaminated with bacteria and/or rapidly growing contaminant fungi. Skin and nails should be disinfected with 70% isopropanol before sampling. Skin samples are gently scraped from the outer edge of a surface lesion using a sterile scalpel. Nail specimens may be submitted as either scrapings or cuttings and occasionally as a complete nail. The best nail specimen is crumbly material from the nail bed. A sterile scalpel blade, small curette, or scissors is used to harvest the nail.
A Wood's lamp (ultraviolet radiation from a mercury-vapor source) can be used to detect infected hairs. Hairs infected with such fungi as Microsporum species and Trychophyton schoenleinii fluoresce when a Wood's lamp is shone on the scalp. Sterile forceps should be used to pluck the hair for culturing. These hair, skin, and nail specimens should be transported to the laboratory in sterile petri dishes, or screw top containers. A potassium hydroxide (KOH) wet mount should be requested. Specimens should not be refrigerated.
Blood cultures are helpful to detect disseminated fungal infections. Blood from patients that are septic (growing organisms in their blood) can harbor a wide variety of fungal pathogens as well as opportunistic saprophytes. Yeast are adequately recovered from any of several automated bacterial blood culture systems in use in microbiology laboratories, but filamentous fungi may require specialized techniques for isolation and fungal blood cultures should be specified. A lysis-centrifugation system may be employed which lyses red and white blood cells that may be harboring fungi. After lysis, centrifugation concentrates the organisms before culturing on fungal media.
Cerebrospinal fluid for fungal culture should ideally be of a volume of one to two milliliters or more. The specimen is transported to the lab immediately in a sterile, leak-proof tube. The second or third tubes collected in the lumbar puncture are preferred for any microbiology culture. An India-ink preparation and latex agglutination for cryptococcal antigen test should be requested along with fungal culture.
Other sterile body fluids, deep tissue samples and bone marrow aspirates should be collected by the physician in a sterile manner and transported to the lab in sterile syringes or screw-top tubes. A minimum of 0.5 milliliters of fluid is required, but any size tissue is acceptable. Bone marrow aspirates require sterile anticoagulant.
Urine to be cultured for fungus should be collected by the clean-catch, midstream (CCMS) void method into a sterile screw-top container. First-voided morning urine specimens are preferred, as they are more concentrated. Twenty-four-hour or quantitative urine samples are not acceptable for culturing.
Abscess fluids and wound drainage may be cultured for fungus. Actual tissue from the wound site will greatly increase the likelihood of recovering a fungal pathogen. Urogenital and fecal specimens may occasionally be sent for fungal culture but are generally screened only for yeast. Swabs are acceptable for these cultures, as well as for throats and wounds, but the medical staff should be aware that only yeast, no molds, would be isolated.
The care of patients after the collection of a specimen varies with the specimen type. Scrapings of skin or nails, plucking of hair, urine or stool collection and sputum production should involve little or no discomfort or subsequent care. Urogenital and throat swabs may be momentarily uncomfortable. Collection of some respiratory-type specimens such as bronchoscopy or bronchial
Following the sterile collection of blood for fungal cultures, firm pressure is applied to the draw site for a few minutes, followed by a bandage. Collection of bone marrow aspirates, deep tissue biopsies and sterile body fluids are invasive procedures performed by trained physicians observing sterile procedures. The patient should be monitored afterward to avoid hemorrhage at the site of aspiration or biopsy. Lumbar puncture for the collection of CSF requires special aftercare. The patient should lie prone for four to twelve hours to avoid headaches and the aspiration site is monitored for swelling or bleeding. Any of the invasive procedures may result in some normal aches for one to two days.
Complications for the patient whose specimen is being sent for fungus culture is dependent on the type of specimen being collected. Drawing of blood cultures can sometimes result in hematoma (bruising) at the draw site and should be done by a well-trained phlebotomist who is familiar with sterile drawing procedures. More invasive procedures such as tissue biopsy, aspiration of bone marrow and other body fluids may be complicated by hemorrhage or infection of the sampling site. Complications for lumbar puncture for CSF collection may include numbness, tingling or pain in the legs.
