Module 3.3 Systemic Opportunistic Mycoses PDF

BIO 125 Chapter 3.3 Systemic/Opportunistic Mycoses OUTLINE Medical Microbiology DISEASE I. Mycoses a. Systemic Mycoses II. Systemic opportunistic mycoses a. Candidiasis b. Cryptococcosis c. Aspergillosis d. Scedosporiosis e. Zygomycosis f. Hyalohypomycosis g. Phaeohypomycosis III. Treatment Ca...

BIO 125 Chapter 3.3 Systemic/Opportunistic Mycoses OUTLINE Medical Microbiology DISEASE I. Mycoses a. Systemic Mycoses II. Systemic opportunistic mycoses a. Candidiasis b. Cryptococcosis c. Aspergillosis d. Scedosporiosis e. Zygomycosis f. Hyalohypomycosis g. Phaeohypomycosis III. Treatment Candidiasis Cryptococcosis Aspergillosis Legend: Lecturer Book Previous Trans To Remember/Trans head note Scedosporiosis (Pseudallescheriasis) Zygomycosis (Mucormycosis) ★ Italicized Font color of transers’ notes • • SYSTEMIC MYCOSES • • • • • • Hyalohyphomycosis MYCOSES Infections that are caused by fungi. Mycoses may be classified as: o Superficial o Cutaneous o Subcutaneous o Systemic The infection is deep within the body or disseminated to internal organs. Systemic mycoses can be further divided into: o True pathogens True pathogenic fungi capable of infecting healthy individuals. o Opportunistic pathogens They infect primarily those individuals who have predisposing conditions, such as immunodeficiency or debilitating diseases (also called the systemic opportunistic mycoses [discussed further below]). Phaeohyphomycosis • • • • CAUSATIVE ORGANISMS Candida, Debaryomyces, Kluyveromyces, Meyerozyma, Pichia, etc. Cryptococcus spp. Especially C. Neoformans/C. Gattii Aspergillus fumigatus complex, A. Flavus, complex, A. Terreus complex etc. Scedosporium and Lomentospora Rhizopus, Mucor, Rhizomucor, Lichtheimia etc. Penicillium, Paecilomyces, Beauveria, Fusarium, Scopulariopsis etc. Cladophialophora, Exophiala, Bipolaris, Exserohilum etc. INCIDENCE Common Rare/Common Rare Rare Rare Rare Rare CANDIDIASIS Causative agent: Candida albicans (80–90%cases) Candida is a normal inhabitant of skin, GIT, oral and vaginal cavities (normal flora) Opportunistic endogenous infection Infection of the following: o Skin o Mucosa o Internal organs MORPHOLOGY • • Ovoid or spherical budding yeast cell, 3-5μm in diameter Produces pseudohyphae (elongated filamentous cells joined end to end resembling hyphae) in both culture and tissue SYSTEMIC OPPORTUNISTIC MYCOSES Occur almost exclusively in debilitated patients Produced by relatively non-pathogenic or contaminant fungi Host: immunological defense mechanisms are weakened by: o Endogenous causes: cancer, leukemia o Exogenous causes: immunosuppressive therapy, AIDS Important cause of morbidity and mortality in hospitalized patients Figure 1. Candida albicans Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 1 of 12 Species of Candida Oropharyngeal candidiasis • • • • • • • C. Albicans C. Stellatoidea C. Tropicalis C. Krusei C. Parapsilosis C. Glabrata C. Viswanathii • • • • • • Opportunistic endogenous infection Predisposing factors: o Diabetes o Immunodeficiency o Malignancy o Prolonged administration of antibiotics o Patients on immunosuppressive drugs o Intravenous catheters Primary or secondary o In primary oral candidiasis, where the condition is confined to the mouth and perioral tissues. o In secondary oral candidiasis, where there is involvement of other parts of the body in addition to the mouth. Acute, subacute or chronic to episodic Localized o Mouth, throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, or the gastrointestinal tract Systemic o Septicemia, endocarditis and meningitis In healthy individuals o Due to impaired epithelial barrier functions o Occur in all age groups, but are most common in the newborn and the elderly o Candida usually remain superficial and respond readily to treatment. Systemic candidiasis o Patients with cell-mediated immune deficiency, o Receiving aggressive cancer treatment, immunosuppression, or transplantation therapy. • PATHOGENESIS • • • • • • • Thrush, glossitis, stomatitis, and angular cheilitis Rarely seen in healthy adults Up to 5% of newborn infants & 10% of the elderly Often associated with severe immunological impairment due to diabetes mellitus, leukemia, lymphoma, malignancy, neutropenia, and HIV infection Other predisposing factors o Use of broad-spectrum antibiotics, corticosteroids, cytotoxic drugs, and radiation therapy White plaques → milk curd form on the buccal mucosa o Less commonly on the tongue, gums, the palate or the pharynx. o Symptoms may be absent; may cause burning or dryness of the mouth, loss of taste, and/or pain on swallowing. Figure 2. Oral candidiasis in new born (left) & in a immunocompromised patient (right). Figure 3. Oral candidiasis in new born CLINICAL MANIFESTATIONS • • • An iatrogenic, nosocomial infection Endogenous in origin Clinical manifestations: o Heroin addicts: cutaneous, ocular and arthritic manifestations o Leukemia patients: fever, rash and myalgia associated o Candida cholecystitis o Pancreatic abscesses o Candida prostatitis o Epiglottitis o Osteomyelitis o Osteroarticular candidiasis Bio 125 Medical Microbiology The explanation for the proceeding section was lifted from the book to better explain the images that were directly taken from the ppt. Cutaneous candidiasis • • This include invasion of the skin, which occurs when the skin is weakened by trauma, burns, or maceration. Intertriginous infection (infection caused by two skin areas