Fungal staining technique helps in clear visualization for studying the morphological characteristics of fungi that aid in detecting and identifying fungi, thus playing a crucial role in diagnosing fungal infections. Fungal staining techniques can be broadly categorized as wet mount preparation and differential staining. 

A wet mount is a fungal staining technique performed by suspending the specimen in the reagent solution and observing it in the microscope. Potassium hydroxide (KOH) wet mount, lactophenol cotton blue (LPCB) stain, India ink stain or Nigrosin stain, calcofluor white stain, PHOL stain, neutral red stain, and diazonium blue b (DBB) stain are some of the wet mount technique for identifying fungus.

Differential staining is a fungal staining technique that uses more than one stain. The color contrast aids in identification of the organisms. The different types of differential stains used for fungus identification are hematoxylin and eosin (HE) stain, May-Grunwald Giemsa (MGG) stain, diff-quick stain, periodic acid-Schiff (PAS) stain, Gridley’s fungal stain, Gromori’s methenamine silver stain, Mayer’s mucicarmine stain, Masson-Fontana (MF) stain, Schmorl’s melanin stain, Toluidine blue O stain, acridine orange stain, and fluorescent-antibody stain.  Gram stain and modified acid-fast stain are also useful to identify fungal isolates.

Potassium Hydroxide (KOH) Mount

Potassium hydroxide (KOH) is a strong alkali, and it digests and dissolves the tissue material in the skin, hair, nail, or sputum sample for the clear observation of fungi. Depending on the clinical specimens, the potassium hydroxide wet mount can be prepared on a slide or tube. 

It is the primary screening method for identifying fungal elements in clinical samples. KOH wet mount helps to diagnose dermatophytosis (ringworm), mucormycosis, chromoblastomycosis, Blastomyces dermatitidis infection, etc.

Read more: KOH wet mount preparation.

Lactophenol Cotton Blue 

Lactophenol cotton blue (LPCB) wet mount preparation is a widely used routine laboratory technique. Phenol acts as the disinfectant and kills the fungus, lactic acid preserves the fungal morphology, glycerol prevents drying, and cotton blue stains the outer wall of the fungus. It is performed in two ways: standard tease mount and cellophane tape.

LPCB wet mount detects and identifies the fungus from the culture medium. LPCB stains the fungal elements in blue color.

Read more: LPCB wet mount

Gram stain

Gram staining is the differential method for detecting bacteria, but it also helps identify fungi. Fungi appear as Gram-positive (purple color). While examining clinical specimens, detecting fungal elements (hyphae, yeasts, or mycelia) helps us diagnose fungal infections.

There are two modifications of the Gram stain for staining the tissues. 

• Weigert’s modification of the Gram stain helps to identify Pneumocystis cysts. 
• Brown and Brenn’s modification of the Gram stain is used to identify Nocardia, Actinomyces, and Microsporidium spp. 

India Ink and Nigrosin Stain

India Ink and Nigrosin stain are the acidic dyes used in negative staining. Either India ink or Nigrosin stain helps to stain capsules. Nigrosin stain is preferred over India stain because it can last more than one year, and no carbon particles will appear in the solution. 

These stains detect capsule-forming fungi such as Cryptococcus neoformans which causes cryptococcal meningitis. The capsule appears as a clear halo against the dark background.

Calcofluor White Stain

Calcofluor white stain is the fluorescent dye that stains the chitin present in the cell wall of fungi. It is a water-soluble, colorless dye and a fluorescent whitener. It fluoresces light-blue in color when exposed to UV light in a fluorescent microscope. 

Calcofluor white staining is used to detect hyphae, pseudohyphae, and yeast. Under the fluorescent microscope observation, the fungus fluoresces green. It also helps to detect non-culturable fungi like Pneumocystis jirovecii. 

Hematoxylin and Eosin Stain 

Hematoxylin stains the cell’s nuclei in blue, and eosin stains the cytoplasm in pink. H and E stain helps to differentiate the nuclear and cytoplasmic parts. Hematoxylin stains the nuclear components like the heterochromatin and nucleoli. Eosin stains the cytoplasmic components like collagen and elastic fiber, muscle fibers, and red blood cells.

Hematoxylin and Eosin stain is used to examine the frozen sections of tissues, fine needle aspirates, and paraffin fixed embedded tissue. It can be useful in examining the biopsy sample when cancer is suspected. H and E stain helps visualize hyphae, yeast cells (nucleus), the double refractive cell wall of Blastomyces, and the spherule of Coccidioides. 

May-Grunwald Giemsa Stain

May Grunwald-Giemsa stain is made from two stains, May Grunwald stain, and Giemsa stain. May Grunwald stain consists of methylene blue and eosin. Giemsa stain consists of methylene blue, eosin, and azure B.

May-Grunwald Giemsa stain detects the Histoplasma capsulatum in the peripheral blood smear. This staining technique stains the blood and bone marrow smears. Differential counting of the blood cells can be one. It helps to visualize the chromosomes and identify the mast cells. 

Diff-Quik Stain

Diff-Quick Stain consists of fixative, stain solution I, and stain solution II. This is a ‘dip’ stain and uses the Coplin jars. It is based on the modification of the Wright Giemsa stain. Diff-Quick stain is better than Wright Giemsa Stain because the 4 min procedure completes in just 15 seconds.

Diff-Quick stain is useful for rapid staining and differentiation of the smears, like blood and vaginal smears. Fungi appear in as dark blue color. 

Periodic Acid-Schiff Stain 

The periodic acid-Schiff (PAS) is a staining technique that identifies the presence of glycogen, polysaccharides, and mucin in the tissue. These tissue sections may be formalin-fixed, paraffin-embedded, or frozen. PAS stains the nuclei blue and fungi in deep pink or magenta color.  

PAS stain is useful for identifying Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, and Blastomyces in tissue samples because their cell walls and capsules contain a significant amount of carbohydrates.

Gridley’s Fungal Stain

Gridley’s Fungal stain is like the PAS stain but uses chromic acid as the oxidizing agent. This technique combines Bauer, Schiff, and aldehyde fuchsin techniques. 

Gridley’s fungal stains the fixed tissue sections. It stains the mycelia and conidia in deep purple color and elastic tissue and mucin background in the yellow color. It does not stain the actinomycetes. 

Gomoris Methenenamine Silver Stain

Gomori’s methenamine silver stain (GMS) is useful for the demonstration of the polysaccharide content of the fungus in tissue sections. The most widely used stain is Grocott’s modification of the GMS stain, known as Grocott’s Gomoris Methenenamine Silver Stain. GMS stain can stain degenerated and non-viable fungi too. It stains the fungi and bacteria black, mucopolysaccharide dark grey, and tissue pale green.

GMS stain identifies the Pneumocystis jiroveci, which causes the pneumocystosis.

Mayer’s Mucicarmine Stain 

Mayer’s Mucicarmine stains the mucin, a secretion produced by different epithelial cells. Epithelial cells produce mucin in excess amounts during carcinoma or in some inflammations. 

Mayer’s Mucicarmine stain detects the Cryptococcus and Rhinosporidium spp. in tissue. It stains the Cryptococcus spp. deep rose red, nuclei black, and the tissue in yellow color. In the case of rhinosporidiosis, mucicarmine stain the sporangium and endospores. 

Masson-Fontana Stain

Masson-Fontana (MF) stain detects a cell’s argentaffin granules and melanin. It is useful for examining the frozen or paraffin-embedded tissue sections. Melanin is the brown, black pigment that is also present in the human body’s hair, skin, retina, and iris. Argentaffin granules are present in carcinoid tumors. Mosson-Fontana stains the melanin and argentaffin granules in black color and nuclei in red. 

Masson-Fontana (MF) stain detects melanin-containing dematiaceous (phaeoid) fungi like Curvularia lunata and Bipolaris hawaiiensis in tissue. 

Schmorl’s Melanin Stain

Schmorl’s Melanin stain is the melanin-based staining method that detects the presence of the reducing substances in the tissues like melanin, keratin, keratohyalin, etc. Melanin is present in the cell wall of the dematiaceous (phaeoid) fungi. This technique uses the reducing properties of melanin to stain granules in blue-green color. It stains the nuclei in pinkish-red and cytoplasm a pale-pink color. 

Schmorl’s melanin stain is useful for diagnosing phaeohyphomycosis in tissue biopsy. 

Toluidine Blue O Stain

Toluidine Blue is a metachromatic dye. It stains the frozen tissue section rapidly in about 10-20 seconds. Toluidine blue O stain stains the cysts of Pneumocystis jirovecii as reddish blue or dark purple against a light blue background. It detects only the cyst form, not the trophozoite form. 

Toluidine blue O stain is useful for detecting the Pneumocystis jirovecii in the lung biopsy and bronchoalveolar lavage (BAL) fluid. 

Acridine Orange Stain

Acridine orange stain is the fluorochrome dye that binds the cells’ nucleic acids. When fungi are stained with the acridine dye and viewed under ultraviolet light, RNA fluoresces orange-red and the DNA as green. The differential staining of the DNA and RNA requires a pH of 6. Acridine orange stains the human epithelial cell, inflammatory cell, and background debris, which appear pale green to yellow color.

Acridine orange stain is useful in demonstrating the fungi in tissue sections and smear rapidly. It is also useful for identifying the fungus Aspergillus spp., Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Rhinosporidium seeberi in tissue sections.

Fluorescent-Antibody Stain

Fluorescent-antibody stain is an antigen detection test in which the antibody is tagged with the fluorochrome. It detects fungal antigens in pus, blood, cerebrospinal, and paraffin sections of formalin-fixed tissues.

The fluorescent antibody is useful in detecting Sporothrix schenckii, Aspergillus spp., Blastomyces dermatitidis, Cocciodioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, etc. 

PHOL Stain 

PHOL stain is derived from the scientist’s name Pal, Hasegawa, Ono, and Lee. The LPCB and PHOL have similar functions but differ in the stain’s composition. In PHOL stain, formalin replaces phenol, and methylene blue replaces cotton blue. 

