Fungi of medical importance_Lect 4.pdf

FUNGI OF MEDICAL IMPORTANCE [email protected] Fungi as infectious agents Fungi are a large group of unicellular and multicellular eukaryotes They live as saprobes, symbionts, and/or parasites They make up a kingdom of their own due to their distinct morphological, biological,...

FUNGI OF MEDICAL IMPORTANCE [email protected] Fungi as infectious agents Fungi are a large group of unicellular and multicellular eukaryotes They live as saprobes, symbionts, and/or parasites They make up a kingdom of their own due to their distinct morphological, biological, and molecular features. 2 Classification of Fungi: Yeasts, Molds, Dimorphic ✓ Traditionally, the kingdom Fungi is separated into two groups: yeasts and filamentous fungi, based on their macroscopic and microscopic characteristics. ✓ Human diseases resulting from fungal infections, primarily by yeasts and molds, are termed mycoses (singular: mycosis). ✓ Infectious fungi occur in groups based on the virulence of the pathogen and the degree of invasion: systemic, subcutaneous, cutaneous, or superficial. ✓ The fungi of medical importance to human pathology are divided into two groups: true pathogens and opportunistic pathogens 3 Molds ✓ Multicellular fungi with filamentous structures called hyphae. ✓ They form a network of hyphae known as mycelium. ✓ They reproduce by producing spores (sexual or asexual). ✓ Colonies often appear fuzzy or powdery due to spore production. 4  ASSIGNMENT ONE In chronological order discuss the life cycle of yeasts and molds. 5 Yeast Oral thrush/oral candidiasis ✓ They are unicellular fungi. ✓ Reproduce asexually by budding or binary fission. ✓ Typically appear as smooth, creamy colonies on culture media. Have two genus Candida species: ✓ Common cause of infections such as oral thrush and vaginal yeast infections. Cryptococcus neoformans ✓ Causes cryptococcosis, primarily affecting the lungs and central nervous system. 6 7 Fungi versus yeast 8 Dimorphic fungi Can exist in two different forms: 1. yeast form at temperatures (35-37°C) 2. mold forms at environmental temperatures (25-30°C). They exhibit temperature-dependent morphology. Examples: Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis Usually upon mammalian infection, the filamentous form converts to yeast form when the fungus adapts to their body temperature 9 Pathogenic fungi They are pathogens that can cause infections to humans or other animals There are about 300 human pathogenic fungi Pathogenic fungi are present with a wide variety of phenotypes in the environment. Classification according to WHO ✓ Critical priority Eg. Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans. ✓ High priority Eg. Nakaseomyces glabrata (Candida glabrata), Histoplasma spp, Mucorales, Fusarium spp., Candida tropicalis, Candida parapsilosis. ✓ Medium priority Eg. Scedosporium spp., Lomentospora prolificans, Coccidioides spp., Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, Paracoccidioides spp. 10 Types of Pathogenic fungi True pathogens: True fungal pathogens can invade and grow in healthy hosts due to their ability to cycle between two morphological and physiological forms. ✓ they grow as molds with hyphae at 30ºC and as yeasts at 37ºC. ✓ This is called thermal dimorphism Treatment: amphotericin B is the principal drug prescribed for most all systemic mycoses with fluconazole as a second choice. Eg. Blastomyces, Coccidioides, Histoplasma, Paracoccidioides, and Sporothrix. 11  Opportunistic pathogens: pathogens that tend to cause serious disease only in people with abnormal host defenses. Many opportunistic fungal pathogens do not require a host and they are non- communicable, with the exception of some dermatophytes and Candida Eg. Aspergillus, Candida, Cryptococcus, Penicillium, Pneumocystis, and the zygomycetes are opportunistic. 12 Factors Contributing to Fungal Pathogenicity ✓ Fungi can enter the body, portal of entry, by various routes such as respiratory and cutaneous. Once in the body, fungi can tolerate higher temperatures (thermal dimorphism) and low oxygen tensions of the body. Different fungi produce a variety of virulence factors. ✓ Adhesion Factors - Surface adhesins (e.g., mannoproteins) - Biofilm formation ✓ Enzymatic Factors (Eg., Proteases, Lipases, Phospholipases, Keratinases) 13 ✓ Morphological Factors - Dimorphism (ability to switch between yeast and mold forms) - Hyphal growth (tissue penetration) ✓ Immune Evasion Strategies -Capsule formation (e.g., Cryptococcus neoformans) - Antioxidant production (e.g., catalase, superoxide dismutase) - Immune modulation (e.g., inhibition of macrophage activation) ✓ Toxin Production - Mycotoxins (e.g., aflatoxins by Aspergillus species) ✓ Environmental Adaptation - Thermotolerance (growth at body temperature) - Ability to utilize various carbon sources - Stress response mechanisms (e.g., heat shock proteins) 14 ✓ Genetic Factors - Gene mutations and variations - Horizontal gene transfer ✓ Nutrient Acquisition - Siderophore production (iron acquisition) - Enzyme