that rub or touch together) o Occurs in moist, warm parts of the body such as the axillae, groin, and intergluteal or inframammary folds o It is most common in obese and diabetic individuals. Chapter 3. Introduction to Mycology 2 of 12 Figure 7. Diaper rash in newborn Figure 4. Candidiasis in the groin mimicking tinea • Satellite lesion o Superficial candidal skin infections appear as a red flat rash with sharp, scalloped edges. Smaller patches of similar-appearing rash, known as "satellite lesions" or "satellite pustules," are usually nearby. Candidal invasion of the nails • Invasion of the nails and around the nail plate causes onychomycosis o Onychomycosis is a painful, erythematous swelling of the nail fold resembling a pyogenic paronychia, which may eventually destroy the nail. Figure 5. Satellite lesion • Interdigital involvement between the fingers follows repeated prolonged immersion in water o Most common in homemakers, bartenders, cooks, and vegetable and fish handlers. Figure 8. Paronchyia Vulvovaginitis and balanitis • • Yeast invasion of the vaginal mucosa leads to vulvovaginitis, characterized by irritation, pruritus, and vaginal discharge. Balanitis is an infection or inflammation of the skin on the head (glans) of the penis. Figure 6. Interdigital candidiasis (left) and candidiasis between toes mimicking tinea (right) • Before newborns establish a balanced microbiome, they are susceptible to extensive diaper rash and skin infection caused by Candida. Figure 9. Vulvovaginal candidiasis Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 3 of 12 Figure 10. Balanitis Chronic mucocutaneous candidiasis (CMC) • • It is a rare but distinctive clinical manifestation characterized by the formation of granulomatous candidal lesions on any or all cutaneous and/or mucosal surfaces. The patients may develop chronic, raised, and crusty highly disfiguring keratitic lesions on the skin, oral mucosa, and scalp. Figure 12. Neonatal candidiasis Systemic candidiasis • • • • • Figure 11. CMC Neonatal candidiasis • • • • Candidemia can be caused by indwelling catheters, surgery, intravenous drug abuse, aspiration, or damage to the skin or gastrointestinal tract. In most patients with normal innate immune responses and circulating neutrophils, the yeasts are eliminated and candidemia is transient. Patients with compromised innate phagocytic defenses may develop occult lesions anywhere, especially the kidney, skin (maculonodular lesions), eye, heart, and meninges. Candidal endocarditis is frequently preceded by the deposition and growth of the yeasts and pseudohyphae on prosthetic heart valves or vegetations and the formation of recalcitrant biofilms. Kidney infections are usually a systemic manifestation, whereas urinary tract infections are often associated with Foley catheters, diabetes, pregnancy, and antibacterial antibiotics. 3rd most common cause of late – onset sepsis in NICU (high risk) Bimodal frequency: o Early – onset (within 3 days of birth) o Late – onset (7 – 60 days & later) Median gestational age: 27.5weeks Mortality in neonatal patients: 25 – 60% Figure 13. Esophageal and gastrointestinal candidiasis. Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 4 of 12 Figure 14. Pulmonary candidiasis Figure 18. Endocarditis, myocarditis, and pericarditis. Figure 15. Candida peritonitis Figure 19. Endopthalmitis due to Candida. Osteoarticular candidiasis • • SUMMARY OF CLINICAL GROUPS AND/ OR PREDISPOSING FACTORS FOR INVASIVE CANDIDIASIS Figure 16. Urinary tract candidiasis. Laparoscopic view within the urinary bladder with extensive fungal plaques. Figure 17. Hepatic and splenic candidiasis (white circles). Occurs when Candida spp. Proliferate in the joints or bones via hematogenous seeding. Areas most often seeded are intervertebral discs and knee joints • • • • • • • • • • • • Neutropenia (especially >7 days) Hematological malignancy Solid tumor malignancy Postsurgical intensive care patients Prolonged intravenous catheterization Broad-spectrum or multiple antibiotic therapy Diabetes mellitus Parental nutrition Severe burns Neonates Corticosteroid therapy Intravenous drug abuse • A positive direct microscopy from a sterile site, especially a tissue biopsy → considered significant, even if unable to culture the yeast. LABORATORY DIAGNOSIS Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 5 of 12 • Demonstration of pseudohyphae in scrapings or smears → considered significant o Provided the clinical manifestations support the diagnosis o Note: pseudohyphae will not be observed in smears when C. Glabrata is involved, and the diagnosis will require additional supporting evidence. IDENTIFICATION • • • • • • • Figure 20. Candida albicans in corn meal agar. • • • • Figure 21. Budding yeast cells and pseudohyphae seen in stained smear or KOH. Characteristic: globose to elongate yeast-like cells or blastoconidia Reproduce: multilateral budding Most species are also characterized by the presence of welldeveloped pseudohyphae o Maybe absent: the genus Torulopsis, Arthroconidia, Ballistoconidia Colony pigmentation always absent. Fermentation, nitrate assimilation and inositol assimilation may be present or absent o All inositol positive strains produce pseudohyphae CRYPTOCOCCOSIS Chronic, subacute to acute pulmonary, systemic or meningitic disease Inhalation: infectious propagules (basidiospores and/or desiccated yeast cells) from the environment. Primary pulmonary infections o No diagnostic symptoms o Usually subclinical Disseminated o Predilection for the central nervous system, o Skin, bones and other visceral organs may also become involved. C. Neoformans and C. Gattii: the principle pathogenic species Cryptococcus albidus and C. Laurentii also implicated CLINICAL MANIFESTATIONS • • • Encapsulated basidiomycete yeast-like fungus Predilection for the respiratory and nervous system of humans and animals. Two species, C. Neoformans and C. Gattii o Distinguishable biochemically and by molecular techniques. Pulmonary cryptococcosis Figure 22. 