PHOL stain is useful in examining the fungal isolates and Prototheca species.

Neutral Red Stain 

Neutral Red Stain is a water-soluble dye. It can pass through the intact plasma membrane and the lysosome of viable cells can store it. When cell membrane and lysosomes are damaged, dye uptake decreases. 

Neutral Red Stain is used to differentiate the viable and non-viable fungus. The microscopic observation and the fungal culture are done to diagnose dermatophytosis. Sometimes fungus can be observed during direct microscopic observation, but it may not grow in the culture medium. In such cases, a neutral stain helps to know if the fungus is viable because it will stain the viable cell red and won’t stain the non-viable cell.

Diazonium Blue B stain

Diazonium Blue B stain (DBB) is useful in differentiating the species of two genera of fungi, basidiomycetes, and ascomycetes. DBB positive will stain dark-red or violet red, and DBB negative won’t stain. 

Diazonium Blue B stain differentiates  Trichosporon spp (basidiomycetes) from the Geotrichum spp. (ascomycetes). Trichosporon spp. is DBB positive and Geotrichum spp. is DBB negative.

References 

1. Chick, E. W., & Chick, E. W. (1961). Acridine Orange Fluorescent Stain for Fungi. Archives of Dermatology, 83(2), 305–309. https://doi.org/10.1001/archderm.1961.01580080135015
2. Ebenye, C. M. (2012). A case of disseminated histoplasmosis detected in peripheral blood smear staining revealing AIDS at terminal phase in a female patient from Cameroon. Case Reports in Medicine, 2012. https://doi.org/10.1155/2012/215207
3. Gridley, M. F. (1953). A Stain for Fungi in Tissue Sections. American Journal of Clinical Pathology, 23(3_ts), 303–307. https://doi.org/10.1093/ajcp/23.3_ts.303
4. Haque, A. (2010). Special Stains Use in Fungal Infections. Connection, 187–194. http://www.dako.com/us/28829_connection_14.pdf#page=187
5. Kaplan, W. (1973). Direct fluorescent antibody tests for the diagnosis of mycotic diseases. Annals of Clinical Laboratory Science, 3(1), 25–29.
6. KAPLAN, W., & IVENS, M. S. (1960). Fluorescent antibody staining of Sporotrichum schenckii in cultures and clinical materials. The Journal of Investigative Dermatology, 35(3), 151–159. https://doi.org/10.1038/jid.1960.99
7. Lowrey, T. (1986). A modified sulfation-toluidine blue technique for the demonstration of fungi in tissue sections: A brief report. Journal of Histotechnology, 9(1), 23–24. https://doi.org/10.1179/his.1986.9.1.23
8. Naka, W., Hanyaku, H., Tajima, S., Harada, T., & Nishikawa, T. (1992). Application of Neutral Red Staining for Evaluation of the Viability of Dermatophytes in Human Skin Scales. Nippon Ishinkin Gakkai Zasshi, 33(2), 207–211. https://doi.org/10.3314/jjmm.33.207
9. Piaton, E., Fabre, M., Goubin-Versini, I., Bretz-Grenier, M. F., Courtade-Saïdi, M., Vincent, S., Belleannée, G., Thivolet, F., Boutonnat, J., Debaque, H., Fleury-Feith, J., Vielh, P., Egelé, C., Bellocq, J. P., Michiels, J. F., & Cochand-Priollet, B. (2016). Guidelines for May-Grünwald–Giemsa staining in haematology and non-gynaecological cytopathology: recommendations of the French Society of Clinical Cytology (SFCC) and of the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP). Cytopathology, 27(5), 359–368. https://doi.org/10.1111/cyt.12323
10. Schnadig, V. J., & Woods, G. L. (2009). Histopathology of fungal infections. Clinical Mycology, 79–108. https://doi.org/10.1016/B978-1-4160-5680-5.00005-0
11. Walker, D. H., & McGinnis, M. R. (2014). Diseases Caused by Fungi. In Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms. Elsevier Inc. https://doi.org/10.1016/B978-0-12-386456-7.01710-X
12. West, K. L., Proia, A. D., & Puri, P. K. (2017). Fontana-Masson stain in fungal infections. Journal of the American Academy of Dermatology, 77(6), 1119–1125. https://doi.org/10.1016/j.jaad.2017.02.052

Aspergillus is a fungus found ubiquitously in the environment. Aspergillus is derived from the Latin word “Aspergere,” which means “to scatter”. In 1729, Micheli defined the genus Aspergillus. They are saprophytic mold and are found in decaying organic matter. They grow commonly as the molds on the substrate surface as the contamination in the bread and potatoes. More than 200 species of Aspergillus are found in nature, out of which only about 20 species can cause human disease. Out of these 20, the three most common pathogenic agents in about 95 percent of the cases are Aspergillus flavus, A. fumigatus, and Aspergillus niger. 

Aspergillus spp. causes aspergillosis, a systemic fungal infection that occurs both in the immunocompromised and the immunocompetent individuals. The sources of infection from the Aspergillus spp. are soil, air (inhalation of spores), water/storage tanks in hospitals, food, compost, decaying vegetation, fireproofing materials, ventilation, air conditioning systems, and computer fans. It exists as the mold form only. Aspergillus has septate hyphae that form the V-shaped dichotomous branches. The color of the colony on the culture media may be yellow, brown, or black, depending upon the species and its growth condition. There are different species of Aspergillus. They are

• A. flavus
• A. fumigatus
• A. niger
• A. terreus
• A. glaucus
• A. nidulans
• A. oryzae
• A. versicolor
• A. clavatus
• A. philaliseptus

Classification

• Kingdom: Fungi
• Phylum: Ascomycota
• Class: Eurotiomycetes
• Order: Eurotiales
• Family: Trichocomaceae
• Genus: Aspergillus
• Species: fumigatus, niger, clavatus, etc.

Aspergillus flavus

A. flavus is a thermotolerant fungus and can grow even in 48℃. It causes infection in the cereals, grains, and legumes. In immunocompromised patients, it causes otitis, keratitis, sinusitis, and pulmonary and systemic infections. After A. fumigatus, it is the second leading causative agent of aspergillosis. Since its spore’s size is big (3-6 µm), it gets deposited in the upper respiratory tract causing fungal sinusitis. It also causes cutaneous infection and non-invasive fungal pneumonia. A. flavus produces aflatoxin, which can cause acute hepatitis, hepatocellular carcinoma, and neutropenia in humans. 

Colonies of the A. flavus are velvety, yellow to green or brown. The reverse side is golden to red-brown. The reverse side is golden to red-brown. The conidiophores are of variable length, rough, pitted, and spiny; phialides are single and double, cover the entire vesicle, and point out all directions. 

Aspergillus fumigatus

A. fumigatus is a thermophilic fungus, and it can grow at 55℃ and survive up to 70℃. Due to the small size of the conidia, spores reach the alveoli in the lungs. It can germinate to hyphae and cause endothelial damage. It produces gliotoxin, inhibiting phagocytosis, which can evade the immune defense mechanism. Aspergillus fumigatus can infect the skin, eye, and other organs, causing aspergilloma and allergic bronchopulmonary aspergillosis. In the immunocompromised condition, aspergillosis can be fatal, leading to death. 

Colonies of the A. fumigatus are velvety or powdery at first, turning to smoky green. The reverse side is white to tan. The conidiophore is smooth, phialides single (uniseriate), usually covering the upper half vesicle parallel to the axis of a stalk. 

Aspergillus niger 

A. niger is also known as the black mold and causes infection in food and vegetables. In immunocompromised patients, it can cause invasive pulmonary aspergillosis. People exposed to horticulture are more likely to inhale its spores. A. niger can also cause otomycosis (infection of the ear). Some of the strains of Aspergillus niger also produce the mycotoxins called ochratoxin A.

Colonies of the A. niger are woolly at first, white to yellow, then dark brown to black. The reverse side is white to yellow. The conidiophore is of variable length; phialides are biseriate, i.e., arranged in two rows, covering the entire vesicle, forming a radiate head.

Aspergillus terreus  

A. terreus is also called the Aspergillus terrestrius. It is a fungal pathogen of humans, animals, and plants. In potatoes, it causes foliar blight disease. A. terreus has intrinsic AmB resistance in the invitro and invivo conditions. This intrinsic resistance mechanism has challenged antifungal therapy for the treatment of fungal infections. Morphologically A. terreus can be distinguished from the other Aspergillus spp. by aleuroconidia which is produced both in invitro and invivo conditions. Similarly, on the other hand, despite being a pathogenic fungal, Aspergillus terreus is being used in cancer therapeutics. It consists of anticancer bioactive compounds such as polyketides. Aspergillus terreus produces the statin group of polyketides (e.g., lovastatin), which has therapeutic value.

Colonies of the A. terreus are usually velvety cinnamon brown. The reverse side is white to brown. The conidiophores are short and smooth, phialides are in two rows, compactly columnar, and conidia are very small, i.e., 2 µm.

Aspergillus glaucus 

A. glaucus is a xerophilic fungus capable of growing at different temperatures. It is osmotolerant fungi and grows in a sugar concentration of 60 %. It can grow as outdoor fungi in winter and even in dry areas with low moisture content. Aspergillus glaucus can produce mycotoxin. It can cause pneumonitis and also dermatitis. 

Colonies of the A. glaucus are green with yellow areas, occasionally brown. The reverse side is yellowish to maroon. The conidiophores are variable in length and smooth. The phialides are single and radiate very loosely columnar.

Aspergillus nidulans 

A. nidulans (Emericella nidulans) are called homothallic fungi because they can fertilize themselves even without a mating partner. It has industrial importance as it produces enzymes like cellulases, hemicellulases, laccases, lipases, proteases, etc. 