secretion for nutrient breakdown ✓ Resistance Mechanisms - Antifungal drug resistance (e.g., efflux pumps) - Biofilm-associated resistance ✓ Virulence Factors - Toxins and metabolites that damage host tissues - Secreted effector proteins 15 Host defense mechanisms against fungi Key aspects of host defenses against fungal infection include: ✓ anatomical barriers -skin -mucosal surfaces ✓ innate and adaptive immune responses ✓ nutritional immunity (eg., the use of Transferrin and Lactoferrin to sequester iron from fungi) 16 Organization of Fungal Infections Fungal infections are organized into: 1. True pathogen: Systemic Mycoses, Subcutaneous Mycoses, Superficial Mycoses 2. Opportunistic Mycoses 17 Systemic Infections by True Pathogens They are located in a geographic region, disturbed spores are inhaled, they cause primary pulmonary infection (PPI), can become systemic in susceptible hosts, can infect the skin and cause granulomatous lesions (formation of granulomas during infections), and can elicit long-term immunity. Eg., Histoplasmosis: Ohio Valley Fever Histoplasma capsulatum Distributed worldwide, H. capsulatum causes histoplasmosis (Ohio Valley fever). Associated with humans who practice agriculture. ✓ Most prevalent in eastern and central regions of the United States. 18 ✓ Approximately 500,000 cases per year, several thousand of which require hospitalization, and a small number being fatal. ✓ Infection and Pathogenesis of Histoplasma Infection is related to soil disturbance and guano (accumulated excrement of bats, seabirds, seals); inhaled conidia (spores) produce primary pulmonary infection (PPI) that may progress to systemic involvement of a variety of organs and chronic lung disease. ✓ AIDS patients and children are most susceptible. ✓ Yeast form grows intracellularly in macrophages. Most severe systemic forms occur in immuno-compromised patients and children. ✓ Liver and spleen enlargement, anemia, circulatory collapse, and death are possible consequences. 19 Diagnosis and Control of Histoplasmosis ✓ Appears as “fish-eye” yeast intracellularly in macrophages. ✓ Most exposed to the yeast recover, but those with systemic infections are treated with amphotericin B. 20 Coccidioidomycosis: Valley Fever Coccidioides immitis has recently demonstrated the greatest virulence of all mycotic pathogens. ✓ It is present in alkaline soils in semi-arid, hot climates. ✓ C. immitis has free-living arthroconidia (spores existing independently in the environment, rather than within a host organism) which when inhaled can lead to coccidioidomycosis (Valley fever) - ~100,000 cases/year 21 Infection and Pathogenesis of Coccidioidomycosis ✓ The arthrospores are lightweight and are easily inhaled. ✓ In 60% of cases PPI is unapparent; in 40% it is accompanied by cold-like symptoms such as fever, chest pains, cough, headaches, and malaise. ✓ ✓ AIDS patients are most susceptible. 22  Diagnosis and Control of Coccidioidomycosis ✓ Based on the highly distinctive spherules seen in body fluid/tissue samples. ✓ Antigen tests have also been used ✓ Patients with disseminated disease (a disease that has spread from the initial site of infection) require amphotericin B. 23 Blastomyces dermatitidis Dimorphic morphology of B. dermatitidis follows that of other true pathogens Causative agent of blastomycosis. Mild PPI disease is accompanied by cough, chest pain, hoarseness (strained quality of the voice), and fever. Severe, chronic blastomycosis can progress to the skin and numerous other organs. Subcutaneous nodules can erupt to the skin surface Chronic systemic blastomycosis of the spleen, liver, and urogenital tract can last for weeks to years and eventually destroy the host defenses 24 Cutaneous Mycoses ✓ Dermatophytoses are fungal infections of non-living epidermal tissues (stratum corneum) and its derivatives (hair and nails); they are known variously as tineas or ringworm. ✓ Caused by species in the genera Tichophyton, Microsporum, and Epidermophyton – all adapted to keratinized epidermis (skin, hair, and nails) NB: Infections are communicable among humans and animals and are facilitated by moist, chafed skin. 25 Treatment: topical antifungal agents containing tolnaftate, miconazole, or thiabendazine are applied regularly for several weeks. Griseofulvin is prescribed in severe cases. Alternate: oral or topical terbinafine HCl (Lamisil ) 26 Pneumocystis jiroveci (previously known as P. carinii) P. jiroveci is a small, unicellular fungus that causes Pneumocystis pneumonia (PCP) – the most prominent opportunistic infection in AIDS patients ✓ This form of pneumonia forms secretions in the lungs that block breathing and can be rapidly fatal if not controlled with medication. Treatment: traditional antifungal drugs like amphotericin B are ineffective. ✓ Primary treatment is cotrimoxazole, a combination of sulfamethoxazole and trimethoprim (Bactrim or Septra ). ✓ Therapy should be applied even if disease appears mild or is only suspected. ✓ Pentamidine is another treatment, but has many side-effects. 