10% KOH mount showing the presence of budding yeast cells and pseudohyphae in a skin scraping and a PAS stained smear showing the presence of budding yeast cells and pseudohyphae in a urine specimen. Figure 244. Chest radiograph showing pulmonary cyptococcosis Central Nervous System Figure 23. Typical moist colonies of Candida. Bio 125 Medical Microbiology • Most clinical manifestation: Chapter 3. Introduction to Mycology 6 of 12 o o Dissemination to the brain and meninges Meningitis, meningoencephalitis or cryptococcoma • expanding • Most commonly include papilledema and optic atrophy due to raised intracranial pressure. Other ocular signs of cryptococcosis are uncommon and usually occur as a result of dissemination. Cutaneous cryptococcosis • Ulcerated lesions or cellulitis o May resolve spontaneously or with systemic antifungal treatment Other forms • • • • • • Pyelonephritis or prostatitis Adrenal cortical lesions Endocarditis Hepatitis Sinusitis Localized esophageal lesions LABORATORY DIAGNOSIS Clinical Material Figure 25. Ulcerated lesions and cellulitis • • • • • • • Cerebrospinal fluid (CSF) Biopsy tissue Sputum Bronchial washing Pus Blood Urine Figure 26. Nodular and ulcerated skin lesion caused by C. Neoformans Cryptococcosis of Bone • • • • Figure 28. Indian ink preparation of CSF showing a typical yeast cell C. Neoformans surrounded by a characteristic wide gelatinous capsule. Up to 10% of disseminated cases May involve bony prominences, cranial bones and vertebrae. Lesions: lytic without periosteal reaction and symptoms of dull pain on movement are reported. Occasional cases of arthritis have also been reported, mostly involving the knee joint. Figure 29. Tissue sections showing typical yeast cells of C. Neoformans Figure 267. Bone Cryptococcosis Ocular Cryptococcosis Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 7 of 12 In immunocompromised patients, especially those with leukemia, stem cell transplant patients, and individuals taking corticosteroids, the conidia may germinate to produce hyphae that invade the lungs and other tissues CLINICAL MANIFESTATIONS Pulmonary Aspergillosis • • • Figure 31. Bird seed agar plate showing the typical brown color effect seen with C. Neoformans CAUSATIVE AGENTS • • • • Cryptococcus albidus Cryptococcus laurentii Cryptococcus neoformans Cryptococcus gattii ASPERGILLOSIS • • • • • • In the lungs, alveolar macrophages are able to engulf and destroy the conidia. However, macrophages from corticosteroid-treated animals or immunocompromised patients have a diminished ability to contain the inoculum. In the lung, conidia swell and germinate to produce hyphae that have a tendency to invade preexisting cavities (aspergilloma or fungus ball) or blood vessels. Mycotoxicosis Allergy and sequelae Colonization Invasive, inflammatory, granulomatous, narcotizing disease of lungs, and other organs Systemic and fatal disseminated disease. Aspergillus fumigatus complex, A. Flavus complex, A. Niger complex, A. Nidulans and A. Terreus complex. Aspergillosis is a spectrum of diseases that may be caused by a number of Aspergillus species. Allergic reactions in hypersensitized hosts Development of ige antibodies to the surface antigens of Aspergillus conidia elicits an immediate asthmatic reaction upon subsequent exposure Aspergilloma • Occurs when inhaled conidia enter an existing cavity, germinate, and produce abundant hyphae in the abnormal pulmonary space. Invasive aspergillosis • Following inhalation and germination of the conidia, invasive disease develops as an acute pneumonic process with or without dissemination. • Patients at risk are those with lymphocytic or myelogenous leukemia and lymphoma, stem cell transplant recipients, individuals taking corticosteroids and AIDS patients with CD4 cell counts less than 50 CD4 cells/μl are predisposed to invasive aspergillosis. • Figure 30. Mucoid colonies of C. Neoformans Figure 32: Pulmonary Aspergillosis Disseminated Aspergillosis • Disseminated disease → highly immunocompromised hosts A fumigatus is the most common human pathogen, but many others, including Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus lentulus may cause disease. This mold produces abundant small conidia that are easily aerosolized. Following inhalation of these conidia, atopic individuals often develop severe allergic reactions to the conidial antigens. Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 8 of 12 Figure 33: A. Hemorrhagic lesions (arrows) 7A. Minimal punctate enhancement (arrows) 7B. White matter lesions that are hyperintense which consisted of hemorrhagic, necrotizing encephalitis with numerous branching hyphae Figure 35: Cutaneous Aspergillosis Figure 34: Endophthalmitis Aspergillosis of the Paranasal Sinuses • Two types of paranasal sinus aspergillosis are generally recognised: o (1) A non-invasive "aspergilloma" form, primarily seen in non-immunosuppressed individuals. Predisposing factors include a history of chronic sinusitis and poorly draining sinuses with excessive mucus. o (2) An invasive form, usually seen in the immunosuppressed patient. This form has a similar clinical setting to that seen in rhinocerebral zygomycosis; and symptoms include fever, rhinitis and signs of invasion into the orbit. Cutaneous Aspergillosis • • • A rare manifestation Usually result of dissemination from primary pulmonary infection in the immunosuppressed patient. Primary cutaneous aspergillosis also occur, o Usually as a result of trauma or colonisation. o Lesions: erythematous papules or macules with progressive central necrosis. Bio 125 Medical Microbiology LABORATORY DIAGNOSIS Clinical Material • Patients with pulmonary disease o Sputum o Bronchial washings o Tracheal aspirates • Patients with disseminated disease o Tissue biopsies Direct Microscopy Figure 36: Methanamine silver stained tissue section -> dichotomously branched, septate hyphae (top) and a conidial head of A. Fumigatus (bottom) Chapter 3. Introduction to Mycology 9 of 12 Figure 37: Lung tissue showing fungus balls of hyphae of A.fumigatus; Conidal heads forming in an alveolus Figure 39: Aspergillus spp. Colonies on culture media • • Mostly consist of a dense felt of erect conidiophores Conidiophores terminate in a vesicle covered with either a single palisade-like layer of phialides (uniseriate) or a layer of subtending cells (metulae) which bear small whorls of phialides (the so-called biseriate structure). Culture • • • • • Specimens should be inoculated onto primary isolation media, like Sabouraud's dextrose agar; usually fast growing White, yellow, yellow-brown, brown to black or shades of green Species are common environmental airborne contaminants o Positive culture from non-sterile specimen, such as sputum -> not proof of infection. Detection of Aspergillus (especially A. Fumigatus and A. Flavus) in sputum cultures, in patients with appropriate predisposing conditions, o Likely to be of diagnostic importance and empiric antifungal therapy should be considered. Patients with invasive pulmonary aspergillosis have often negative sputum cultures -> making a lung biopsy a prerequisite for a definitive diagnosis. Figure 40: Conidiophores of Aspergillus spp. On culture • • Serology • • Figure 38: Aspergillus spp. On media Bio 125 Medical Microbiology The vesicle, phialides, metulae (if present) and conidia form the conidial head. Conidia are one-celled, smooth or rough-walled, hyaline or pigmented and are basocatenate, forming long dry chains which may be divergent (radiate) or aggregated in compact columns (columnar). Some species may produce Hülle cells or sclerotia. Immunodiffusion tests for the detection of antibodies to Aspergillus species o Diagnostic value of allergic, aspergilloma, and invasive aspergillosis. o Should never be used alone, o Must be correlated with other clinical and diagnostic data. Antigen tests: for Aspergillus from blood, urine and CSF o (1→3)- β-D- glucan test Detects a wide variety of fungal pathogens: Aspergillus, Candida, Fusarium, Trichosporon and several Commercial kits: fungitec G, Fungitell o Aspergillus galactomannan ELISA test (Platelia® Aspergillus ELISA kit) MOST wisely used Specificity of 89-93%; sensitivity of 61-71%; NPV of 9598%; PPV of 26-53% Serial screening twice weekly for optimal diagnosis is recommended Chapter 3. Introduction to Mycology 10 of 12 Meningitis Brain abscesses Endophthalmitis Disseminated systemic disease CAUSATIVE AGENTS • • • • • • Aspergillus spp. Aspergillus flavus complex Aspergillus fumigatus complex Aspergillus nidulans complex Aspergillus niger complex Aspergillus terreux complex Lomentospora prolificans infections • • SCEDOSPORIOSIS (PSEUDALLESCHERIASIS) Sceoporium and Lomenstospora infection • • • • • Spectrum of disease similar in terms of variety and severity to those caused by Aspergillus. Infections are results of: o Inhalation of air-borne conidia or o Traumatic implantation of fungal elements (penetrating injury) Vast majority of infections are mycetomas Remainder: infections of the eye, ear, central nervous system, internal organs and more commonly the lungs. The etiological agents: o Scedosporium apiospermum o Scedosporium aurantiacum o Scedosporium boydii o Lomentospora prolificans Scedosporium apiospermum, Scedosporium Scedosporium aurantiacum infections • • • boydii and Non-invasive colonization of the external ear and pulmonary colonization in patients with poorly draining bronchi or paranasal sinuses and "fungus ball" formation in pre-formed cavities are similar to those seen in Aspergillus. Invasive infections in normal patients: o Caused by traumatic implantation o Most Common in normal patient: Mycetoma -> fungus exists in tissue as resistant microcolonies Followed by penetrating joint injuries, especially to the knee, resulting -> arthritis and osteomyelitis. Clinical manifestations include: • Mycotic keratitis • Non-mycetoma like cutaneous and subcutaneous infections Invasive infections: o Patients receiving treatment with corticosteroids and immunosuppressive therapy for organ transplantation, leukemia, lymphoma, systemic lupus erythematous or Crohn's disease. o Diseases include: Invasive sinusitis Pneumonia Arthritis with osteomyelitis Cutaneous/subcutaneous granulomata Bio 125 Medical Microbiology • • • • Spectrum of clinical manifestations are similar to that described above for Scedosporium Disseminated