Colonies of the A. nidulans are typically plain green in color, with dark red-brown cleistothecia developing within and upon the conidial layer. The reverse side may be olive to drab-gray or purple-brown. Colonial heads are short, columnar, and biseriate. Conidiophores are usually short, brownish, and smooth-walled. Conidia are globose and rough-walled. 

Clinical Features of Aspergillus 

Aspergillus spp. causes aspergillosis. Aspergillosis is a granulomatous, necrotizing, and cavitary disease of the lungs, often with the hematogenous spread to other organs. Tissue invasion or allergic disease occurs in this condition. Depending upon the modes of infection, it can be a primary or secondary infection. Aspergillus spp. can cause superficial infection of skin, nails,  external auditory canal, burn eschar, and paranasal sinuses. The various types of clinical conditions caused by Aspergillus spp. are:

• Pulmonary Diseases
• CNS Aspergillosis
•  PNS Aspergillosis
• Aspergillus Endocarditis
• Cutaneous Aspergillosis
• Miscellaneous Forms

1. Pulmonary Diseases

Pulmonary disease is the disease of the lungs and has following categories:

a) Allergic Aspergillosis

Aspergillus spp. causes bronchial asthma. It results in pulmonary eosinophilia or extrinsic allergic alveolitis. It occurs mainly in atopic persons. The chronic form of the disease is asthma with eosinophilia. It causes decreased lung function and progressive lung damage. The important characteristics of pulmonary disease are lung consolidation and fleeting shadows on chest x-rays.

i) Allergic Bronchopulmonary Aspergillosis (ABPA)

ABPA  is caused either by heavy or repeated exposure to spores of Aspergillus spp. In this condition, conidia germinate, and hyphae colonize the bronchial tree.  It results in allergic alveolitis and causes breathlessness, fever, and malaise.  It then produces the plugs of mycelium (mucous plug)  that occludes the lumen. The formation of the mucous plug is the diagnostic feature of the disease. This mucous plug may be coughed out.

ii) Obstructive Aspergillosis

Obstructive aspergillosis causes a progressive cough, no wheezing sound, eosinophilia, and hypoxia.

b) Aspergilloma

People with already preexisting lung cavities are at risk of aspergilloma. The fungus can settle and grow in the cavity. The fungus multiplies and forms the ball in it. Aspergilloma is also called the fungus ball. The colonization of the fungus occurs in the preexisting cavity. Aspergilloma is of various sizes and solitary. Approximately its diameter is 8-10 cm. Radiological diagnosis shows the air crescent or Monod’s sign because it looks like the crescent of air at the upper margin. It is usually asymptomatic or may have a moderate degree of cough and sputum production.

c) Invasive Aspergillosis

At first, the fungus develops pneumonia and then disseminates to other organs like the GI tract, kidney, liver, brain, heart, and other organs producing abscesses and necrotic lesions. There is a widespread growth of fungus in the lung tissue. It can disseminate further to the kidneys and brain. Risk groups include acute leukemia patients, solid-organ transplantation, AIDS, neutropenia, and transplant recipients.

d) Chronic Necrotizing Pulmonary Aspergillosis (CNPA)

In the immunocompromised host, CNPA or semi-invasive pulmonary aspergillosis may create its cavity. Then it grows as a non-invasive organism.

2. Central Nervous System (CNS) Aspergillosis

It is a fungal infection in which aspergillosis occurs in the central nervous system. Most cases occur by the hematogenous dissemination from pulmonary or gastrointestinal focus. The causative agent of CNS aspergillosis are  Aspergillus fumigatus,  A. flavus, A. terreus and A. versicolor. 

 Clinical manifestations of CNS aspergillosis are 

• Abscesses to granulomas
• Rhinocerebral form to meningitis
• Intracranial mass lesions (solitary or multiple)
• Ventriculitis

 The cranial and intracranial invasion also occurs following the infection within the sinuses. The different  clinical syndromes observed are:

• Encephalitis
• Meningoencephalitis
• Stroke like syndrome
• Intracranial space-occupying lesion
• Skull base syndrome
• Intra-orbital space-occupying lesion 

3. Paranasal Sinuses (PNS) Aspergillosis

 PNS Aspergillosis is the colonization and invasion of the paranasal sinuses by Aspergillus spp. Its types are: allergic, non-invasive, invasive, and fulminant.

•  Allergic form: It is similar to ABPA. It combines Type I and Type III hypersensitivity to Aspergillus antigens.
•  Non-invasive form: It results from the formation of aspergilloma. It behaves as chronic sinusitis.
•  Invasive form: It behaves like malignant neoplasia. It is slowly progressive and locally destructive.
•  Fulminant form: It is angioinvasive and rapidly destructive. The infection spreads from bone to the orbit of the eye and brain.

4. Aspergillus Endocarditis

In immunocompromised patients and persons with prior cardiac surgery, Aspergillus species may cause endocarditis. The large fungal vegetation occurs on heart valves. Fever and multiple embolic strokes can occur. Successful treatment depends on effective antifungal therapy and surgical removal of the infected tissue.

5. Cutaneous Aspergillosis

It is an infection of the skin and occurs by direct inoculation from trauma or surgery. It may be inoculated from objects like arm boards and external catheter sites. Primary cutaneous aspergillosis includes nodules, molluscum-like papules, plaques, and ulcers. Secondary cutaneous aspergillosis usually occurs on the chest wall. Cutaneous aspergillosis occurs in neutropenic cancer patients, neonates, and HIV patients. 

Superficial infections: Aspergillus flavus and Aspergillus fumigates colonize the paranasal sinuses, i.e., sinusitis, external ear (otomycosis)

6. Miscellaneous Forms

The other forms are keratomycosis, endophthalmitis, onychomycosis, mastoiditis, and osteomyelitis.  

Laboratory Diagnosis of Aspergillosis

The presumptive assumption of the fungus is made based on the colony morphology, and for the definitive identification, microscopic observation of hyphae and the conidial head is done. The conidial head consists of conidiophores. 

Sample

For the laboratory diagnosis of aspergillosis, the specimen of choice is sputum, sinus drainage, bronchial washing, bronchoalveolar lavage (BAL) fluid, a biopsy of the infected area (lung), and skin scraping.

1. Direct Examination

For the direct examination of the Aspergillus spp. 10 %, KOH wet mount is prepared. Then microscopic observation of the Aspergillus spp. is done. The hyphae of the Aspergillus spp. are hyaline and septate. Its diameter is 3-6µm, and dichotomous branching (type of branching in which two branches are approximately equal in size).

A biopsy is also taken in the case of fungal granuloma. For the examination, biopsy material should be kept in a tube of KOH. It is then incubated overnight at 37°C. For the histological examination of the biopsy material, the preferable staining methods are Hematoxylin and Eosin, Periodic Acid-Schiff, and Grocott-Gomori’s methenamine silver stain. Hyphae appear branched at a nearly 45-degree angle. Finger-like appearance is seen in the branching elements. In invasive aspergillosis, a proliferation of hyphae is seen throughout the tissue. It occurs in parallel or radial arrays. Aspergilli colonizing pulmonary cavity lesions grow as tangled masses of hyphae. 

The chronic infection may exhibit atypical hyphal features. Swelling may be seen in this condition, up to 12µm in diameter. Similarly, there may be an absence of conspicuous septa in this condition, as seen in the Aspergillus fungus balls. 

2. Fungal Culture

Different media used for the fungal culture are Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA), Czapek Dox Agar, and Malt extract agar. Inoculation is done in SDA agar with antibiotics and without cycloheximide at 25°C and 37°C, respectively. The isolate can be subcultured on the Czapek Dox agar and 2% Malt Extract Agar and incubated at 25°C. For the induction of the sporulation, PDA can be used. Culture should be examined after 48 hrs of inoculation. Then it should be examined daily for a week, twice a week for further four weeks, before discarding the plate. Colonies of different Aspergillus spp. produces different colors.

Colony morphology of Aspergillus spp. 

Species	Colonies	Reverse
A. flavus	Velvety, yellow to green or brown	Golden to red-brown
A.fumigatus	Velvety or powdery at first, turning to smoky-green	White to tan
A. niger	Woolly white to yellow at first, turning to dark brown to black	White to yellow
A. terreus	Velvety cinnamon brown	White to brown
A. glaucus	Green with yellow areas, occasionally brown	Yellowish to maroon
A. nidulans	Plain green in color with dark red-brown cleistothecia	Olive to drab-gray or purple-brown

Microscopic observation of Aspergillus spp.

Species	Phialides	Color of Conidia
A. fumigatus	Uniseriate	Grey, green or blue-green
A. flavus	Biseriate or Uniseriate	Yellow to green
A.niger	Biseriate	Black
A.terreus	Biseriate	Orange to brown
A. nidulans	Biseriate	Dark green

 Aspergillus spp. is a common laboratory contaminant. So, to confirm the relevance of positive culture, their quantitation is necessary. For this, 6-consecutive morning samples are required, out of which should show the same fungal growth in the 50% sample.