27 Subcutaneous Mycoses Sporothrix schenckii ✓Sporotrichosis (rose gardener’s disease) is caused by S. schenckii, a free- living fungus that accidentally infects the appendages and lungs. ✓Lymphocutaneous variety occurs when contaminated plant matter penetrates the skin (by a thorn prick for example) and the pathogen forms a local nodule, then spreads to nearby lymph nodes, where it can erupt to the skin surface. 28 Opportunistic Mycoses Candida albicans ✓ Candidiasis is caused by C. albicans and other Candida species that normally reside in the mouth, vagina, and skin. ✓ Infection predominates in cases of lowered resistance (infants, AIDS, drug therapy4) and arises from normal flora. ✓ Thrush occurs as a thick, white, adherent growth on the mucous membranes of mouth and throat ✓ Vulvovaginal yeast infection is a painful inflammatory condition of the female genital region that causes ulceration and whitish discharge. ✓ Sexually transmissible. 29 ✓Male urogenital tract infections have similar pathology. ✓Cutaneous candidiasis occurs in chronically moist areas of skin (e.g. diaper rash) and burn patients. Treatment: therapy for superficial mucocutaneous infection consists of topical antifungal agents (azoles and polyenes). ✓Recurrent bouts of vulvovaginitis are managed by over-the-counter topical azole ointments. 30 C. albicans Pap stain 31 Cryptococcus neoformans ✓ Cryptococcosis is caused by C. neoformans, a widespread yeast ✓ Common infection occur in AIDS, cancer, and diabetes patients. ✓ Infection of lungs (by inhalation) leads to cough, fever, and lung nodules. ✓ Dissemination to meninges and CNS can cause severe neurological disturbance and death. Treatment: systemic infection requires immediate treatment with amphotericin B and fluconazole over a period of weeks or months 32 Field stain of Cryptococcus species in the lungs 33 Mechanism of fungal infection 34 Diagnosis of Fungal Infections Clinical presentation and history Laboratory methods - Microscopy and staining techniques - Culture methods - Serological tests - Molecular methods (PCR, DNA sequencing) 35 Laboratory diagnosis of fungal infection ✓ Microscopic Examination Direct Microscopy: Examines clinical specimens (e.g., skin scrapings, sputum) for fungal elements. -Eg., KOH Preparation: Specimens are treated with potassium hydroxide (KOH) to dissolve human cells, making fungal elements more visible. - Calcofluor White Staining: Binds to fungal cell walls, fluorescing under UV light. - India Ink Preparation: Used for detecting Cryptococcus neoformans in cerebrospinal fluid. 36 ✓ Culture -Eg., Sabouraud Dextrose Agar (SDA): Commonly used for growing fungi. It contains nutrients that support fungal growth and antibiotics to inhibit bacterial growth. - Potato Dextrose Agar (PDA): Supports the growth of a wide variety of fungi. - Brain Heart Infusion (BHI) Agar: Used for fastidious fungi and dimorphic fungi. - Incubation Conditions: Fungi are incubated at different temperatures (e.g., 25°C and 37°C) to promote the growth of both mold and yeast forms. 37 ✓ Histopathological Examination -Eg., Tissue Biopsy: Involves taking a sample of infected tissue for microscopic examination. - Hematoxylin and Eosin (H&E) Staining: General stain for tissue architecture and cellular detail. - Periodic Acid-Schiff (PAS) Staining: Highlights fungal cell walls in pink/red. - Gomori Methenamine Silver (GMS) Staining: Fungal elements stain black against a green background. 38 ✓ Serological Tests Antibody Detection: Identifies host antibodies against fungal antigens. - Eg., ELISA (Enzyme-Linked Immunosorbent Assay): Detects specific antibodies in patient serum. - Antigen Detection: Identifies fungal antigens in body fluids. - Cryptococcal Antigen Test: Detects Cryptococcus neoformans antigen in cerebrospinal fluid or serum. - Galactomannan Test: Detects Aspergillus antigen in serum. 39 ✓ Molecular Methods -Eg., Polymerase Chain Reaction (PCR): Amplifies fungal DNA from clinical specimens for identification. - Real-Time PCR: Provides rapid and quantitative detection.Sequencing: Determines the exact sequence of fungal DNA for species identification. - Internal Transcribed Spacer (ITS) Sequencing: Commonly used for fungal identification. ✓ Biochemical Tests -Eg., Yeast Identification Kits: Identify yeast species based on carbohydrate assimilation and fermentation patterns. - Urease Test: Differentiates Cryptococcus (urease-positive) from other yeasts. 40 ✓ Imaging Techniques Eg., Radiography (X-rays, CT scans, MRI): Helps visualize deep-seated infections and assess the extent of fungal involvement. ✓ Antifungal Susceptibility Testing -Eg., Broth Microdilution: Determines the minimum inhibitory concentration (MIC) of antifungal agents. - Disk Diffusion: Measures the zone of inhibition around antifungal-impregnated disks placed on an agar plate inoculated with the fungal isolate. ✓ Fluorescence in situ Hybridization (FISH)FISH: Uses fluorescent probes that bind to specific fungal RNA sequences, allowing visualization of fungi in clinical specimens. 41 THANK YOU 42