disease o Immunosuppressed patients • Especially those with prolonged neutropenia and post- transplantation therapy. Colonization of the external ear, paranasal sinuses, and lung, including "fungus ball" Cases of onychomycosis and mycotic keratitis Localized invasive infections: o Septic arthritis and osteomyelitis • Following penetrating injuries to joints • An emerging clinical problem • 80% of the reported cases Culture identification is important LABORATORY DIAGNOSIS Clinical Material • • Pulmonary disease: – Sputum, bronchial washings and tracheal aspirates Subcutaneous and disseminated disease: – Tissue biopsies from patients Direct Microscopy • • • (a) Sputum, washings and aspirates make wet mounts in either 10% KOH & Parker ink or Calcofluor white and/or Gram stained smears; (b) Tissue sections should be stained with H&E, GMS and PAS digest. Note: hyphal elements of Scedosporium boydii and Scedosporium prolificans are indistinguishable from those of Aspergillus hyphae and may be missed in H&E stained sections. Examine specimens for branched, septate hyphae. Figure 41: branching septate hyphae of Scedosporium spp. On microscopy • Presence of branching septate hyphae in any specimen, from a Chapter 3. Introduction to Mycology 11 of 12 • patient with supporting clinical symptoms should be considered significant. Biopsy and evidence of tissue invasion is of particular importance. Remember culture is necessary for a specific identification of the causative agent. • • Culture characteristics and microscopic morphology are important, especially conidial morphology Arrangement of conidia on the conidiogenous cell and the morphology of the conidiogenous cell, in this case an annellide. CAUSATIVE AGENTS • • • • • • • Figure 42: Tissue biopsy showing Scedosporium spp. Culture • • Fast growing, greyish-white, to olive-grey to black with a suede-like to downy surface texture. S. Apiospermum, S. Boydii, S. Aurantiacum and L. Prolificans are common soil fungi o Positive culture from a non-sterile specimen, such as sputum or skin, needs to be supported by direct microscopic evidence in order to be considered significant. o A positive culture from a biopsy or aspirated material from a sterile site should be considered significant. o Culture identification is the only reliable means of distinguishing these fungi from Aspergillus species. • Aspergillosis immunodiffusion tests o Valuable in the diagnosis of pseudallescheriasis. o Present reagents are not commercially available and antigenic extracts have to be made in the laboratory. ZYGOMYCOSIS (MUCORMYCOSIS) Primitive, fast growing, terrestrial, largely saprophytic fungi with a cosmopolitan distribution. Some 665 species have been described although infections in humans and animals are generally rare. Medically important orders and genera include: 1. Mucorales and Mortierellales, causing subcutaneous and systemic zygomycosis (Mucormycosis) - Rhizopus, Lichtheimia, Rhizomucor, Mucor, Cunninghamella, Saksenaea, Apophysomyces, Cokeromyces and Mortierella. 2. Entomophthorales, causing subcutaneous zygomycosis (Entomophthoromycosis) - Conidiobolus and Basidiobolus. CLINICAL MANIFESTATIONS • Figure 43: Scedosporium spp. Appearance on culture media Serology Scedosporium apiospermum Scedosporiun aurantiacum Scedosporium boydii Lomentospora prolificans • Zygomycosis in the debilitated patient o Most acute and fulminate fungal infection known. o Typically involves the rhino-facial-cranial area, lungs, gastrointestinal tract, skin, or less commonly other organ systems. o Associated with: Acidotic diabetes Starvation Severe burns Intravenous drug abuse Other diseases • Leukemia and lymphoma • Immunosuppressive therapy • Use of cytotoxins and corticosteroids • Therapy with desferrioxamine (an iron chelating agent for the treatment of iron overload) • Other major trauma o Infecting fungi have a predilection for invading vessels of the arterial system Embolization and subsequent necrosis of surrounding tissue. A rapid diagnosis is extremely important if management and therapy are to be successful. Rhinocerebral Zygomycosis • Identification Predisposing factors include: o Uncontrolled diabetes mellitus or acidosis o Steroid induced hyperglycemia Especially in patients with leukemia and lymphoma, renal transplant Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 12 of 12 o Concomitant azathioprine. treatment with corticosteroids and • May originate from any of the previous discussed diseases o Especially in severely debilitated patients with hematological malignancies, burns, diabetes or uremia. CNS Zygomycosis • • Intravenous drug abuse. Traumatic implantation o Both leading to brain abscess. LABORATORY DIAGNOSIS Figure 44: Rhinocerebral zygomyosis showing involvement of the palate • Infections usually begin in the paranasal sinuses following the inhalation of sporangiospores and may involve the orbit, palate, face, nose or brain. Pulmonary Zygomycosis • • Predisposing conditions: o Hematological malignancies o Lymphoma and leukemia, or severe neutropenia, o Treatment with cytotoxins and corticosteroids, desferrioxamine therapy o Diabetes and organ transplantation. Infections result by inhalation of sporangiospores into the bronchioles and alveoli leading to pulmonary infarction and necrosis with cavitation. Clinical Material • • • • • • Direct Microscopy • • Gastrointestinal Zygomycosis • • • Skin scrapings from cutaneous lesions Sputum and needle biopsies from pulmonary