3. Immunodiagnosis

In the case of aspergilloma, a high percentage of patients demonstrates precipitating IgG antibodies. In case of allergic bronchopulmonary aspergillosis:

•  Skin test reaction is positive for Aspergillus antigens.
•  Elevated level of IgE
•  Specific IgE and IgG precipitating antibodies to Aspergillus spp. in serum.

i. Detection of Antibody

 An immunodiffusion test is done using commercial or laboratory-prepared antigens. Biotin-avidin-linked enzyme immunoassay (BALISA) is a sensitive enzyme-linked immunosorbent assay that uses biotin-avidin amplification systems. It is used for the demonstration of antibody isotypes. Antigens used are the crude culture filtrate, cell wall, or cytoplasmic extracts of A. fumigatus.

ii. Detection of Antigen

This test is useful for the detection of the antigen in immunosuppressed patients. In that condition, demonstrable antibodies may be absent in them. In invasive infection, antigen detection is useful. Various tests can detect the soluble antigen in the serum, urine, body fluids, and within-host phagocytic cells. They are:

•  Latex agglutination test
•  RIA
•  ELISA and BALISA

 For the demonstration of the Aspergillus antigens in the cerebrospinal fluid, western blot analysis was done by Ray and colleagues.

iii. Molecular Techniques

 Different molecular techniques for the detection of the Aspergillus spp. are DNA probes, DNA sequencing, PCR (real-time PCR), and nested PCR assay

iv. Molecular Typing

Molecular typing is based on the phenotypic and genotyping characters. Typing based on the phenotyping characters is based on antigens, enzymes, morphology, and biochemistry. Typing based on genotyping methods involves analysis of genomic DNA, mitochondrial DNA (mtDNA), and ribosomal DNA (rDNA). Restriction fragment length polymorphism (RFLP) involves restriction endonucleases that cleave the DNA on specific sequences.

v. Detection of Fungal Metabolites

For the detection of the fungal metabolites, G-test is done. G-test helps to detect circulating β-(1,3)-D-glucan. Aspergillus galactomannan (the cell wall content) is used as an indicator mannitol as a marker diagnosis of invasive aspergillosis.

vi. Skin Tests

 A skin test is done for patients with suspected allergic bronchopulmonary aspergillosis, atopic dermatitis, or allergic asthma. The 0.1 ml antigen (1000 PNU/ml aspergillin) is given intradermally for this test.

 Results:

•  Type I hypersensitivity: Erythema and wheal within 1 hour.
•  Type III: Arthus reaction within 4-10 hours develops.
•  Type IV reaction: induration >5mm diameter after 24 hours.

4. Animal Pathogenicity

Rabbits and mice are model animals for testing the pathogenicity of the Aspergillus spp. Conidia are injected intravenously, which results in granulomatous lesions in various organs, especially in kidneys.

Prevention  

Mostly immune-compromised individuals and persons with severe lung diseases are at risk of the infection. To prevent aspergillosis, the best way is to avoid exposure to the Aspergillus spp. Some of the approaches one can consider to reduce exposure to fungal infections are as follows:

• Avoid direct and close contact with the soil or dust, such as during fieldwork or gardening. While going to the construction sites close to the soil, wear a mask properly. It will help to reduce the chances of inhalation of the conidia directly from the vegetation.
• To improve the quality of air, use HEPA filters.
• Use soap and water to clean the skin injury exposed to the soil.

Treatment

For the treatment of the fungal infection caused by Aspergillus, different antifungal agents such as voriconazole, itraconazole, or amphotericin B are used. Voriconazole is found to be more effective than amphotericin B.

• Aspergilloma: Itraconazole, Amphotericin B
• Invasive aspergillosis: Voriconazole
• ABPA: Steroids with anti-fungal agents
• Allergic aspergillosis: Corticosteroids with sodium cromoglycate   

References

1. Abdin, M. Z., Ahmad, M. M., & Javed, S. (2010). Advances in molecular detection of Aspergillus: An update. Archives of Microbiology, 192(6), 409–425. https://doi.org/10.1007/s00203-010-0563-y
2. Cadena, J., Thompson, G. R., & Patterson, T. F. (2016). Invasive Aspergillosis: Current Strategies for Diagnosis and Management. Infectious Disease Clinics of North America, 30(1), 125–142. https://doi.org/10.1016/j.idc.2015.10.015
3. Chander, J. (2018). Textbook of Medical Mycology (Fourth edition). Jaypee Brothers Medical Publishers Ltd.
4. Sharma, S., Dubey, S. K., Kumar, N., & Sundriyal, D. (2013). “Monod” and “air crescent” sign in aspergilloma. BMJ Case Reports, 1–2. https://doi.org/10.1136/bcr-2013-200936

 

Dermatophytes are keratinophilic and keratinolytic fungi. They can affect all keratinized body areas (hair, skin, and nails) and cause cutaneous mycosis (dermatophyte infection or dermatophytosis).

Dermatophytosis spreads by direct or indirect contact with the infected animal or humans. Indirect transfer can occur from the swimming pool floor, showers, brushes, etc. Direct transfer can occur by the viable fungus in the keratin fragments of skin, hair, and nails. The three clinically significant dermatophytes are Trichophyton, Microsporum, and Epidermatophyton.

1. The genus Trichophyton is capable of invading the hair, skin, and nails.
2. The genus Epidermophyton involves the skin and nails only (not hairs).
3. The genus Microsporum involves only the hair and the skin (not nails).

Mnemonics to remember which dermatophyte does not affect what:

Trichophyton: Infects all three (so Tri)– i.e. Skin, Nail and Hair

Epidermophyton does not affect Hair (REMEMBER “E” is close to ‘H’ alphabetically and it does not infect near ones)

Microsporum does not affect Nails as ‘M’ is close to ‘N’ (similar scenario of not infecting neighbors)

Dermatophytes cause Tinea or ringworm infections. The common symptoms of dermatophytes are irritation, erythema (redness of the skin), edema (swelling), and vesiculation (formation of the vesicle or cyst). In the Tinea pedis, infection is mainly confined to the toe’s clefts. An infected nail gets discolored, thickened, raised, and friable (easily reduced to powder). Most nail infections are due to the T. rubrum.

Classification of Dermatophytes

Dermatophytes are classified into three genera based on the conidia’s morphology and formation: Trichophyton (T), Microsporum (M), and Epidermatophyton (E). Conidia is the structure of asexual reproduction. Many dermatophyte species produce two types of asexual spores: multi-celled macroconidia and single-celled microconidia (arthroconidia).

Dermatophytes are classified into three groups based on their habitat: soil (geophilic species), animals (zoophilic species), and man ( anthropophilic species).

• Anthropophilic organisms: T. rubrum, T.interdigitale, T. tonsurans, T. violaceum, M.audouinii, M. ferrugineum.
• Zoophilic organisms: M. canis ( originating from cats and dogs), M. nanum (originating from pigs), T.equinum (originating from horses), T. verrucosum ( originating from cattle)
• Geophilic organisms: M. gypseum, M. fulvum

Pathogenesis of Dermatophytes

Virulence factors of dermatophytes are arthroconidium (asexual spores), enzymes (keratinase, protease, lipase), and mannan of the fungal cell wall.

Adherence

Arthroconidium (asexual spores produced by dermatophytes) adheres to the surface of the tissue. Trichophyton rubrum adheres to the epithelial cells by the carbohydrate-specific adhesions, which are expressed on the surface of the spores. Fibrillar projections have been observed in the T. mentagrophytes during the adherence phase. At the skin surface, long and sparse (thin) fibrils connect fungal arthroconidia to keratinocytes.

The arthroconidium then germinates into hypha, and hypha enters into the stratum corneum (the outermost layer of the epidermis). In the pathogenesis of dermatophytoses, the initial interaction between the arthroconidia and the stratum corneum occurs 3-4 hours after contact.

Penetration

After the adherence, dermatophytes need to obtain the nutrients for their survival. To use the nutrients, the proteins, starch, cellulose, and lipids are degraded into smaller compounds. Dermatophytes then secrete various enzymes like proteases, lipases, elastases, collagenases, phosphatases, and esterases to degrade the complex compounds into simple ones. Keratinases degrade the keratin present in the host tissues into oligopeptides or amino acids. Proteolytic enzymes degrade the proteins of the skin. It helps in the penetration of the stratum corneum.

Development of host response

Dermatophytes have mannan, which suppresses the inflammatory response. It also inhibits the proliferation of the keratinocytes, which allows for establishing the persistent infection.

Clinical features

Dermatophytes cause dermatophytosis, which is also known as the tinea or ringworm. The clinical presentation of the infection depends on the species, strain of fungus, size of the inoculum, anatomical site, and immune status of the host.

1. Tinea capitis

Tinea capitis is the infection of the shaft of scalp hairs caused by the fungus Trichophyton spp. and Microsporum canis. The infection starts on the scalp, where the dermatophytes grow down the keratinized wall of the hair follicles. The infected hair appears dull and grey. T. capitis presents two clinical forms: inflammatory and non-inflammatory.

• Inflammatory: Kerion, favus, and agminate folliculitis
• Non-inflammatory: Black dot, seborrheic dermatitis-like, and grey patch

Kerion

It is the severe form of dermatophytosis which causes deep, suppurative lesions on the scalp. Follicles may be seen with the discharging pus. It is usually caused by the T. verrucosum and T. mentagrophytes, zoophilic dermatophytes. In children, one of the common clinical forms of tinea infection is kerion celsi.

Favus

The word favus is derived from the Latin word favus which means the honeycomb-like appearance. Favus is caused by T. schoenleinii, and it forms the cup-like crusts around the infected follicles. The fungal growth occurs within and around the hair follicle, which produces waxy, honeycomb-like crusts on the scalp. It may lead to alopecia (loss of hair) and scarring.

Black dot
Black dot is usually caused by the T. tonsurans and T. violaceum. It attacks the hair shaft by endothrix type invasion with the abundant sporulation inside the hair and breakage of the hair near the surface of the scalp. It results in a black-dot appearance within an area of smooth scalp surface.

2. Tinea corporis

Tinea corporis is caused by Trichophyton rubrum. It is the infection of the body’s glabrous (non-hairy) skin and is caused by tinea glabrosa or tinea glabrata circinata. It is characterized by erythematous scaly lesions and annular, sharply marginated plaques with a raised border that may be single, multiple, or confluent.

3. Tinea pedis (Athlete’s foot)

Tinea pedis is also known as the athlete’s foot because it is seen in individuals who wear shoes for a long period. The warmth and the moisture facilitate the establishment of the fungus.

It causes the infection of the foot, toes, and interdigital web spaces. In the toe webs, scaling, fissuring, maceration, and erythema are associated with the itching and burning sensation. The small vesicles rupture and the thin fluid is discharged. Then there are the chances of secondary infection as the skin will become macerated and peel. Lymphangitis and lymphadenitis will occur in the case of secondary infection.

