CLINICAL MYCOLOGY 1 DR SONNIE P. TALAVERA Candidiasis, Cryptococcosis, and Other Infections Caused by Yeast The Genus Candida • most important of the yeast pathogens – increased use of immunosuppressive therapies – the use of broad-spectrum antibiotics – aging of the population, yeasts have assumed a large place among nosocomial pathogens • fourth most common cause of hospital-acquired blood stream infections in the United States, • C. albicans accounting for more than 50% of infections followed by Candida glabrata and the less common species C. tropicalis, Candida parapsilosis, C. krusei, and Candida lusitaniae Risk Factors • limited in extent and severity if the host is normal. • part of the normal flora • infections occur as an opportunity. • excessive moisture predisposes an individual to Candida infection. • Severe and invasive disease -- host defenses are compromised. • Diabetes, immunosuppressive diseases or therapies, and neutropenia as a result of disease (AIDS) • treatment with high-dose chemotherapy • Bloodstream infections, including fungal endocarditis, --- use of indwelling vascular lines Clinical Diseases • C. albicans is by far the most common pathogen • non-albicans species has increased – invasive infections caused by C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. lusitaniae • Resistance to imidazole antibiotics (e.g., ketoconazole, fluconazole, or itraconazole) • C. glabrata and C. krusei, – C. lusitaniae, -- resistance to amphotericin Cutaneous disease • most frequent infection • erythematous lesions of the skin with a creamy, white exudate or scaling • Moist conditions predispose to infection, -- diaper rash or skin folds (intertrigo) • Common sites • groin (a form of jock itch), • between fingers and toes, • under the female breast • axilla. • Workers who must immerse their hands in water for long periods of • hands, nails (onychomycosis), or the nail bed (paronychia). • chronic cutaneous disease is an uncommon manifestation in patients with defective cellular immunity Oral candidiasis • Oral candidiasis is usually manifest as creamy white patches overlying erythematous buccal mucosa (thrush) • dysphagia -- heavy infections. • Fissuring at the corners of the mouth is common • a common initial infection in patients infected with HIV
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CLINICAL MYCOLOGY 1DR SONNIE P. TALAVERACandidiasis, Cryptococcosis, and Other Infections Caused by Yeast
The Genus Candida• most important of the yeast pathogens
– increased use of immunosuppressive therapies
– the use of broad-spectrum antibiotics– aging of the population, yeasts have
assumed a large place among nosocomial pathogens
• fourth most common cause of hospital-acquired blood stream infections in the United States,
• C. albicans accounting for more than 50% of infections followed by Candida glabrata and the less common species C. tropicalis, Candida parapsilosis, C. krusei, and Candida lusitaniae
Risk Factors• limited in extent and severity if the host is
normal. • part of the normal flora • infections occur as an opportunity. • excessive moisture predisposes an individual to
Candida infection. • Severe and invasive disease -- host defenses are
– diabetes, – antimicrobial therapy, – pregnancy, – sexual activity
• Vaginal burning and itching, dyspareunia • discharge that is classically curd-like • difficult to eradicate.
Urinary tract infection• difficult to diagnose • frequently recovered from the urine as a result
of vaginal contamination or colonization of the bladder in patients with indwelling catheters, especially when systemic antibiotics have been administered
• Severe infection of the upper urinary tract, including necrosis of the renal papillae,
• The histologic response to Candida infection is commonly purulent
• Abscesses are frequently present. • Candida infection is granulomatous • Vascular invasion-- Aspergillus or the
zygomycetes • sections stained with the methenamine silver
stain or the periodic-acid–Schiff technique.Laboratory Diagnosis
• Specimens for culture should be taken from the affected organs and from lesions
• Modern continuous-monitoring blood culture systems detect most clinically significant yeast
• Tissue specimens, scrapings, or swabs from the mouth or vagina should be inoculated onto primary isolation media with and without cycloheximide.
• filamentous extensions from the edges of the colony (feet) is pseudohyphae are being produced
• C. glabrata and Cryptococcus spp. do not form pseudohyphae
• The extent of the mycologic evaluation depends on the clinical setting and the specimen type.
• Candida spp. are frequently isolated from the respiratory and urinary tracts
• preliminary report of C. albicans/C. dubliniensis may be issued if the germ tube test is positive
• yeast morphology using cornmeal agar to confirm the presence of chlamydoconidia,
• single agar plate within 24–48 hours• Molecular methods • inability to grow at 42°C, --chlamydoconidia, • sugar assimilation pattern can -- distinguish
these two species• Complete identification requires
assimilation tests• rapid assimilation trehalose test for the
identification of C. glabrata--common cause of invasive disease
• At present, immunologic tests cannot be recommended for diagnosis of candidiasis.