lesions Nasal discharges Scrapings and aspirates from sinuses in patients with rhinocerebral lesions Biopsy tissue from patients with gastrointestinal and/or disseminated disease. Zygomycetous fungi o Clearly visible in direct microscopic or histopathological mounts o Often difficult to grow in culture from clinical specimens. Rare entity Usually associated with: o Severe malnutrition (particularly in children) o Gastrointestinal diseases which disrupt the integrity of the mucosa. Primary infections probable result following the ingestion of fungal elements and usually present as necrotic ulcers. (a) Scrapings, sputum and exudates should be examined using 10% KOH & Parker ink or Calcofluor mounts; (b) Tissue sections should be stained with H&E and GMS. Examine specimens for broad, infrequently septate, thin-walled hyphae, which often show focal bulbous dilations and irregular branching. Cutaneous Zygomycosis • Local traumatic implantation of fungal elements through the skin, especially in patients with: o Extensive burns o Diabetes or steroid induced hyperglycemia o Trauma Figures 45&46: Stained specimen of a zygomycete Figure 44: Ulcerated cutaneous zygomycosis Disseminated Zygomycosis Bio 125 Medical Microbiology • As a rule, a positive direct microscopy, especially from a sterile Chapter 3. Introduction to Mycology 13 of 12 o site, should be considered significant, even if the laboratory is unable to culture the fungus. • The isolation of any zygomycete fungus -> regarded as potentially significant. In patients without predisposing conditions, the isolation of a zygomycete from a non-sterile site, (skin or sputum, must be interpreted with caution, especially in the absence of direct microscopy. Serology • • There are currently no commercially available serological procedures for the diagnosis of zygomycosis. Some laboratories have developed ELISA tests for the detection of antibodies to Zygomycetes. Figure 47: Stained specimen of a zygomycete Identification • • • Tissue morphology in zygomycosis showing distinctive infrequently septate thin walled hyphae with focal bulbous dilation and irregular branching, typical for those species belonging to the Mucorels • • Fast growing fungi Characterized by primitive coenocytic (mostly aseptate) hyphae. Asexual spores include o Chlamydoconidia, o Conidia and sporangiospores contained in sporangia borne on simple or branched sporangiophores. Sexual reproduction is isogamous producing a thick-walled sexual resting spore called a zygospore. Figure 48: GMS stained tissue section from a lung showing typical zygomycete hyphae and by chance a sporangium of Lichteimia corymbifera Culture • • • Primary isolation media: o Sabouraud's dextrose agar. *Most zygomycetes are sensitive to cycloheximide (actidione) and this agent should not be used in culture media. Fast growing, white to grey or brownish, downy colonies. Figure 51: Conidia appearance of Zygomycetes • • Most isolates are heterothallic i.e. Zygospores are absent, Based primarily on sporangial morphology. o The arrangement and number of sporangiospores, shape, colour, presence or absence of columellae and apophyses, o Arrangement of the sporangiophores and the presence or absence of rhizoids. Figure 49&50: Zygomycete appearance on culture • • • Common laboratory contaminants Zygomycetes are infrequently isolated in the clinical laboratory. In patients with any of the predisposing conditions described especially diabetes or immunosuppression and/or clinical symptoms: Bio 125 Medical Microbiology Figure 52&53: Micro and macroscopic appearance of Zygomycetes • Growth temperature studies (25, 37, 45C) can also be helpful. Chapter 3. Introduction to Mycology 14 of 12 • Laboratory identification of some zygomycetous fungi, especially Apophysomyces elegans and Saksenaea vasiformis may be difficult or delayed because of mold's failure to sporulate on the primary isolation media or on subsequent subculture onto potato dextrose agar. Sporulation may be stimulated by the use of nutrient deficient media, like cornmeal-glucose-sucrose-yeast extract agar, Czapek Dox agar, or by using the agar block method on water agar. • • • • • • • CAUSATIVE AGENTS • • • • • • • • • Lichtheimia corymbifera Apophysomyces elegans Cunninghamella bertholletiae Mortierella wolfii Mucor sp. Rhizomucor pusillus Rhizopus arrhizus Rhizopus sp. Saksenaea vasiformis INFECTIONS CAUSED BY ENTOMOPHTHORACEOUS FUNGI Caused by species of two genera Basidiobolus and Conidiobolus. • Infections are chronic, slowly progressive and generally restricted to the subcutaneous tissue in otherwise healthy individuals. • Other characteristics that separate these infections from those caused by mucoraceous fungi • A lack of vascular invasion or infarction andproduction of a prolific chronic inflammatory response, often with eosinophils and Splendore-Hoeppli phenomena around the hyphae. • Zygomycosis caused by Basidiobolus ranarum • Chronic inflammatory or granulomatous disease generally restricted to the subcutaneous tissue: o Limbs o Chest o Back o Buttocks • Primarily occurring in children and with a predominance in males. • Initially, lesions appear as subcutaneous nodules -> develop into massive, firm, indurated, painless swellings o Freely movable over the underlying muscle, are attached to the skin which may become hyperpigmented but not ulcerated. • Chronic inflammatory or granulomatous disease Typically restricted to the nasal submucosa Characterized