Further, it will cause the Moccasin or Sandal ringworm. In this condition, the infection extends from the sole to the side of the foot. It is also known as the one-hand-two feet syndrome because it causes infection in the one-hand and two feet of the patient. In the chronic infection, the sole becomes hyperkeratotic, often covered by fine scales. The causative agent of the athlete’s foot is Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermatophyton floccosum. Tinea pedis is divided into four types: interdigital, hyperkeratotic, ulcerative, and vesicular.

4. Tinea barbae

Tinea barbae is the ringworm infection of the beard and mustache area of the face. It is also called barber’s itch or tinea sycosis. In this condition, erythematous patches can be seen on the face, which shows scaling, fragile, lusterless hairs. It has got a tendency to develop folliculitis. The main causative agent of the Tinea barbae are Trichophyton verruosum,Trichophyton mentagrophytes.  Microsporum canis and T. rubrum are occasionally found in it.

5. Tinea manuum

Tinea manum is the palm’s ringworm infection caused by T. rubrum, T. mentagrophytes, and Epidermatophyton floccosum.

6. Tinea unguium

Tinea unguium is the dermatophyte infection of the nail, caused by T. rubrum, T. mentagrophytes, and Epidermatophyton floccosum. 

Causative agents

Tinea is caused by the dermatophytes, which include the fungus of the genus Trichophyton, Microsporum, and Epidermatophyton.

Trichophyton spp.

Trichophyton grows only within dead keratinized tissue. It infects skin, hair, and nail. It produces the keratinolytic protease enzyme, which facilitates the entry of the fungus into the living cells.  The important trichophyton species are Trichophyton rubrum, T. mentagrophytes, T. tonsurans, T. schoneleinii, T. violaceum, T. verrucosum. The Trichophyton rubrum does not perforate the hair in vitro and does not produce urease enzyme, whereas T. mentagrophytes perforates the hair in vitro and produce urease enzyme.

Epidermatophyton spp.

Epidermatophyton floccosum is the causative agent of Tinea cruris and Tinea pedis. It doesn’t invade the hair and grows in the epidermis, i.e., skin and nails. It doesn’t produce the microconidia, whereas another dermatophyte, Trichophyton, and Microsporum produce both the microconidia and macroconidia. Epidermatophyton produces only macroconidia.

Microsporum spp.

Microsporum infects the skin and hair but not the nail. The three species of Microsporum that is mostly pathogenic are Microsporum audouinii, M canis, and M gypseum. Rice grain test is the specific test for Microsporum spp. Microsporum spp. primarily grow on the surface of hair shafts (ectothrix), whereas Trichophyton spp. may grow inside the hair shafts (endothrix) and makes the hair fragile and breaking it or at the surface of the hair follicles.

Laboratory diagnosis of Dermatophytes

Clinical observation and laboratory investigation play a crucial role in diagnosing fungal infection. The sample of choice are the skin, nail, and plucked hairs of short length from the involved sites. 

1. Collection of samples

Hair, nail, and skin samples are taken in a folded square of black paper or the card. When paper is used, it helps to dry out the specimens preventing them from bacterial contamination. It helps to store the fungus for 12 months without losing its viability.

Hairs

For the hair sample collection, instead of cutting, hair is plucked from the edge of the lesion. 

Skin

For the skin sample collection, it needs to be washed well and then scraped from the margin of the lesion onto the folded black paper.

Nail

Nails scrapping is taken from the nail bed or infected areas after discarding the outer layers.

Scales

It includes the hair stubs, the contents of plugged follicles, and skin scales.

Hairbrush sampling

Use the sterile plastic brush and brush it on the scalp. Then inoculate the plastic brush by pressing it in the agar suitable culture media.

2. Wood’s Lamp Examination

It is a device used to diagnose and manage superficial cutaneous fungal infections. It is used for the examination of the scalp and ringworm infection. Most of the infected hair fluorescence when exposed to ultraviolet light. Microsporum audouinii and Microsporum canis fluorescens bright yellow-green, whereas Microsporum gypseum does not fluorescens. Non-fluorescent fungi like T. tonsurans and T. verrucosum are difficult to detect. In Wood’s lamp, positive fluorescence occurs in infected hairs due to using a chemical substance called pteridine. Sometimes false positives may occur if other substances also consist of pteridine. An error may occur when the ointments containing petroleum, jelly, scales, serum, exudates, lint, and dried soaps fluorescens bluish or purple.

Microorganisms	Fluorescence Color
Microsporum audouinii	Bright-green
Microsporum canis	Bright-green
Microsporum ferrugineum	Blue-green
Microsporum distortum	Blue-green
Microsporum gypseum	Dull-yellow
Trichophyton schoenleinii	Dull-green
Malassezia furfur	Golden-yellow
Corynebacterium minutissimum	Coral-red

3. Direct microscopy

Place the specimen on a clean, grease-free slide and add 10-20% KOH. Cover it with the cover slip and examine it after 20 minutes. In the skin or nails, branching arthrospores are seen. In the hair, Trichophyton spp. forms parallel rows of spores outside (ectothrix) or inside (endothrix) of the hair shaft. T. tonsurans and T. violaceum show the endothrix type of hair invasion. T. mentagrophytes show ectothrix type of hair invasion. Hairs infected with the T. schoneleinii show hyphae and air spaces within the shaft.

Read more from this link: KOH wet mount

4. Fungal Culture

The suitable media for the fungal culture is Potato Dextrose Agar (PDA) or Sabouraud’s dextrose agar (SDA) used for culture. Incorporating antibiotics like gentamicin and chloramphenicol in Sabouraud’s media will inhibit the overgrowth of bacteria. Incorporating the cycloheximide will inhibit the overgrowth of the rapidly growing saprophytic fungi. Sabouraud peptone-glucose agar (Emmons’ modification) mixed with cycloheximide and chloramphenicol is commonly used. Its commercial names are Mycobiotic and Mycosel agars. Inoculation is done at 25-30℃ for 1-3 weeks. The fungi are identified based on colonial appearance, color, pigment production, and microscopic observation. For the microscopic observation, lactophenol cotton blue staining is performed.

Morphological Characteristics of Dermatophytes

Dermatophytes	Colony morphology	Microscopic observation
Trichophyton rubrum	Velvety, red pigment on reverse	Tear-drop, microconidia, a few long, pencil-shaped macroconidia
Trichophyton mentagrophytes	White to tan, cottony or powdery, pigment variable	Clusters of microconidia cigar-shaped macroconidia with terminal rat-tail filaments
Trichophyton tonsurans	Powdery to cream or yellow with central furrows	Abundant microconidia, thick-walled, irregular macroconidia
Trichophyton schoenleinii	Smooth, waxy, brownish	Hyphal swellings, chlamydopsores, favic chandelier
Trichophyton violaceum	Very slow growing, waxy, violet to purple pigment	Distorted hyphae, conidia rare
Microsporum audouinii	Velvety, brownish, slow growing	Thick-walled chlamydospores, conidia rare and irregular
Microsporum canis	Cottony, orange pigment on reverse	Abundant, thick-walled spindle-shaped macroconidia with up to 15 septa
Microsporum gypseum	Powdery, buff-colored Yellowish-green	Abundant, thin-walled macroconidia with 4-6 septa
Epidermatphyto floccosum	Slow growing, powdery, greenish-brown	Club-shaped macroconidia in clusters

5. Rice Grain Test

Rice grain test differentiates the M. audouinni from the other Microsporum species. M. audouinii doesn’t grow in the presence of rice grain. Sterile non-fortified rice is inoculated lightly with the hyphae of the test isolate. Then after 10 days of incubation at room temperature, the medium is observed for growth. M. canis and other dermatophytes grow well and usually form many conidia, but M. audouinii does not grow.

6. Dermatophyte Test Medium (DTM)

Dermatophyte Test Medium is used for the presumptive identification of dermatophytes from the other fungal or bacterial contaminants. It helps to distinguish whether the cutaneous lesion is caused by dermatophytes, other fungi, or bacteria. When incubated at 25℃, the dermatophytes turn the medium red, whereas bacteria and other fungi cannot change it. To avoid the false positive result caused by DTM, dermatophyte identification medium (DIM) is also used.

7. Hair Perforation Test

Hair Perforation is the differential test to differentiate the morphologically similar fungal species. It is used to distinguish the Trichophyton rubrum and Trichophyton mentagrophytes. Trichophyton rubrum causes the surface erosion of the hair shaft, whereas Trichophyton mentagrophytes cause wedge-shaped perforation. The hair perforation test also differentiates the Microsporum canis and Microsporum equinum. Microsporum canis perforates the hair, but the Microsporum equinum does not perforate.

8. Urea hydrolysis test

Incubation is done for 2-3 days on Christensen’s Urea Agar. It is used to differentiate between Trichophyton rubrum and Trichophyton mentagrophytes. When the urea is hydrolyzed, the media turns deep red. T. mentagrophytes shows positive results as it produces urease enzyme within 2-4 days, whereas T. rubrum shows negative results.

Treatment

For most skin infections, topical therapy is satisfactory. Oral antifungals are required to treat nail and scalp infections and severe or extensive skin infections. Topical agents include azole compounds, terbinafine, amorolfine, and ciclopirox olamine. Oral griseofulvin is beneficial for scalp, skin, and fingernail infections. Terbinafine and itraconazole have largely replaced griseofulvin for treating nail infections because of their much better cure rates and shorter treatment periods.

Prevention and Control

• Routine inspection of the scalps should be done, and avoid sharing of combs and brushes.
• Barbershop instruments ( combs, brushes, and scissors) must be disinfected.
• Infected clothing, towels, and bedding, these items need to be disinfected after use because there is a chance of transmission of the Tinea corporis and Tinea cruris.
• Prevention of the tinea pedis can be enhanced by using good foot hygiene.