The Genus Cryptococcus• primary pathogen within this genus is C.
neoformans.
– C. neoformans var. neoformans • worldwide in pigeon droppings
or soil with bird excreta– C. neoformans var. gattii –
• tropics and subtropics associated with eucalyptus trees
– sexual stage -- basidiomycete, Filobasidiella neoformans
– Other cryptococci -- nonsterile sites – primary environmental source of C. neoformans
-- droppings of pigeons Risk Factors
• Immunosuppressive therapy • Disease
– HIV infection, 30–50% • Cryptococcus one of the
defining infections for AIDS,- most important risk factor.
• Immunologically competent patients -- no symptoms
– diffuse or localized-- coin lesions, do not calcify.
• Immunocompromised patients – – extensive infection with other infectious
agents, • Pneumocystis (carinii) jiroveci or
cytomegalovirus. • Extrapulmonary disease may appear weeks
after a pulmonary infection. Skin lesions
• result of hematogenous dissemination from the respiratory tract in immunocompromised patients
• single or multiple papules—ulcerate with thin exudate
• Primary cutaneous -- rare in immunocompetent individuals
Bone and joint infection• dissemination from the respiratory tract • Osteolytic lesions
– produced with abscesses soft tissue -- thin exudate with large numbers of cryptococci.
• Less commonly, joint spaces CNS infection
• cryptococcal meningitis– most frequent and most serious foci of
disseminated cryptococcal infection – acute but the presentation is typically
insidious – Headache , changes in mental status
and personality • Basilar meningitis
– cranial nerves, and invasion of the underlying cortex
– hydrocephalus & dec visual acuity. – Fever-- low grade
• acute meningeal irritation – Kernig's and Brudzinski's signs, often
absent.
Pathology of Cryptococcal Infection• histologic response depends -- degree of
encapsulation of the infecting strain. – little or no inflammatory response – cells of the normal tissue are separated
by large numbers of encapsulated cryptococci.
• cerebrospinal fluid – yeast – Large capsules may be seen in positive
India ink preparations. – Short, rudimentary pseudohyphae --rare
in tissue sections. • Poorly encapsulated
– granulomatous inflammatory response, – yeast cells -- cytoplasm of
macrophages. • staining of cryptococcal mucopolysaccharide
with mucin stains • melanin pigment with the Fontana–Masson
stain. • variable size (3–10 μm) – round
Laboratory Diagnosis• blood agar plates 35–37°C
– mucoid appearance to the colonies --Capsules may be diminished
– lack of growth on media that contain cycloheximide.
• Presumptive ID – India ink preparation
rapid urease test
Figure 1 Colonies of Cryptococcus neoformans usually appear mucoid when first isolated. Some strains are poorly encapsulated and lack the mucoid appearance. (Sabouraud dextrose agar)
• Definitive ID-- biochemical testing. • polysaccharide antigen of C. neoformans –
– cerebrospinal fluid and serum, by latex agglutination or EIA
• sensitivity exceeds 90% • assess prognosis and baseline
for treatment. – Large amounts of
antigen
– persistence of antigen following therapy –
• poor prognostic --patients not infected with HIV.
• not useful uniformly with AIDS– serum antigen more sensitive test than
CSF.-- HIV-infected patients – Outside CNS-- poor prognosis HIV
patients. – negative India ink test CSF
• good prognostic sign • does not have the same with
AIDS. The Genus Malassezia
• two most important species – Malassezia furfur -- more common
human pathogen – Malassezia pachydermatis.-- Indian
rhinoceros, ears of dogs, and humans. • M. furfur formerly Pityrosporum ovale or
Pityrosporum orbiculare Risk Factors
• normal flora on the skin of more than 90% of individuals
• cutaneous disease may occur in normal hosts. • Systemic infection
– neonates -- intravenous hyperalimentation with lipid solutions.
– yeast -- skin 37% of infants ICU• risk factors for M. pachydermatis -- similar.