by polyps or palpable restricted subcutaneous masses. Clinical variants: pulmonary and systemic infections o Human infections occur mainly in adults with a predominance in males (80% of cases). o Most cases: reported from the tropical rain forest areas of west, south and central America. Infections begin with unilateral involvement of the nasal mucosa. Subcutaneous nodules develop in the nasal and perinasal regions and progressive generalised facial swelling may occur. Symptoms: o Nasal obstruction o Drainage o Sinus pain LABORATORY DIAGNOSIS Clinical Material • Skin biopsy tissue • Tissue sections should be stained with H&E and GMS. • Examine specimens for broad, infrequently septate, thin-walled hyphae, which often show focal bulbous dilations and irregular branching. Direct Microscopy Figure 55: H&E stained section of infected tissue showing broad, infrequently septate, hyphae surrounded by an eosinophilic sheath [Splendore-Hoeppli phenomena], typical of zygomycosis caused by Basidiobolus ranarum • • • • • • HYALOHYPOMYCOSIS Mycotic infection of man or animals Caused by a number of hyaline (non-dematiaceous) hypomycetes Tissue morphology of the causative organism is mycelial. o This separates it from phaeohyphomycosis The causative agents are brown-pigmented fungi. General term used to group together infections caused by unusual hyaline fungal pathogens that are not agents of otherwise-named infections; such as Aspergillosis. Etiological agents include species of Penicillium, Paecilomyces, Acremonium, Beauveria, Fusarium and Scopulariopsis. Figure 54: Basidiobolus ranarum causing indurated, painless swellings Zygomycosis caused by Conidiobolus sp. Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 15 of 12 Figure 56: Hyalohyphomycosis (Fusarium sp.). Septate hyphae branching in acute or right angles infiltrate the blood vessels and subcutaneous fat (a PAS stain; b Grocott stain—9400) • • • CLINICAL MANIFESTATIONS Ranging from harmless saprophytic colonization to acute invasive disease. Predisposing factors: o Prolonged neutropenia, Leukemia patients or in bone marrow transplant recipients, o Corticosteroid therapy, o Cytotoxic chemotherapy and o To a lesser extent patient with AIDS. The typical patient is granulocytopenic and receiving broadspectrum antibiotics for unexplained fever. Figure 58: . Hyalohyphomycosis (Fusarium sp.). A Skin biopsy-inflammatory infiltrate around blood vessels (H&E 940). B Fungal skin infections— hyphae and yeast-like forms infiltrate the blood vessels and subcutaneous fat (H&E 9400) Culture • Hyaline hypomycetes o Common environmental airborne contaminants o A positive culture from a non- sterile specimen, (i.e. Sputum or skin,) needs to be supported by direct microscopic evidence to be considered significant. o A supporting clinical history in patients with appropriate predisposing conditions is also helpful. o Culture identification is the only reliable mean of distinguishing these fungi. LABORATORY DIAGNOSIS Clinical Material • Skin and nail scrapings • Urine, sputum and bronchial washings • Cerebrospinal fluid, pleural fluid and blood • Tissue biopsies from various visceral organs and indwelling catheter tips Figure 59: Culture of Chrysosporium [left] and Fusarium [right] showing typical colony colour for a hyaline hyphomycete ie any colour except brown, olivaceous black, or black. Figure 57: Lactophenol cotton blue showing thin hyaline septate hyphae Direct Microscopy • Presence of hyaline, branching septate hyphae, similar • To Aspergillus in any specimen, from a patient with supporting clinical symptoms -> considered significant. • Biopsy and evidence of tissue invasion is of particular importance. • Direct microscopy or histopathology does not offer a specific identification of the causative agent. Bio 125 Medical Microbiology Figure 60: a) Case 1: Maceration, erosion, and scales on the left 3 rd interdigital area. (b) Septate hyphae and intercalary chlamydoconidia (potassium hydroxide (KOH) with Parker (c) Basophilic septate hyphae (hematoxylin and eosin stain.) (d) Plate culture of Fusarium petroliphilum, a white felt-like colony with pale-yellow-coloured reverse. (e) Case 2: Maceration and desquamation on the left 3 rd and 4th interdigital area. (f) Septate hyphae and intercalary chlamydoconidia (Parker ink-KOH) (g) Septate hyphae in the upper stratum corneum (Periodic acid-Schiff-stain). Chapter 3. Introduction to Mycology 16 of 12 (h) Plate culture of Fusarium keratoplasticum, a white floccose colony with brown-coloured reverse. Serology • No currently no commercially available serological procedures for the diagnosis of any of the infections classified under the term hyalohyphomycosis. Identification • Culture characteristics and microscopic morphology are important, especially conidial morphology: o Arrangement of conidia on the conidiogenous cell o The morphology of the conidiogenous cell. Figure 61: Digital mucous of myxoid cysts Subcutaneous Phaehypomycosis • • • • • • • • • • • • • • • CAUSATIVE AGENTS Acremonium sp. Beauveria sp. Fusarium sp. Paecilomyces sp. Penicillium sp. Scopulariopsis sp. PHAEOHYPOMYCOSIS Mycotic infection of humans and lower animals caused by a number of dematiaceous (brown-pigmented) fungi Tissue morphology of the causative organism is mycelial. Separates it from other clinical types of disease involving brown-pigmented fungi where the tissue morphology of the organism is a grain (mycotic mycetoma) or sclerotic body (chromoblastomycosis). The etiological agents include various dematiaceous Etiological agents include various dematiaceous hyphomycetes especially species of: o Exophiala o Phialophora o Bipolaris o Exserohilum o Cladophialophora o Verruconis o Aureobasidium o Cladosporium o Curvularia o Alternaria Ajello (1986) listed 71 species from 39 genera as causative agents of phaeohyphomycosis. CLINICAL MANIFESTATION Range from localized superficial infections of the stratum corneum (tinea nigra) to subcutaneous cysts (phaeomycotic cyst) to invasion of the brain. • • • Figure 62: Cutaneous and subcutaneous phaeohyphomycosis of the forearm caused by Exophiala jeanselmei Paranasal sinus Phaehypomycosis • • • Range from localized superficial infections of the stratum corneum (tinea nigra) to subcutaneous cysts (phaeomycotic cyst) to invasion of the brain. Bio 125 Medical Microbiology Sinusitis caused by dematiaceous fungi, especially species of Bipolaris, Exserohilum, Curvularia and Alternaria is increasingly being reported, Patients with a history of allergic rhinitis or immunosuppression. Cerebral Phaehypomycosis • • • Phaehypomycosis • Subcutaneous infections occur worldwide Following the traumatic implantation of fungal elements from contaminated soil, thorns or wood splinters. Most Common agents: Exophiala jeanselmei and Wangiella dermatitidis Cystic lesions occur most often in adults. Occasionally, overlying verrucous lesions are formed, especially in the immunosuppressed patient. • Rare infection, Occurring mostly in immunosuppressed patients following the inhalation of conidia. Cerebral infections caused by Cladophialophora bantiana have been reported in a number of patients without any obvious predisposing factors. This fungus is neurotropic and dissemination to sites other than the CNS is rare Chapter 3. Introduction to Mycology 17 of 12 Figure 64: Cerebral phaehypomycosis LABORATORY DIAGNOSIS CULTURE Clinical Material • • • • Skin scrapings and/or biopsy; Sputum and bronchial washings; Cerebrospinal fluid, pleural fluid and blood; Tissue biopsies from various visceral organs and indwelling catheter tips Direct Microscopy • • • • • • Figure 66:. Haematoxylin and eosin (H&E) stained section showing characteristic, brown-pigmented, septate hyphal elements of Exophiala jeanselmei. • • • • • Skin scrapings, sputum, bronchial washings and aspirates should be examined using 10% KOH and Parker ink or calcofluor white mounts Exudates and body fluids should be centrifuged and the sediment examined using either 10% KOH and Parker ink or calcofluor white mounts Tissue sections should be stained using H&E, PAS digest, and Grocott's methenamine silver (GMS). Presence of brown pigmented, branching septate hyphae in any specimen, from a patient with supporting clinical symptoms should be considered significant. Biopsy and evidence of tissue invasion is of particular importance. Direct microscopy or histopathology does not offer a specific identification of the causative agent. • Primary isolation media: Sabouraud’s dextrose agar. Well recognized as common environmental airborne contaminants, A positive culture from a non-sterile specimen (i.e. Such as sputum or skin) À needs to be supported by direct microscopic evidence in order to be considered significant. A supporting clinical history in patients with appropriate predisposing conditions, is also helpful. Culture identification is the only reliable means of distinguishing these fungi. Figure 67: Cultures showing typical brown, olivaceous black or black colony colour for a dematiaceous hyphomycete. Serology • There are currently no commercially available serological procedures for the diagnosis of any of the infections classified under the term phaeohyphomycosis. Figure 65: Phaeohypomycosis on microscopy • • Direct microscopy of tissue is between chromoblastomycosis Characterized by the presence in planate-dividing, rounded phaeohyphomycosis where the causative organism is mycelial. necessary to differentiate tissue of brown pigmented, sclerotic bodies and tissue morphology of the Identification • Culture characteristics and microscopic morphology are important, especially conidial morphology, the arrangement of conidia on the conidiogenous cell and the morphology of the conidiogenous cell. • Cellotape flag and/or slide culture preparations are recommended. CAUSATIVE AGENTS • • • • • • • • • Alternaria sp. Aureobasidium pullulans Bipolaris sp. Cladophialophora bantiana Verruconis gallopava Curvularia sp. Exophiala sp. Exserohilum sp. Phialophora verrucosa TREATMENT OF SYSTEMIC OPPORTUNISTIC MYCOSES Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 18 of 12 • • • CANDIDIASIS o Mucocutaneous forms – topical nystatin, oral ketoconazole, or fluconazole o Systemic form – Amphotericin B (sometimes in conjunction w/ flucytosine, fluconazole, or caspofungin) CRYPTOCOCCOSIS o Combination therapy of amphotericin B and flucytosine – standard Tx ASPERGILLOSIS o Itraconazole or ampothericin B o Surgery o Invasive form – rapid administration of Amp. B or voriconazole (supplemented with cytokine immunotherapy e.g. G-CSF, IFN-gamma) o Amp. B resistant strains (A. terreus, A. flavus, A. lentulus) – new triazoles, posaconazole o Allergic forms – corticosteroid or disodium cromoglycate • REFERENCES • • • Lecturer’s ppt Jawetz’ Medical Micro 26th ed. Http://www.ijpsi.org/Papers/Vol2(12)/B02120306.pdf Bio 125 Medical Microbiology Chapter 3. Introduction to Mycology 19 of 12