References

1. Brasch, J., Müller, S., & Gräser, Y. (2015). Unusual strains of Microsporum audouinii causing tinea in Europe. Mycoses, 58(10), 573–577. https://doi.org/10.1111/myc.12358
2. GREGORY., P. H. (1935). the Dermatophytes. Biological Reviews, 10(2), 208–233. https://doi.org/10.1111/j.1469-185X.1935.tb00483.x
3. Su, H., Packeu, A., Ahmed, S. A., Al-Hatmi, A. M. S., Blechert, O., Ilkit, M., Hagen, F., Gräser, Y., Liu, W., Deng, S., Hendrickx, M., Xu, J., Zhu, M., & De Hoog, S. (2019). Species distinction in the Trichophyton rubrum complex. In Journal of Clinical Microbiology (Vol. 57, Issue 9). https://doi.org/10.1128/JCM.00352-19
4. Chander, J. (2018). Textbook of Medical Mycology (Fourth edition). Jaypee Brothers Medical Publishers Ltd.
5. profile, V. (2022). Trichophyton rubrum. Thunderhouse4-yuri.blogspot.com. Retrieved 21 August 2022, from http://thunderhouse4-yuri.blogspot.com/2012/02/trichophyton-rubrum.html.

Mycotoxins, the secondary metabolites of fungi, are toxic compounds to animals and humans (mycotoxicosis). 25% of the agricultural products worldwide get contaminated with mycotoxins. The ingestion of mycotoxins causes various acute and chronic biological effects, including carcinomas, mutations, genetic variation, and immunological effects.

Aflatoxins are a group of 16 structurally related mycotoxins that are carcinogenic, teratogenic, and hepatocarcinogenic. The methods generally used for detecting and quantifying aflatoxins are analytical, chromatographic, immunochemical, immunosensors, and spectroscopic techniques.  

Aflatoxin (AF)

• Sources of aflatoxins: Molds like Aspergillus flavus, A. fumigatus, A. parasiticus, Penicillium puberculum, P. citrinin, and P. expansum produces these toxins in foods like corn, peanuts, etc.
• Factors favoring the production: Temperature and grain moisture.
• Types of aflatoxins: B1, B2, G1, G2, M1, and M2 are the family of aflatoxins in food. B1 toxin is the most potent naturally occurring carcinogen. B-type has a characteristic cyclopentane E-ring, and G-type has a xanthone ring instead of cyclopentane. AFM1 and AFM2 are the metabolites of AFB1 and AFB2. If a dairy cow consumes B1 contaminated feed, the excretion of M1 occurs in milk.
• Mechanism of action: Aflatoxins disrupt the breakdown of fats in the liver. Fatty liver or steatosis is a short-term or long-term condition of aflatoxins poisoning. The aflatoxin also inhibits mRNA synthesis by binding to guanine in DNA, leading to cancer.
• Toxicity: AF is more toxic in children than adults. Its susceptibility increases during nutrition deficiency.
• Clinical sign: 
  • It decreases the growth rate.
  • The toxin reduces feed efficiency.
  • Mild anemia. 
  • Increased susceptibility to infectious disease. 

• Treatment:
  • Detoxification using sodium calcium aluminosilicate (HSCAS).
  • Vitamin E and selenium are provided as supplements.

• Prevention: 
  • Inhibition of mold growth.
  • Treatment of grain with anhydrous ammonia for 10-14 days.

Materials for Aflatoxin Testing

Aflatoxin in food after consumption poses a significant health risk due to its severe toxicity. So, aflatoxin testing is very crucial in foods. Due to its low concentration, analytical methods like chromatographic, spectroscopic, electrochemical sensors, and immunochemical techniques are preferred. The sample is suspected food material. 

The materials required for aflatoxin testing depend on the methods used for extraction, detection, and quantification. The materials needed are as follows:

For extraction and purification

• An organic solvent like methanol, acetone, or acetonitrile mixed or not mixed with water.
• Solid matrix and method/water and acetonitrile/water mixture
• Flask, centrifuge tube, or vials and ultrasonic bath
• Supercritical fluid (CO₂)
• Immunoaffinity column for purification

Chromatographic method

• Frits
• Columns
• Flow cells
• Pumps
• Detectors
• Collectors
• Cylinder with gas (gas chromatography)
• Silica gel, aluminum foil, solvent (thin layer chromatography)
• Solvent (high-performance liquid chromatography)

Spectroscopic analysis

• Spectrophotometer

Immunological detection

• Pipettes
• For ELISA (enzyme-linked immunosorbent assay): Coated plate with 96-wells, reader, conjugate, conjugates, and stop solution.
• For RIA (radioimmunoassay): 
• Lateral flow kit

Immunosensor detection 

• QCM (quartz crystal microbalance) sensor
• SPR (surface plasma resonance) sensor
• Electrochemical immunosensor

Methods of Aflatoxin Testing 

The first step of testing aflatoxin is extraction. Then it is followed by purification and concentration. The final step is detection and quantification.

Extraction and Purification (Clean-up)

Extraction is key to better detection and quantification of aflatoxin in food samples. The extraction and purification of aflatoxin can occur in the following ways.

• Liquid-liquid extraction (LLE): Aflatoxin is highly soluble in polar-protic solutions like acetone, chloroform, methanol, and acetonitrile. These can either be mixed with water or used in their pure state. It does not produce clean analytes.
• Liquid-solid extraction (LSE): A fixed amount of solid food particles is mixed using centrifugal force (mixer or vortex) with an extraction reagent, either acetonitrile/water or methanol/water, in varying ratios. It is the most common method, followed by the purification method like IAC (immunoaffinity column). 
• Ultrasound extraction: The mixed sample from LSE containing flask, centrifuge tube, or vial) is dipped into the ultrasonic bath with water. The ultrasound helps quick transfer the toxin from the sample to the extraction reagent.  
• Supercritical fluid extraction (SFE): The use of supercritical fluid CO₂ to extract apolar aflatoxin has shown promising results. However, it is not ideal for polar aflatoxins because it has a low recovery of the toxin and a higher concentration of co-extracts.
• Solid phase extraction (SPE): It is a popular method of clean-up or purification. It uses a C-18 (octadecylsilane) column. Its application is in the immunoaffinity clean-up column (IAC) and SPME (solid-phase microextraction). SPME is compatible with chromatographic techniques, and IAC has columns with antibodies specific to the toxins. SPME has high specificity in foods like nuts, spices, dried fruits, and cereals. In contrast, IAC is useful in pasteurized milk. During IAC, the crude extracted sample is applied to the column. The aflatoxin binds to a specific antibody immobilized in the column. A washing step is required after IAC. 

Detection and Quantification

Various methods have been applied to detect and quantify extracted and purified aflatoxins. They are chromatographic techniques (TLC, HPLC, and GC), Spectroscopic techniques (fluorescence and infrared spectroscopy), immunological techniques (RIA, ELISA, and later flow immunoassay), and immunosensors (electrochemical, optical, and QMC).

Chromatographic Techniques

Chromatographic techniques involve interaction between a mobile and a stationary phase. The mobile phase is usually a liquid, gas, or supercritical fluid that moves around the stationary phase (solid or liquid). The mixture with desired components gets distributed between the two phases (mobile and stationary). The general practice is dissolving the mixture into the mobile phase and applying it as a spot in the stationary phase. The mobile phase carries the mixture along the stationary phase, and the speed of different components of the mixture varies, which is the basis of separation. 

The chromatographic techniques used commonly are thin-layer chromatography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC). 

Thin layer chromatography (TLC)

TLC has been widely used in detecting aflatoxins in food and has found aflatoxins as low as 1-20 ppb (parts per billion). Its stationary phase comprises silica, alumina, or cellulose immobilized in glass or plastic. The mobile phase used for separating aflatoxins is a mixture of the sample with acetonitrile, methanol, and water. The difference in solubility of aflatoxins determines the interaction time between the mobile and stationary phases. Depending on the interaction time and molecular weight of the toxin, it either adheres to the stationary or mobile phase. The longer it adheres to the stationary phase, the later it separates from the mixture and vice versa. TLC helps to detect different types of toxins in a single test. Although it is sensitive, it requires highly trained professionals and lacks precision due to applying samples, plate development, and interpretation errors. 

HPTLC (high-performance thin layer chromatography) has been developed in order to improve the limitation of TLC. The HPTLC is an automated process where sample application, plate development, and interpretation are automatic. HPTLC is currently the most precise and efficient technique for determining mycotoxins.

High-performance liquid chromatography (HPLC)

HPLC is the most common technique used for the separation of organic components. It has an absorbent inside a glass or plastic tube (column) as a stationary phase and an aqueous/organic solvent as a mobile phase. The sample is injected into the stationary phase and flows through it with the help of the mobile phase. A pump provides high pressure for fast movement of the mobile phase through the column (stationary phase). The separation occurs due to the different affinities of the components toward the stationary and mobile phases. The higher the affinity, the longer analyte reacts with the particular phase. If an analyte interacts longer with the mobile phase, it separates faster.

The time taken for the elution (separation followed by removal from the setup) of analytes (mixture) is called retention time. The retention time is detected using UV, fluorescent, or diode array detectors. HPLC is used widely in the determination of aflatoxins. However, it requires rigorous sample purification using immunoaffinity columns and tiresome pre and post-column derivatization for detecting AFB1 and AFG1 due to the use of UV for detection. Hence to overcome the derivatization, HPLC is combined with mass spectroscopy limiting the sample size for generating structural information. But HPLC-MS/MS is bulky and suitable only in laboratory environments.

Gas chromatography (GC)

In GC, the mobile phase is carrier gas, and the stationary phase is liquid coated on inert solid particles confined to a long stainless steel or glass tube (column). The stationary phase needs to maintain an appropriate temperature for separation. The sample is vaporized into its gaseous state and carried by the carrier gas along the stationary phase. Like all chromatographic techniques, the basis of the separation of components is their distribution among the two phases. The sample’s movement depends on the components’ affinity with the stationary phase. The higher the affinity, the slower the movement and the longer it takes to separate and vice versa. Once the separation completes, the separated particles are detected using FID (flame ionization detector), or electron capture detector (ECD), and a mass spectrometer. Since aflatoxins are non-volatile, these may need derivation for detection and are less commonly used. 