Clinical Diseases• asymptomatic lesion -- epidermis in normal
Pityriasis versicolor• common infection of epidermis in --normal
patients• hyper- or hypopigmentation of the skin, trunk
and upper arms. • Fawn-colored macules – m.c. Presentation• ill effects -- purely cosmetic • TX topical application of fungicidal creams or
rinses.Systemic infection
• infants -- IV infusions of lipid. --supports the growth of M. furfur
• often asymptomatic but fever, leukocytosis, and thrombocytopenia
• Pneumonia – m.c. systemic manifestation Pathology of Malassezia Infection
• diagnosis – clinically
– KOH Calcofluor white or other fungal stain enhances detection
• demonstrating yeast and short hyphal forms “ spaghetti and meatballs
biopsied • hyperkeratosis, acanthosis, and
dermal mononuclear infiltrates may be seen.
• rare fatal cases, vasculitis, septic infarcts, and granulomatous inflammation
• lung, liver, and kidney. Laboratory Diagnosis
• Culture -- rarely requested in cutaneous disease• direct observation KOH preparations. • cultures of blood and intravenous catheter tips
from neonates.
Figure 2 Skin scrapings from a lesion of pityriasis versicolor. The yeast and short hyphal forms (spaghetti and meatball appearance) are characteristic of Malassezia furfur. (Lactophenol cotton blue stain; ×400)
• Before inoculation, a drop of sterile olive oil is added to Sabouraud dextrose agar or sheep blood agar.
– 2–3 days, light brown colonies, often with a dry appearance
– M. pachydermatis is not dependent on long-chain fatty acids for growth.
• microscopic examination – measure 3–7 μm in size. – bud is broad-based and the collarettes
of the phialides – distinct dark ring separating the mother
and daughter cells. Other Yeast and Yeast-like Pathogens
oropharyngeal ulcer • cause hoarseness, dysphagia, or
a painful lesion on the tongue or gingiva.
– HIV infection, -- resemble those produced by the virus
• fever, lymphadenopathy, anemia, leukopenia, thrombocytopenia, weight loss, and fatigue
– Solitary cutaneous lesions-- rare. Pathology of Histoplasmosis
• facultative intracellular pathogen• H. capsulatum is found predominantly in
macrophages. • pathologic lesions
• infected macrophages • noncaseating granulomas • caseating granulomas. • similar to Mycobacterium tuberculosis.
• demonstrated by H&E staining • periodic-acid–Schiff and methenamine silver
techniques are more sensitive,
• silver stain -- demonstration of old yeast cells in healed granulomas.
• differential diagnosis – B. dermatitidis, – Penicillium marneffei, – Leishmania species, – Candida spp., especially C. glabrata. – H. capsulatum in caseating granulomas – P. (carinii) jiroveci must be
differentiated. Laboratory Diagnosis
• yeast form in tissue – histology – cytologic preparation
• respiratory secretions, fluids, peripheral blood, bone marrow, or tissue imprints.
– Wet preparations and Calcofluor white • morphology of tissue
– Giemsa staining -- yeast cells is best seen
• 3–5 μm in diameter, single nucleus, and bud with a narrow neck.
technique for yeast phase, blood. • Other clinical specimens -- enriched agar, brain–
heart infusion agar w/ sheep blood, 25–30°C. • Colonies of H. capsulatum
– appear after incubation for 10–14 days – occasionally require incubation for up
to 4 weeks.
– primary isolation medium at 25–30°C, • fluffy and vary from white to
buff-brown in color – diagnostic asexual forms.
• microconidia, -- – resemble B.
dermatitidis. • macroconidia –
– roughened projections from the periphery of the conidia, -- tuberculate
Figure 4 Tuberculate macroconidia with microconidia of Histoplasma capsulatum in culture. These structures are characteristic of the environmental mould form of this dimorphic fungus. (Lactophenol cotton blue stain; ×400; CDC Public Health Image Library)
• Diagnosis – macroscopic appearance of the
colonies, – rate of growth, – growth of H. capsulatum on media
containing cycloheximide, – the presence of yeast forms in tissue – clinical history
• Final ID— – nucleic acid hybridization probe
testing. – molecular techniques (Accuprobe,
GenProbe, San Diego, CA). – Exoantigen testing.
• Serologic tests -- detection of anti-Histoplasma antibodies
• pseudoepitheliomatous hyperplasia of the epidermis overlying the inflammation
• yeast cells are most often found -- microabscesses or within multinucleated giant cells.
