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Introduction of Antifungal Drugs Susceptibility Testing

Antifungal drugs and their susceptibility testing as shown above picture. There are various testing methods like disc diffusion, broth dilution, E-test, and VITEK. Among them, we tested antifungal susceptibility testing (AFST) by the disc diffusion method.

List of common anti-fungal drugs are as follows-

• Clotrimazole
• Econazole
• Miconazole
• Terbinafine
• Fluconazole
• Ketoconazole
• Itraconazole
• Voriconazole
• Posaconazole,
• Ravuconazole
• Amphotericin-B,
• 5-Fluorocytosine,

The Mode of Action of Some Antifungal Agents are as follow-

Amphotericin B binds to plasma membrane creating pores.

Azoles inhibit cytochrome P450 enzymes in the fungal cell 5FC converts to 5FU, incorporated into RNA, abnormal proteins.

Griseofulvin binds microtubule proteins, inhibits cell wall synthesis.

Terbinafine is an ergosterol inhibitor useful for systemic mycosis.

Echinocandins target their action on the fungal cell walls.

Griseofulvin:  Anti-inflammatory properties Inhibits keratolytic action

Antifungal Sensitivity Testing (AFST) can be performed by following methods and they are-

1. Disc diffusion method
2. Broth dilution: Synthetic Media: RPMI 1640 buffered with MOPS (Morpholine propane sulfonic acid) for Candida species and Yeast Nitrogen Broth for Cryptococcus neoformans
3. E-test
4. VITEK

AFST by Disc diffusion method

• It is only applicable for Candida species.
• Other genera are not covered and also have not been used in the studies of the yeast form of the dimorphic fungi.

Requirements for test

• Mueller-Hinton Agar + 2% Glucose + 0.5 mcg/ml Methylene Blue Dye medium (GMB) having  pH 7.2 to 7.4
• Antifungal drugs to be tested
• McFarland Std
• Incubator
• Fungal growth of test organism
• Test tubes
• Sterile cotton swabs

Test procedure

1. Preparation of test inoculum
2. Inoculation of the test organisms into a  plate.
3. Application of Disks to the inoculated agar plate.
4. Incubate the plate.

Observation

•  Examine each plate after 24 to 24 Hr of incubation
• Measure the zone diameter to the nearest whole millimeter at the point at which there is a prominent reduction in growth.
• Pinpoint micro-colonies at the zone edge or larger colonies within a zone are encountered frequently and they should be ignored as shown above picture.

Result Interpretation

Susceptible (S): Infection due to the strain may be appropriately treated with the dose of the antimicrobial agent recommended.

 Susceptible Dose-Dependent (S-DD): It includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels but the response rate may be lower than for susceptible isolates (Only applies when multiple approved dosage options exist)

Resistant (R): Isolates are not inhibited by the usually achievable concentration of the agent with normal dosage schedules.

Importance of AFST

1. Increase of Fungal Infections Several factors have contributed to the increase in fungal infections – most notably, an increasing number of immunosuppressed cases e.g AIDS, cancer, or diabetes, the use of broad-spectrum antibiotics, cytotoxic chemotherapy, and organ transplantation.
2.  Increase in incidence of immunosuppressive states
3. Increasing incidence of invasive mycosis and life-threatening infections as a significant public health issue
4.  Emerging resistance
5. Correlate with in vivo activity and predict the likely outcome of therapy
6.  Provide a reliable measure of the relative activities of two or more antifungal agents.
7. Growing concern about a shortage of effective antifungal agents and an increase in the resistance of fungal pathogens to the existing agents
8.  Among the invasive mycoses, none is more important or common than candidiasis. Candidiasis, specifical candidemia, has been shown in numerous studies to be the most frequent mycotic infection in hospitalized patients and is associated with significant attributable mortality and excess length of hospital stay
9.  Fungal Infections have increased Morbidity and Mortality The increasing incidence of opportunistic severe fungal infections has greatly enhanced the interest in novel methods for in vitro antifungal susceptibility testing, the standardized methodology.

Bibliography

1. https://cmr.asm.org/content/33/3/e00069-19
2. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
3. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
4. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
5. A Textbook of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88997/
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711191/
8. https://academic.oup.com/mmy/article/45/7/569/956049
9. https://www.cdc.gov/fungal/candida-auris/c-auris-antifungal.html
10. https://techlib.biomerieux.com/wcm/techlib/techlib/documents/docLink/