Spectroscopic Techniques

Fluorescence spectroscopy

Aflatoxins are fluorescing compounds that emit light of various wavelengths after absorption. The fluorometric method help quantify aflatoxins in the range of 5-5000 ppb. However, derivatization is required for better analysis, and the limit for detection is slightly higher than the limit of 4 μg/Kg set by European standards. 

Infrared spectroscopy

Infrared spectroscopy depends on the difference in molecular vibrations after irradiation with infrared. Atomic size, bond length, and strength vary from one molecule to another. The absorption rate of infrared radiation by a particular bond depends on the bond type and mode of vibrations. So, different frequency ranges of infrared radiation are passed through the sample during the analysis of compounds using an infrared spectrometer. The absorbed radiant energy by every bond of the sample molecule is then measured. The measurement helps construct the spectrum graph, and no two organic molecules have the same spectrum, helping identify the organic compound. It has been used in determining aflatoxins in peanuts, peanut cakes, and corn kernels.

Immunological Techniques

Immunological techniques involve the specific binding of antibodies and antigens. Various immunological methods have been developed based on the specificity and high-affinity interaction between antibodies and antigens and are expanded to receptor-ligand bonding. These interactions can also be quantified using the absorbance of photons of light energy by spectrophotometer. The other ways of detection and quantification are labeling with radioisotopes, fluorophores, and enzymes. The immunochemical methods used commonly are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunosensors, and ICA (immunocolumn assay). 

Radioimmunoassay (RIA)

RIA relies on the competitive binding of the radiolabeled antigens and non-radiolabeled antigens with available antibodies. The unlabeled antigen competes with the radioactive antigen for binding with a fixed number of antibodies. The amount of radiolabeled antigen and antibody is known, whereas the quantity of non-labeled antigens is unknown, but the amount is inversely proportional to the amount of radiolabeled antigens. RIA is used in the qualitative and quantitative determination of aflatoxins. Although the RIA can analyze multiple samples at once, these require antigens in the pure state, and the radioactive isotope is hazardous and hard to dispose of and store.   

ELISA (enzyme-linked immunosorbent assay)

Another immunochemical method that overcomes the hazardous limitation of RIA is ELISA, where an antigen or antibody is bound with enzymes. The principle of ELISA is similar to that of other immunochemical methods that rely on specific antigen-antibody binding and increased sensitivity due to antigen or antibody linked with an enzyme that combines with a particular substrate. 

ELISA is available in a kit and can easily detect aflatoxins in agricultural products. Although it requires many washing steps, it is cost-effective, can analyze 96 samples simultaneously, and carries no health hazards.

Lateral flow devices (kit)

Lateral flow devices are immunochromatographic assays. The principle is based on the antigen-antibody interaction. These consist of a porous membrane for flowing samples, an absorbent pad for increasing the volume of flowing liquid, and a sample pad to ensure contact between the liquid sample and the membrane. The devices use colloidal gold or gold-coated antibodies to detect the red-colored binding zones. The sample moves through the sample pad due to capillary action. When the sample with aflatoxins reaches the gold particles, these bind with the particle giving the red colored line. A device made by Delmulle et al., can detect 5 μg/Kg AFB1 in pig feed within 10 minutes. These devices are easy to use, cost less, and provide rapid on-site detection.

Immunosensor Techniques for Detection

Immunosenors are biosensors that use antigens or antibodies as recognition components combined with signal transducers like graphite, gold, or carbon. These help to detect binding between complementary species. Based on the signal transduction used, immunosensors are of three types; piezoelectric, optical, and electrochemical. 

Electrochemical immunosensors

These devices use antibodies added in a biorecognition layer for producing electroactive signals that are detectable by transducers resulting in the generation of measurable signals. Aflatoxin analysis can be done using various types of electrochemical immunosensors. The most common type consists of antibodies immobilized to the surface of the glassy carbon electrode, and an enzyme is a biological component used. However, there has also been the construction of non-enzymatic electrochemical immunosensors using chitosan, gold particles, anti-aflatoxin B1 and Fe₃O₄ as electrodes.

Optical immunosensors

Surface plasmon resonance (SPR) is the optical immunosensors used for detecting aflatoxins. It depends on measuring the refractive index produced after binding of the analyte with its biospecific partner (specific antibodies) that is immobilized in the sensor surface. It is used in detected AFB1 using monoclonal or polyclonal antibodies of AFB1. OWLS (optical waveguide light-mode spectroscopy) is another label-free biosensor that relies on fluorescence excitation for measuring the binding events at the surface of the waveguide. OWLS detects aflatoxins and ochratoxins; the detection range is 0.5-10 ng/ml in wheat flour and barley.

Piezoelectric QCMs (quartz crystal microbalances)

These are label-free devices used in detecting antigens. T depends on the change in mass of the electrode when antigens react with the immobilized antibody in the quartz crystal surface. The difference in mass is proportional to the concentration of the antibody-antigen complex. QCM sensor is coated with gold for detecting aflatoxin B1 in the range of 0.5-10 ppb.  

References and further reading

• Abbas, M. (2021). Chromatographic Techniques for Estimation of Aflatoxins in Food Commodities. In (Ed.), Aflatoxins – Occurrence, Detoxification, Determination and Health Risks. IntechOpen. https://doi.org/10.5772/intechopen.98508
• Wacoo, A., Wendiro, D., Vuzi, P., & Hawumba, J. (2014). Methods for Detection of Aflatoxins in Agricultural Food Crops. Journal Of Applied Chemistry, 2014, 1-15. https://doi.org/10.1155/2014/706291
• Miklós, G., Angeli, C., Ambrus, Á., Nagy, A., Kardos, V., & Zentai, A. et al. (2020). Detection of Aflatoxins in Different Matrices and Food-Chain Positions. Frontiers In Microbiology, 11. https://doi.org/10.3389/fmicb.2020.01916
• Eurofins Aflatoxin Testing. Aflatoxin Testing. Retrieved 1 August 2022, from https://www.eurofins.com/food-and-feed-testing/food-testing-services/mycotoxin-testing/aflatoxin-testing/.
• Leszczyńska, J., MasŁowska, J., Owczarek, A., & Kucharska, U. (2013). Determination of aflatoxins in food products by the ELISA method. Czech Journal Of Food Sciences, 19(No. 1), 8-12. https://doi.org/10.17221/6567-cjfs
• Espinosa-Calderon, A., Miguel, L., Francisco, R., Roberto, J., Guevara Gonzalez, R., & Torres-Pacheco, I. (2011). Methods for Detection and Quantification of Aflatoxins. Aflatoxins – Detection, Measurement And Control. https://doi.org/10.5772/28439

Look at the walls around you. Do you see moisture or black/green coloration in those walls? It might signify that your home is turning into a mold breeding ground. Another signal of mold growth in the house is a musty or rotten smell. 

Mold belongs to the kingdom of Fungi or Mycota and is a multicellular eukaryotic microorganism. The damp or moist area is the most suitable habitat for mold growth. Mold growth is typical in the regions that have been flooded recently. Also, mold growth is expected inside household settings with moisture from the leaking roof, pipes, and windows.

Mold growth also requires nutrients like carbon, lipids, and proteins. The dust in the construction material like textile, wallpaper, etc., contains plant and animal residue, which can be a nutrition source. According to CDC, Cladosporium, Aspergillus, and Penicillium species are the common mold that grows indoors. 

Areas in the House Affected

Molds can grow in paper, cardboard, fabric, paint, textiles, tiles, drywall, dust, and things that are in regular exposure to water and retain moisture. The most commonly affected areas of houses are as follows:

• Moist walls 
• Roof after constant raining
• Air conditioning ducts 
• Carpets
• Furniture
• Water heater
• Bathroom tiles
• Kitchen sinks

Signs of Mold Growth

Spores of mold enter indoors with the help of open windows, doorways, vents, and heating and air-conditioning systems. Excessive mold growth shows various signs. The signs are as follows:

• Smell: A musty or earthy smell can be a big indication of mold growth. Mold produces some chemical compounds during its life cycle, which gives the damp stench similar to old books.  
• Black or green spots: The mold colony appears green or black. That is why the areas that are affected by mold turn green or black colored. 
• Regular flare-ups of allergy and asthma: The mycotoxin released by mold triggers different respiratory problems in patients with a history of respiratory conditions. 
• Bubbling in paint: One of the important signs of mold infestation is bubbling in wall paint.  
• Distortion of wallpaper: If your wall is covered with wallpaper, it may look distorted or changed. 
• Discolored tiles: The normal color of bathroom discolors into moldy or black, greenish color. 

Risk of Mold Growth

Mold growth in the houses can carry massive damage to properties; sometimes, one may even need to evacuate their home. The growth also has enormous health risks, especially in immunocompromised people.

Damage to Property

• Decaying of furniture: The prolonged growth of mold in wooden furniture ruins it beyond repair. Likewise, the growth in metal furniture causes rusting, eventually degrading the furniture and not being suitable for use.
• Destruction of walls: The mold growth can multiply and spread from affecting a small area to the entire wall. This rapid growth leads to the collapse of the walls, which may need to be replaced entirely. 

Health Risk

People with chronic respiratory conditions like asthma, pulmonary fibrosis, etc., are at risk of severe health issues due to the fungal toxins and spores produced by the growing molds. They may face health issues like:

• Aspergillosis (fungus ball in the lungs due to prolonged exposure to Aspergillus fumigatus)
• Difficulty in breathing
• Allergic reactions
• Chronic cough
• Constant headache
• Skin rashes
• Memory loss and difficulty in focusing (due to toxins produced by some molds)

Detecting Mold Growth

Detecting mold growth in your home is quite simple. Just look for musky or rotten smells and blackening around the affected areas. But detecting the level of growth is a time-consuming process. A do-it-yourself (DIY) kit is available for detecting mold growth indoors. The kit consists of the following things:

• A Petri dish
• A microbial culture media

Materials like painter’s tape and a marker for labeling the sample are required along with the kit.