• H&E, PAS or methenamine silver stains. – thick-walled yeast cells measure 8–15
μm in diameter – broad-base during the budding process. – diagnosis of blastomycosis --
characteristic yeast forms in tissues by pathologist
Laboratory Identification• Direct demonstration of the yeast cells
– tissue sections – wet preparations of aspirated fluids and
imprints of tissues. – Calcofluor white staining may enhance
the detection of the yeast. • Growth -- more rapid than Histoplasma;
– fluffy white to buff colonies, – microconidia resemble those of H.
capsulatum – macroconidia are not formed. – Conversion to the yeast phase -- 37°C
with hemoglobin and cysteine containing agar
• nucleic acid probe testing colony material – – preferred method for confirming ID
(Accuprobe, GenProbe,). • in situ hybridization protocol –
– ID of yeast-like organisms in tissue sections
– using specific and pan fungal oligonucleotides -- 18S and 28S rDNA ( sensitivity 90% and specificity 97%).
Figure 5 Mould form of Blastomyces dermatitidis in culture. The lollipop appearance of the conidium on a conidiophore is characteristic of the environmental mould form for this dimorphic fungus. (Lactophenol cotton blue stain; ×400)
Figure 6 Budding yeast cells of Blastomyces dermatitidis in culture. When cultures are incubated at 37°C, large, broad-based budding yeast with a double-contoured all are detected which are characteristic for the yeast phase of this dimorphic fungus. (Lactophenol cotton blue stain; ×400)
• complement fixation – used only as an adjunct to culture of the
fungus.• Blastomyces antigen test
– available in a reference laboratory
The Genus Coccidioides• C. immitis --sole etiologic agent of
coccidioidomycosis. • two genetically
– C. immitis clades, one called the California clade (C. immitis)
– non-California clade (Coccidioides posadasii)
• soil-dwelling fungi , LOCATION – southern portions of California – southwestern United States
(espsouthern Arizona, southern New Mexico, and west Texas),
– northern Mexico, – Central and South America (especially
Venezuela) – Arizona -- dramatic increase in cases
over the years, \occurring > 65 years and HIV infection
• C. immitis and C. posadasii -- agents of bioterrorism
– potential bioweapons – strict federal regulations -- possess and
transport and report confirmed infections
Risk Factors• Residence in an endemic area is the primary risk
factor – arthroconidia of the mould phase are in
the soil and easily disseminated through the air
– Utah 2001 • epidemic pulmonary
coccidioidomycosis -- group of archeology workers.
– California October 2001. • outbreak in individuals
attending the World Championship of Model Airplane Flying,
• Genetic factors – frequency of severe and disseminated
infection, • Race
– black people and Filipino people > white people.
– Asians, Native Americans, and Mexicans • Sex
– Adult women > men -- develop erythema nodosum
– disseminated infection -- adult men >in women.
• Immunosuppressive infection or disease -- impt risk factor , HIV patients
Clinical Diseases Primary coccidioidomycosis
– most frequently asymptomatic– indicated by the high prevalence of
positive results of skin tests for the coccidioidal Ag in endemic areas.
Symptomatic disease– fever with cough or chest pain, or both,
mimic bacterial pneumonia – Erythema nodosum and erythema
multiform-- good prognostic signs. – Solitary pulmonary nodules -- primary
pulmonary infection. Disseminated infection
– affects the skin, skeletal system, and meninges.
– papules, ulcers, draining sinuses, and subcutaneous abscesses.
Clinical Diseases• Arthritis most
– often results from involvement of adjacent bones.
– • Meningitis
– acute but is more commonly indolent and chronic.
Pathology of Coccidioidomycosis• granulomatous, with or without caseation. • Developing spherules found in macrophages
and multinucleated giant cells – Endospores within the spherules
measure 2–5 μm in size – Spherules measure up to 200 μm in
diameter, • differential diagnosis
– nonbudding forms of B. dermatitidis or C. neoformans.
Laboratory Diagnosis• Spherules -- sputum or bronchoalveolar lavage
fluid—dec sensitivity• Calcofluor white staining with KOH – inc
sensitivity. • C. immitis
• grows rapidly (less than 1 week) on media with or without cycloheximide
• appear on blood agar plates • arthroconidia
• highly infectious and easily transmitted by aerosolization.
• Infection of laboratory workers is a major concern, -- handled in a biosafety cabinet.
• (along with H. capsulatum) as a risk group 3 biological agent:
• agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may not be available ( Department of Health and Human Services, 1999 ).
• colonies of the Coccidioides are extremely variable in appearance,
• ranging from velvety or cottony to granular or powdery.
• arthroconidia • alternating barrel-shaped
arthroconidia with empty disjuncture cells are distinctive of C. immitis
• differentiated Trichosporon, Geotrichum, and Gymnoascaceae.
• Confirmation ID—
• most commonly by the nucleic acid hybridization probe test.
