Mycology

CHAPTER SEVEN: MYCOTOXINS AND LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS

Written by MicroDok

7.1        MYCOTOXINS

Mycotoxins are exotoxins produced by fungi. The area of microbiology that studies fungi and the toxins they produce (i.e., mycotoxins) is known as mycotoxicology. The disease condition provoked by the intake of mycotoxins in human or animal hosts is generally known as mycotoxicoses. Mycotoxins are pharmacologically active secondary metabolites produced by toxin-producing (pathogenic) fungi in food including stored grains and cereal crops. These exotoxins have the potential to stimulate lethal reactions in humans or animals that consume such toxin-infested food. When taken in large doses and allowed to accumulate in the body system, mycotoxins can elicit several acute and chronic intoxifications and other physiological reactions in human hosts. Mycotoxins are mainly secreted by some specific fungal species especially moulds and some mushrooms such as Aspergillus species, Penicillium species, Fusarium species, Claviceps species and Acremonium coenophialum (Table 7.1). Apart from secreting potent harmful toxins, most Aspergillus species especially A. niger are common contaminants found in the microbiology; and they are notorious in colonizing culture media plates that are not properly stored (Figure 7.1). Mushrooms (for example, Amanita species) produce a variety of mycotoxins; and the disease condition they cause is known as mycetismus. Mycetismus is known as mushroom poisoning; and it results from the consumption of toxic substances (released as secondary metabolites) from wild mushrooms. It can also occur following contact with wild mushrooms. Diarrhea, vomiting, abdominal pain, fever and nausea are some of the signs of mycetismus.

The main route of entry of mycotoxins into the body is via the ingestion of mycotoxin-contaminated foods. Most fungal mycotoxins are produced in dried food products such as nuts, cereals, maize and other grains and food products especially when stored poorly. Other groups of food especially fruits (for example, oranges, apple, mangoes) as well as vegetables are not left out as they could be contaminated by fungal toxins. Mycotoxins are heat-stable or resistant to heating, and thus most food preparation techniques such as boiling may have little or no effect on their in vivo effectiveness. Thus, it is critical to avoid fungal infestation of food products instead of exposing them to conditions that encourage the growth of moulds in them. Moisture and humid conditions are important factors that stimulate the production of mycotoxins in stored food or grains.

Table 7.1: Synopsis of mycotoxins produced by fungi

Organism

Mycotoxin produced

Effect

Aspergillus flavus Aflatoxins

 

Aflatoxins possess mutagenic, teratogenic, immunosuppressive and carcinogenic activity.
Penicillium species Ochatoxin A and patulin

 

Ochatoxin A is a carcinogenic and protein synthesis inhibitor. Patulin promotes apoptosis in cells.
Fusarium species Fumonisins, deoxynivalenol, zearalenone These mycotoxins promote cellular damage and oxidative stress in cells. They also possess carcinogenic activity.
Acremonium coenophialum Ergopeptine alkaloids

 

Ergopeptine alkaloids cause abortion, gangrene, convulsion, and suppression of lactation in animals such as sheep and cattle.
Amanita phalloides Amatoxins Amatoxins are toxic lethal peptides that cause severe vomiting and diarrhea. Degenerative changes occur in the kidney and liver; and death may occur few days after intoxifications.
Adapted with modification from: Hussaini Anthony Makun (2013). Mycotoxin and food safety in developing countries. InTech Publishers, Rijeka, Croatia. Pp. 77-100.

Figure 7.1: Illustration of Aspergillus niger (arrow) growing on Sabouraud dextrose agar (SDA) plate. MicroDok. 

Utmost care is required in the processing and preparation of food meant for human consumption because of the ubiquity of fungal spores in the natural environment. Fungal spores including their toxic mycotoxins are normally produced during the normal metabolic activities of fungi. High humidity and moist environment as well as low temperature conditions are some of the environmental factors that encourage the growth of moulds in food products. Fungi easily contaminate food products especially crop plants because fungi are ubiquitous in the soil. And stored food products such as grains are mostly affected by these environmental fungi that also have some pathological effects because of their ability to produce mycotoxins – which causes mycotoxicoses in the consumers.