Procedure for Detecting Mold Growth

The procedure for detecting the mold growth by yourself is as follows:

1. Close the windows and doors to prevent cross-contamination.
2. Pour the microbial culture media into the Petri dish and label the lid with the sample name.
3. Then, leave the Petri dish in the suspected area by opening the lid and the open end facing upward for at least 48 hours (it may vary based on the company used).
4. Leave the room and seal off the area using painter’s tape for the duration the Petri dish is kept there. 
5. Close the lid of the Petri dish after 48 hours and label the collection date.
6. Then, place the Petri dish in a dark area like a dresser, drawer, or closet.
7. After that, check for mold growth after two days. If there is growth present, it is similar to the growth of mold in food items. If there is no growth after two days, leave the dish in the dark area longer and check every alternative day.

Solution for Removing Mold Growth    

The growth of mold is controllable. The removal process involves cleaning the affected area thoroughly before the growth spreads to another site. Household items are washed with clean water, soap, and detergents. Hard surfaces like tiles and walls are cleaned with bleach. 

Things to Consider While Using Bleach

1. Mix no more than 1 cup of laundry bleach in a gallon (3.785 liters) of water.  
2. Use gloves, rubber boots, and goggles while cleaning the surface.
3. Ventilation in the area of cleaning is crucial.
4. Follow the company’s manual thoroughly.  

How to Prevent Regrowth of Mold?

According to the environment protection agency’s guide, the following things help prevent mold growth in your home:

1. Keep the humidity level in your house as low as possible.
2. Ensure the rooms in your house are well ventilated.
3. You should constantly clean the walls and roof and repair any leakage.
4. Use paints that have mold inhibitors.
5. Clean the flooded room before the 24-hour mark.
6. Remove infected carpets and furniture.
7. Clean bathrooms with disinfectant products. 

References 

• WHO guidelines for indoor air quality : dampness and mould. Euro.who.int. (2009). Retrieved 25 July 2022, from https://www.euro.who.int/__data/assets/pdf_file/0017/43325/E92645.pdf.
• Basic Facts about Mold and Dampness. Centers for Disease Control and Prevention. (2022). Retrieved 25 July 2022, from https://www.cdc.gov/mold/faqs.htm.
• Araujo, K. (2013). How, where, and why mold grows in our homes, and what to do about it. – The Martha’s Vineyard Times. The Martha’s Vineyard Times. Retrieved 25 July 2022, from https://www.mvtimes.com/2013/09/10/how-where-why-mold-grows-our-homes-what-do-about-17199/.
• A Brief Guide to Mold, Moisture and Your Home | US EPA. US EPA. Retrieved 25 July 2022, from https://www.epa.gov/mold/brief-guide-mold-moisture-and-your-home.
• McIntosh, J. (2019). Mold in the home: how big a health problem is it?. Medicalnewstoday.com. Retrieved 25 July 2022, from https://www.medicalnewstoday.com/articles/288651#outlook.
• Mundorf, D., Taylor, G., & Vila, B. (2022). How to Test for Mold in Your Home. Bob Vila. Retrieved 25 July 2022, from https://www.bobvila.com/articles/how-to-test-for-mold/.
• You Can Control Mold. Centers for Disease Control and Prevention. Retrieved 25 July 2022, from https://www.cdc.gov/mold/control_mold.htm.

Antifungal drugs are the agents that kill or stop fungal growth and are applied to treat or prevent fungal infections(mycoses). A proper antifungal drug selectively eliminates fungal pathogens from a host with minimal toxicity to the host.

The efficiency of antifungal drugs lagged due to the cellular structure of fungi. Antibiotics work well in bacteria because they are prokaryotes with many different structural and metabolic targets. Fungi are eukaryotes, so the toxic agents of fungi are also harmful to the host. In addition, the fungi grow slowly, and most are multicellular, which makes them difficult to quantify.

However, the mechanism of action of most antifungal drugs is targeting ergosterol, an essential component of the fungal cell membrane. Instead of ergosterol, the mammalian cell membrane possesses cholesterol. Advances have been made to develop new antifungal agents and to understand the existing ones.

• Fungistatic agent: The agent that inhibits fungi’s growth and reproduction but does not necessarily kill them is called a fungistatic agent. The fungal growth resumes when such agent is removed from the environment.

• Fungicidal agent: The agent that kills fungi is called a fungicide agent.

Chemical Classification and Mechanism of Action of Antifungal Drugs

Antifungals can be grouped into various classes based on their chemical composition and the site of action.

Polyenes

These contain alternating conjugated double bonds that constitute a part of their macrolide ring structure. Polyenes are derived from Streptomyces species; these are broad-spectrum and fungicide drugs. These drugs act directly on the fungal cell membrane by interacting with ergosterol. Amphotericin, nystatin, and pimaricin are major drugs in this category.

• Amphotericin B combines and gets inserted within a cell membrane. It leads to the formation of micropore that causes the leakage of cellular components, and ultimately the cell dies. However, it is associated with numerous side effects. Adverse effects, like suppression of glomerular filtration, can be reduced, especially by administrating sodium chloride.
• Nystatin and pimaricin (natamycin) follow the exact mechanism of action as polyenes. Like polyenes, these are also toxic to the host, hence limited to topical use.

Azoles

Drugs of this group have five-membered organic rings that contain two or three nitrogen molecules (the imidazole and the triazoles, respectively). These are fungistatic and broad-spectrum drugs.

Drugs like fluconazole, itraconazole, posaconazole, and voriconazole belong to the Triazoles, whereas cotrimoxazole, ketoconazole, econazole, and miconazole belong to the Imidazole class.

Azoles inhibit cytochrome P450 – dependent enzymes (particularly C14-demethylase). These enzymes help in the biosynthesis of ergosterol. Hence this drug inhibits fungal cell growth.

Allylamine and Morpholine

Allylamines like naftifine and terbinafine inhibit ergosterol biosynthesis at the level of squalor epoxidase. These are the structural analog of squalene. The accumulation of unsaturated hydrocarbons reduces ergosterol concentration in the fungal cell membrane leading to cell death. The morpholine drug amorolfine inhibits the same pathway at a later step.

Antimetabolite

5-Fluorocytosine or flucytosine is the fluorine analog of cytosine. It is a narrow-spectrum drug and acts as an inhibitor of DNA and RNA synthesis via the intracytoplasmic conversion of 5-fluorocytosine to 5-fluorouracil. It is subsequently converted to 5-fluoro-deoxy uridylic acid monophosphate. This non-competitive inhibitor of thymidylate synthetase then interferes with fungal nucleic acid synthesis. Hence, metabolic activities incorporate these drugs into fungal DNA and RNA and disrupt nucleic acid and protein synthesis.

Griseofulvin

Heterocyclic benzofurans like griseofulvin also act against fungal infections, especially dermatophytes. Griseofulvin is the metabolic by-product of Penicillium griseofulvum. Such drugs are fungistatic and interfere with mitosis.

These are ineffective against Candida albicans and do not disturb normal gut bacterial flora.

Echinocandins

Echinocandins inhibit the synthesis of fungal cell wall polysaccharides- a new mode of action. It interferes with synthesizing the beta-1,3-D-glucan polymer in the fungal cell wall and results in the loss of rigidity leading the fungal cell disruption, cellular osmotic instability, and cell death. It is usually applied against Candida species.

Others

Sordarins, a new class of antifungal drugs, inhibit fungal protein synthesis. Topical antifungals, like, ciclopirox, inhibit the transport of essential elements in the fungal cell; this disrupts DNA, RNA, and protein synthesis. These drugs are active against dermatophytes and Candida species.

Tolnaftate distorts hyphae and stunts mycelial growth in susceptible fungi.

Drawbacks of Using Antifungal Drugs

• Side Effects

Antifungal side effects vary depending on the drug type, strength/dose, and the type of pathogenic fungus. Allergy, skin reactions, liver damage, anaphylaxis, and nephrotoxicity are major side effects.

Due to the host toxicity, antifungal drugs are used in limit.

• Antifungal drug resistance

After the treatment of fungal diseases, some clear up within a few weeks, whereas some need months. Drug resistance may arise if the proper dose and period of drugs are not considered. Most antifungal drug resistance occurs because of the extended period or incomplete use of drugs or receiving too low doses. As a result, fungus no longer responds to treatment.

Mechanisms of antifungal drug resistance can be alteration in the drug target, alteration in sterol biosynthesis, reduction in intracellular concentration of target enzyme, and overexpression of the antifungal drug target.

References

• Denver et al. (2011). Hugo and Russell’s Pharmaceutical Microbiology. 8^th edition. A John Wiley & Sons, Ltd. Publication. Page no. 191-193.
• Dixon DM & Walsh TJ (1996). Antifungal Agents. In: Baron A, editor. Medical Microbiology. Galveston (TX): The University of Texas Medical Branch at Galveston. 4^th edition. Chapter 76. https://www.ncbi.nlm.nih.gov/books/NBK8263/
• Ghannoum MA & Rice LB(1999). Antifungal Agents: Mode of Action, Mechanisms of Resistance, and Correlation of These Mechanisms with Bacterial Resistance. Clin Microbiol Rev. DOI 10.1128/cmr.12.4.501 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88922/
• Neofytos et al. (2009). Pharmacology of infections. In Pharmacology and Therapeutics. https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/systemic-antifungal-agent
• Odds, F., Brown, A., & Gow, N. (2003). Antifungal agents: mechanisms of action. Trends In Microbiology, 11(6), 272-279. https://doi.org/10.1016/s0966-842x(03)00117-3
• Martinez-Rossi, N., Peres, N., & Rossi, A. (2008). Antifungal Resistance Mechanisms in Dermatophytes. Mycopathologia, 166(5-6), 369-383. https://doi.org/10.1007/s11046-008-9110-7