• complement fixation test – • useful for assessing the extent and
prognosis of coccidioidomycosis
Figure 7 Spherules of Coccidioides immitis in tissue. The observation of these large structures in tissue is diagnostic for coccidioidomycosis. (Hematoxylin and eosin stain, ×100)
Figure 8 Spherules of Coccidioides immitis in tissue showing endospore release. At maturity, the spherule ruptures and releases endospores which in turn mature to become spherules. When seen in tissue, these immature endospores may be confused with other smaller yeast. (Gomori's methenamine silver stain; ×1000)
• Antibodies are detected approximately 2–6 weeks after infection.
– height of the titer – SPREAD of disseminated disease
– rising titer bodes – suggest poor outcome.
• Skin testing does – disseminated infection may
demonstrate anergy to the skin test antigen.
The Genus Sporothrix• Sporothrix schenckii, • two varieties:
– S. schenckii var. schenckii • present worldwide in soil,
plants, and decaying vegetation • numerous outbreaks, with one
major outbreak occurring among florists and nursery and forestry workers who handled contaminated sphagnum moss, Wisconsin 1988
• mode of entry -- traumatic implantation (vs. inhalation),
– 4th m.c. cause of disseminated opportunistic infection in AIDS pt. in Thailand, southern China, and other parts of south-east Asia where it is endemic
– environmental source for this fungus is still unknown
– recent reports suggest -- bamboo rats – unique among the penicillia in being
dimorphic, • producing in tissue yeast-like
cells that are oval or cylindrical and may have crosswalls
Dermatophytoses• Dermatophytic fungi -- common and important
causes of human morbidity – mild and rarely do cause invasive
Risk factors• result from contact with the relevant source of
the fungus, -- clue to the diagnosis. • face in farmers who lean against their cows as
they milk the animals -- Trichophyton verrucosum,
• epidemic of cutaneous fungal infection among American troops during the Vietnam War
• become more frequently -- direct contact between the athletes, such as wrestling
Clinical Diseases• infection of the epidermis, hair, and nails • referred to as ringworm (or tinea),
– advancing, serpiginous nature of the lesions (especially evident on the skin).
• Locations – tinea capitis (scalp), – tinea barbae (beard), – tinea corporis (trunk and limbs), – tinea pedis (foot), – tinea cruris (groin), – tinea unguium (nail, which is also called
onychomycosis). • Incidence
– Epidermophyton floccosum infects the skin only,
– Microsporum spp. infect the scalp and skin,
– Trichophyton spp. infect the skin, scalp, and nails.
• most common cause of tinea capitis
• M. canis -- common cause of scalp infection, particularly in children
• T. mentagrophytes and T. verrucosum --deep infection of the hair follicles occurs, a boggy inflammatory process called a kerion
• T. mentagrophytes, T. rubrum, and M. canis --commonly found in tinea corporis and tinea pedis.
– T. rubrum -- chronic and intractable• E. floccosum -- common cause of tinea cruris.
Laboratory Diagnosis• Tx of dermatophyte infections is not directed by
the specific ID, • demonstration of hyphae in skin scrapings (KOH
preparation) is frequently only diagnostic technique performed before the initiation of therapy
• size and morphology of hyphae -- dermatophytes
• Confirmation of the species – assessing the probable source of the
infection, – assessing for chronic, relapsing
infections • Skin scrapings, nail, and hair -- KOH
preparations, w/ Calcofluor white – Microsporum audouinii, M. canis, or
Microsporum ferrugineum • cause the hairs to fluoresce
with a UV light (Wood's lamp). • Microscopic examination --appearance and
location of the arthroconidia. – ectothrix colonization,
• M. canis and M. audouinii infection,
• arthroconidia are external to the hair shaft.
– Endothrix invasion, • T. tonsurans, • arthroconidia within the hair
shaft. – favic infection,
• hyphae, air bubbles, or tunnels and fat droplets are present in the intrapilar area.