Validity of results
Fungus cultures are fraught with false-positive and false-negative test results and the medical staff must interpret culture results in light of the patient's health status. Any direct microscopic examination may be falsely called negative, giving the physician and patient the early impression that no fungal infection exists. The false-negative direct examination may be due to examiner error through inexperience or lack of thoroughness. The portion of the specimen selected to be examined may have very few organisms, or the organisms may be missed amongst background debris in the smear. Likewise, a direct microscopic examination may be called falsely positive when an examiner misinterprets background artifacts as fungal elements in a preparation. This happens with the widely used KOH preparation and thorough training and experience is essential. A false-positive direct examination may cause antifungal treatment to be initiated unnecessarily.
It is important to be aware that falsely negative fungal cultures may result if the patient has already had antifungal
Dermatophyte—A fungus that infects only epidermal tissues in which keratin is abundant: hair, skin and nails.
Dimorphic fungus—A fungus capable of existing in both a yeast and mold form, generally as a yeast at body temperature and mold at room temperature. Some dimorphic fungi cause disease in humans.
Fungal elements—Microscopic structures of fungi that may be observed in specimens or from positive cultures: yeast, budding yeast, hyphae, pseudohyphae, spores, mycelium.
Hyphae—Multicellular filaments or tubular projections composing the mycelium of a mold.
Mycelium—A mass of loosely intertwined hyphae extended out from a mold colony.
Mycosis—Any disease caused by a fungal infection.
Opportunistic pathogen—An organism that normally is present in the environment or as part of the normal flora of a host without causing disease, but which causes disease when the host becomes debilitated.
Pseudohyphae—chains of budding yeast that adhere together and appear to look like hyphae during microscopic examination.
Spore—A small, reproductive body of a fungus that is capable of sprouting new hyphae.
Yeast—A single-celled, rounded fungi that usually reproduce by budding.
treatment. Inhibitory agents within the patient's tissues and specimens may suppress the growth of organisms in culture.
False positive cultures result when a fungus contaminates a culture. Certain species of fungi are known to routinely be contaminants of fungal cultures; thus a positive culture report should lead to a discussion between the patient's doctor and the microbiology laboratory or infectious disease specialists, if necessary. Repeating the culture is sometimes indicated.
The fungal diseases, or mycoses, have been categorized as superficial or cutaneous, subcutaneous, and systemic,
The subcutaneous mycoses include a diverse group of infections that are characterized by the formation of a lesion at the inoculation site, often the result of injury. Generally, the fungus grows slowly in the subcutaneous tissues at the site, causing gradual spreading of the lesion, but does not disseminate to distant parts of the body. Examples of subcutaneous mycoses include chromoblastomycosis, mycetoma, and sporotrichosis.
The systemic mycoses are those fungal diseases involving the internal organs, often spreading from an initial lung infection. They may become widely disseminated and involve any organ system where they can produce abscesses and granulomas (inflammatory nodules). Prior to effective antifungal therapy, these disseminated mycoses were almost invariably fatal. Traditionally, this group included dimorphic fungi from the genera Blastomyces, Histoplasma, Coccidioides and Paracoccidioides, as well as the yeast Cryptococcus. Recently, Penicillium marneffei has been added to this group.
Opportunistic pathogens comprise another category of mycosis, which is becoming increasingly common. Opportunistic infections are caused by organisms normally found in the environment, or as part of the normal body flora, which do not cause illness unless the host becomes debilitated. Opportunists cause infection in patients that are immunocompromised, either by underlying disease or such immunosuppressive agents as steroids or cytotoxic drugs. Examples of such opportunists are Aspergillus species, which are among the most ubiquitous molds in the human environment. These fungi may inadvertently cause infection in a hospital setting through such portals as dialysis bags, air conditioning ducts and airborne particles associated with construction. Another frequent opportunist is Candida albicans, a yeast commonly carried in the mouth, vagina and intestinal tracts of healthy humans. C. albicans is the causative agent of oral thrush, vaginal yeast infections and occasionally systemic disease.