7.2       LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS/DISEASES

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 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 (Table 7.2). 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. Clinical specimens for mycological investigations should be aseptically collected based on the prevailing hospital sample collection procedures in order to get optimum result.

Table 7.2: Isolation media used to recover fungi from clinical specimens

Media*

Description & function

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 and it does not incorporate 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.
Cornmeal agar Cornmeal 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; cornmeal 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. Cornmeal 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.
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 with antibiotic medium is different from the traditional SDA without antibiotics, 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 cornmeal agar, PDA is used to induce spore formation in sporulating fungi. PDA also increases pigment production in fungi during culture.
*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. The list of culture media in this table is not exhaustive as there are several growth media available for the cultivation and identification of fungi in the microbiology laboratory. 

For skin scrapings; the site of sample collection should be cleaned and wiped with cotton wool soaked in 70 % ethanol to remove dirt’s or dust and oil. The disinfected site should be allowed to dry before collecting the skin scraping. Skin scrapings should be collected aseptically and in such a manner that excludes bleeding. The scrapings should be collected in a clean white paper or container.

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 media.

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. Woods lamp can be used to identify infected areas of the scalp and other skin parts where hairs should be obtained from.

For body fluids (blood, CSF, urine, 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.

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. It 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; and thus lack the ability to produce the skin pigment known as melanin. Such dead cells lack the ability to produce melanin; and they lose their colour and turn white.

Most fungal specimens (for example, skin scrapings, nail clippings and hair) are processed 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.

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 (which differentiates Nocardia species from other aerobic Actinomyces); Indian ink stain (which reveal the capsules of C. neoformans in CSF specimens); fluorescent antibody stain (which is used to detect fungi in tissues or fluid specimens); Periodic Acid-Schiff (PAS) stain (which is used to stain the polysaccharide layer in the fungal cell wall) and Giemsa Stain (which is used for staining blood and bone marrow samples). Other 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. Biochemical tests and skin tests also exist for the laboratory diagnoses of human mycoses.

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). 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 medium and the organism being sought for. 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. Different culture media are available for the cultivation and identification of fungi in the microbiology laboratory (Table 7.2). 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 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 spore 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. 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.

7.3       TREATMENT OF FUNGAL INFECTIONS

Fungal infection is treated using antifungal agents. Some of the antifungal agents used for the treatment of mycoses in humans include polyenes (for example, amphotericin B and nystatin) which are cidal in action and binds to the fungal ergosterol membranes to disrupt the integrity of the fungal cell membrane; azoles (for example, itraconazole, ketoconazole, voriconazole, fluconazole, miconazole) which are static in action and inhibit the synthesis of ergosterol; griseofulvin, which is static in action and inhibits fungal growth by binding to microtubules during mitosis; 5-fluorocytosine or flucytosine, which is an antimetabolite and inhibits nucleic acid synthesis and protein synthesis; echinocandins (for example, caspofungin, micafungin) which are static in action and inhibits the synthesis of chitin and glucan in fungal cell wall; and allylamines (for example, terbinafine) which are static in action and inhibit the synthesis of ergosterol like the azoles.  

7.4       PREVENTION AND CONTROL OF FUNGAL INFECTIONS

The prevention and control of mycoses is largely dependent on avoiding exposure to fungal spores or conidia and limiting contact with natural reservoirs of most fungal organisms. An intact immunity is vital to the prevention of fungal infections in humans. Thus, people should avoid some risk factors such as prolonged antibiotic usage and other factors that weakens the body’s natural defense against fungal infection. People who work in certain type of occupation such as farmers, horticulturists, construction workers and forest workers should wear protective footwear and face mask to avoid traumatic introduction of pathogenic fungi into the body as well as the introduction of fungal spores into the body via the nostrils. While some fungal infections are non-communicable, others such as superficial mycoses (for example, ringworm) are infectious; and avoidance of body contact or sharing of towels, soap and bathtub with infected people is critical to the prevention of most superficial mycoses in human population. Fungicides and other antimicrobial agents should be used on the natural reservoirs of fungi (for example, soil) to avoid the aerosolization of their spores which when inhaled can lead to mycoses in humans. People working in dusty areas should always wear protective face mask to avoid the inhalation of fungal spores.

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MicroDok

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