Figure 10 Hyphae of a dermatophyte are demonstrated in a scraping from the edge of a tinea lesion. The presence of thin-segmented hyphae in this clinical setting establishes the diagnosis of dermatophytosis but does not define the specific etiology. (KOH preparation; ×400)
Figure 11 31 A fluffy white Microsporum canis colony (left) has the characteristic lemon yellow pigment, which may also be seen on the reverse of the colony (right). (Sabouraud dextrose agar with cycloheximide)
Figure 12 Thick-walled, roughened, tapered, spindle-shaped macroconidia of Microsporum canis with thin, septate hyphae. (Lactophenol cotton blue stain; ×400)
Figure 13 Thick-walled, smooth macroconidia of Microsporum gypseum. (Lactophenol cotton blue stain; ×400)
Figure 14 34 Fluffy white colonies of Trichophyton rubrum with diffusible red pigment, which may also be seen on the reverse of the colony. (Potato dextrose agar)
Figure 15 Fluffy white colony with radial folds of Trichophyton mentagrophytes. (Sabouraud dextrose agar with cycloheximide)
Figure 16 A flat colony showing pale yellow coloration with a reddish-brown pigmentation at the periphery characteristic of Trichophyton tonsurans. Radial furrowing of the thallus as seen is also common. (Sabouraud dextrose agar with cycloheximide)
Figure 17 The suede-like texture and yellowish green color are characteristic of Epidermophyton floccosum. (Sabouraud dextrose agar with cycloheximide)
• Sabouraud dextrose agar – recovery of dermatophytic fungi and is
also e for isolating C. albicans, • All dermatophytic fungi have septate, hyaline
hyphae and produce macroconidia and/or microconidia in various combinations.
– Arthroconidia and chlamydoconidia may also be produced
– but macroconidia, and especially the microconidia, --more impt for ID.
• combination of both micromorphologic and phenotypic testing is used to differentiate the dermatophytes
• Microsporum species – produce characteristic macroconidia, --
key diagnostic structures. – M. audouinii,
• a classic anthropophilic agent of ringworm,
• often does not produce diagnostic structures,
– M. canis, • most frequently isolated
zoonotic species of this genus, • produces a characteristic lemon
yellow pigment in culture -- intensified by growth on potato flake agar.
• macroconidia – Microsporum gypseum,
• the most common isolated geophilic species
• macroconidia • Trichophyton
– most commonly isolated dermatophytic fungi:
• T. rubrum • T. verrucosum, • T. mentagrophytes • T. tonsurans
– colonies • fluffy, granular or, less
commonly, glabrous appearance.
– Macroconidia,. • thin-walled, smooth, and
contain variable numbers of septa.
– Microconidia are formed more commonly.
– Trichophyton agars
• helpful in the characterization of nutritional requirements among members of the genus.
• media allow for the assessment of dependency on inositol, thiamine, or both compounds together.
– T. rubrum • intensely red pigment
enhanced by culture on potato flake agar or cornmeal agar with 1% dextrose.
– T. mentagrophytes • Production of urease within 3–5
days – hair penetration test can be used as a
differential test. • T. mentagrophytes --wedge-
shaped defects in hairs • T. rubrum --grows on the
outside of the hair without penetration.
– Epidermophyton – – E. floccosum,
• anthropophilic fungus • important cause of tinea cruris • Colonies, which are initially
brownish yellow, gray, or khaki brown, become velvety and folded as they mature
• E. floccosum produces no microconidia, but distinctive macroconidia
– smooth external walls, with up to four crosswalls and a club shape
• they tend to ‘flocculate,'. • molecular approaches have also
recently been initiated to study the dermatophytes
Aspergillosis, Fusariosis, and Other Mycoses Caused by Hyaline Fungi
• Large group of saprophytic fungi, frequently encountered in the clinical laboratory
• some of the most important pathogenic moulds.
• moulds are collectively classified --hyaline • disease -- hyalohyphomycosis. • human disease -- immunosuppressive therapies • human disease are classified
• Eurotiales (Aspergillus, Paecilomyces, and Penicillium),
• Microascales (Scedosporium and Scopulariopsis)
• Hypocreales (Cylindrocarpon, Fusarium, and Trichoderma).
• Aspergillus spp. and Fusarium spp., • most commonly associated with
invasive human disease.
The Genus Aspergillus• Aspergillosis -- genus Aspergillus. • wide spectrum of infections in the normal host
– mycotoxicosis, • intoxication by ingestion of
Aspergillus toxin – allergic manifestations,
• associated with Aspergillus spore exposure, respectively.
• superficial infection; compromised host – localized noninvasive infection with
tissue damage or foreign body obstruction;
– invasive infection • 1ST Aspergillus fumigatus -- most common with
human diseases, • 2ND Aspergillus flavus, although in some
institutions A. flavus has emerged as the most common species detected
• 3RD either Aspergillus niger or Aspergillus terreus – Resistance of A. terreus to amphotericin
B Risk Factors
• Hyphomycetes -- fungi to which all are exposed on a regularly.
– resident in vegetation of all types and ubiquitous in the environment.