In the last two decades there has been a striking increase in the incidence of opportunistic infections, not only because the number of immunocompromised patients, such as AIDS patients, is increasing, but also because some modern treatments permit the proliferation and invasion of vital organs by opportunistic molds and yeast. Such things as prolonged use of indwelling catheters, prosthetic heart valves, and immunosuppressive drugs for organ transplants have contributed to the increase.
Health care team roles
Members of the health care team play essential roles in the diagnosis of a fungal infection. Doctors and nurses must be aware of when to suspect an infection in general and fungal infections in particular. Signs of any infection include fever, chills, headache, loss of appetite, bad odor, persistent cough, localized pain, burning or bloody urination, diarrhea, pus or discharge from a wound or lesion, and elevated white blood cell count. The very young patient (less than one year), the very old, and the debilitated or immunocompromised patient are more susceptible to infection.
Predisposing factors for fungal infection include patients who have received broad-spectrum antibiotics, which eradicate the normal bacterial flora and allow fungi to overgrow. Steroid treatment decreases the body's normal protective inflammatory response to invading fungi. Cytotoxic drugs used during cancer chemotherapy suppress the immune system and make the patient more susceptible to fungi in their environment. Patients who have very low white blood cell counts (less than 1,000 cells per cubic millimeter), or such underlying disorders as AIDS, pneumonia, diarrhea, leukemia, hepatitis, or transplanted organs are more prone to fungal infections.
Skin conditions that may indicate fungal infection include scaling of skin, blisters of hands or feet, scaling of scalp with broken-off hairs, and discolored toenails or fingernails. Fungus infections of the cornea are more common in farm workers, contact lens wearers, topical steroid users, and those who have corneal injury due to such organic material as a tree branch.
Certain fungal infections are more common in particular areas of the world and the medical staff needs to be aware of what fungi are endemic (constantly present) in their area and also inquire about a patient's travel history. For example, Histoplasma capsulatum, the cause of histoplasmosis, is endemic in the Ohio and Mississippi River valleys of the United States.
The medical staff also has a critical role in selecting the appropriate specimen for fungus culture and should be knowledgeable about their institution's requirements
The mycology section of the microbiology laboratory has the important role of notifying the medical staff promptly of positive direct examination results as well as positive culture results. The cytology and surgical pathology laboratories must work closely with the microbiology laboratory as these former laboratories may detect fungal elements with their tissue stains.
The experienced microbiology technologist's knowledge of common fungal contaminants is invaluable to the doctor. A discussion between the doctor and the lab is often helpful in distinguishing between a contaminant and an opportunistic infection. Fungi that would have once been disregarded as contaminants on a culture are now known to be responsible for serious disease in debilitated hosts.
Barnett Lammon, Carol. "Assisting with Diagnostic Procedures." In Clinical Nursing Skills. Philadelphia: W.B. Saunders Company, 1995.
Forbes, Betty A., Daniel F. Sahm, and Alice S. Weissfeld. "Laboratory Methods in Basic Mycology." In Bailey & Scott's Diagnostic Microbiolgy, 10th ed. St. Louis, MO: Mosby, Inc., 1998.
Larone, Davise H. Medically Important Fungi, A Guide to Identification, 3rd ed. Washington, D.C.: American Society for Microbiology, 1995.
Nettina, Sandra M. "Infectious Diseases." In The Lippincott Manual of Nursing Practice, 7th ed. Philadelphia: Lippincott, 2001.
Padhye, A.A., et al. "Biosafety considerations in handling medically important fungi." Medical Mycology 36, Suppl 1 (1998): 258-65.
Marianne F. O'Connor, MT, MPH
Fungal Culture News
Table Of Contents
- Mycology, the study of fungi
- Safety precautions
- Direct examination of specimens
- Culturing specimens
- Identification of positive cultures
- Susceptibility testing
- Specimen collection
- Specimen types
- Validity of results
- KEY TERMS
- Health care team roles