• Nosocomial infections are not uncommon • most important risk factors for invasive
aspergillosis – immunosuppressive disease, – high-dose chemotherapy with or
without transplantation, – solid organ transplantation
• Neutropenia is a prominent predisposing factor for disease
• Invasive disease -- rare, • Wound infection
– extensive burn wounds AND exposed to conidia
• Fungus balls – lung of patients with COPD,
bronchiectasis, or old cavities caused by tuberculosis
Clinical Diseases• spectrum of invasive disease caused by
Aspergillus species is broad. • low pathogenicity in humans, • immunocompromised patients
expanded vesicles as small globes projecting above the vegetative mycelium.
• conidiophore is smooth, relatively short (300–500 μm), -- expanded into a flask-shaped vesicle.
• phialides develop in a single row (uniseriate), and most commonly develop only on the upper two-thirds of the vesicle parallel to the axis of the conidiophore
• conidia are roughened (echinulate) and round to oval in shape.
• A. flavus. – second most important pathogen– colonies grow rapidly – velvety yellow to green appearance
once fruiting heads develop – conidiophore is long (400–700 mm),
ending in a vesicle that may be elliptical or round.
– conidiophore is rough and stubbly-- single or double (biseriate) row of phialides occurs over the whole vesicle or the upper three quarters -- Round to oval conidia appear yellow-green en masse and measure 3–4.5 μm.
• Aspergillus terreus – production of a cinnamon brown colony – microscopic characteristics that include
short conidiophores (< 250 μm) and biseriate phialides.
• gold standard for diagnosing invasive aspergillosis -- positive culture from sterile tissue with histologic evidence of mycelial invasion,
ELISA for the detection of galactomannan antigenemia
• rapid diagnosis of invasive aspergillosis.
• sensitivity less than 50% to 95% and specificity less than 70% to 100%, suggesting that it will not replace careful microbiologic and clinical evaluation
– Variables • antimicrobial therapy
(piperacillin–tazobactam and amoxicillin–clavulanate can cause a false-positive result)
• age (false-positive results are more common in children than in adults)
• Molecular methods – culture confirmation of Aspergillus
species – direct detection of fungal DNA in clinical
material
Figure 18 A fruiting head of Aspergillus is demonstrated within a pulmonary mycetoma (fungus ball). The phialides and conidia are well demonstrated. Although the hyphae of Aspergillus are not etiologically diagnostic, demonstration of the fruiting head documents the presence of this genus. (Hematoxylin and eosin stain, ×400)
Figure 19 The fruiting head of Aspergillus flavus is uniseriate and biseriate. The phialides extend around much of the vesicle with a roughened (spiny) appearance of the conidiophore present. (Lactophenol cotton blue stain; ×400)
The Genus Fusarium• number of infectious diseases
– keratomycosis – burn wounds – invasive disease
• considered the second most common pathogenic moulds causing invasive disease
• histopathologic
– identical to invasive aspergillosis (angioinvasive and acute branching-septate hyphae).
– fungemia • Colonies of Fusarium
– develop rapidly, – producing a fluffy aerial mycelium,
which often has a pink, lavender, or salmon color.
• first recognized in outbreaks of pulmonary disease -- malnourished infants in Eastern Europe during World War II (called ‘interstitial plasma cell pneumonia')
• major cause of life-threatening pneumonia in 60–80% of HIV-infected adults
• one of the AIDS-defining illnesses • less than 10% of patients because
• availability of highly active antiretroviral therapy,
• use of anti-PCP prophylaxis• improved awareness of the infection
• Concurrent infection with other agents, cytomegalovirus or C. neoformans, is common.
• fatal if untreated. • most common extrapulmonary sites of infection
-- the thyroid, liver, bone marrow, lymph nodes, and spleen
Pathology• definitive diagnosis --demonstration of cysts or
trophozoites within clinical specimens
• open-thorax lung biopsy -- one of the most sensitive tests
• sputum and bronchoalveolar lavage -- reliable specimens for diagnosis
• H&E • widening of the alveolar septa with an
infiltrate of mononuclear cells and a foamy exudate within the alveolar spaces (sometime referred to as honeycombed exudates)
• exudate consists of aggregated cysts and trophozoites(methenamine silver stain).
• cysts, which are 5–6 μm , brown to black cup-shaped or crescent-shaped
• Pneumocystis cysts do not bud, Laboratory Diagnosis
• cell cultures • standard for diagnosis is staining
bronchoalveolar lavage fluid with an immunofluorescent monoclonal antibody
– directed against surface epitopes for Pneumocystis cysts and trophozoites
• development of sporozoites within a cyst, -- sporozoites are released.
• fluorescein-conjugated monoclonal antibodies -- specific and sensitive
• more sensitive in histochemical stains • Polymerase chain reaction assays -- testing
samples with low organism burden,
Figure 22 Cysts of Pneumocystis jiroveci in a smear from bronchoalveolar lavage. (Methenamine silver stain; ×400) (Courtesy of Dr Russell K. Brynes, CDC Public Health Image Library)
Figure 23 Histopathologic changes in protothecosis of the skin and mucous membrane of the nose due to Prototheca wickerhamii. Septation of the algal cells leads to multiple intracellular bodies and structures known as morula. (Courtesy of Dr William Kaplan, CDC Public Health Image Library)
Mycoses Caused by Dematiaceous Moulds• also called the black moulds • fascinating and complex group of fungi –
– formation of a dark pigment due to melanin in the cell walls of hyphae or conidia or both
• soil and plant saprophytes • rare causes of disease in humans • emerging fungal pathogens
Clinical Diseases• eumycotic mycetoma (pl. mycetomata) • chromoblastomycosis,
– predominantly in hosts with normal immune systems,
• phaeohyphomycosis, – immunocompromised patients
• diagnosed on the basis – unique histologic picture of the fungus
in tissue – isolation of the fungus in culture – morphologic evaluation of the
reproductive structures. • classification of black moulds. • Dematiaceous moulds, • Stachybotrys chartarum,
– not associated with invasive disease, – ‘sick building syndrome,'
Mycetoma• localized, chronic granulomatous infection of
the cutaneous and subcutaneous tissues • formation of a sinus tract and frequent
involvement of the contiguous bones. • confined to the hands or feet (also called
‘Madura foot') – farmers, field hands, … in contact with
– demonstration of sclerotia (also referred to as grains or granules) in a tissue biopsy, in draining exudates from a sinus tract, or in material aspirated from an unopened sinus ( Fig. 60-38 ).
• grains are microscopically composed of broad, interwoven, septate hyphae 2–5 μm in diameter
– associated with a dark pigment and generally called black grains,
– sclerotia of nondematiaceous fungi consist of nonpigmented hyphae called white grains.
• Sabouraud dextrose agar, 4 weeks or longer to grow
– gross colony morphology and pigmentation
• micromorphologic characteristics of the reproductive structures
• physiologic tests such as carbohydrate and nitrate utilization may aid in ID.
• molecular techniques Chromoblastomycosis
• chromomycosis • chronic infection of the skin and soft tissue
– presence of muriform fungal structures called sclerotic bodies within the infected tissues.
– painless verrucous plaque or nodule at the site of inoculation with healing areas of the lesion evident
– formation of scar materials. • most common etiologic agents are the black
moulds – Fonsecaea pedrosi, Phialophora
verrucosa, and Cladophialophora carrionii,
– round, nonhyphal, brown cells (sclerotic bodies) in tissues.
• Culture and recognition of the morphologic features
Pheohyphomycosis• from the Greek word phaeo, meaning ‘dark' • fungi that develop in tissue in the form of dark-
walled dematiaceous septate mycelial elements'
• cause of subcutaneous and systemic diseases • pigmented septate hyphae
– chromoblastomycosis and eumycotic mycetoma are distinguished from pheohyphomycosis by the appearance in tissue of sclerotic bodies and mycotic granules, respectively.
– indolent subcutaneous lesion, – devastating, destructive infection that is
almost always fatal. – solitary asymptomatic subcutaneous
nodule or cyst • most common dematiaceous fungi Exophiala
jeanselmei, Wangilla dermatitidis, and Phialophora
• Systemic pheohyphomycosis, • more common infection, especially in
individuals with chemotherapy induced neutropenia.
• pheohyphomycosis from 1966–2002, • Cladophialophora bantiana accounted
for 48% cerebral disease • Ramichloridium mackenzei 13% of cases
• 72 cases of disseminated pheohyphomycosis, • leukemia most common underlying condition, • ,most common pathogen was Scedosporium
dermatitidis) (7%) • mortality was 79%. • tissue, the presence of irregularly swollen,
septate hyphal forms with yeast-like structures that stain with a melanin-specific Masson–Fontana stain is presumptive for the diagnosis of pheohyphomycosis.
Figure 24 Histologic appearance of ‘black grain mycetoma' due to Madurella mycetomatis. (Gridley stain, ×1000) (Courtesy of Dr Libero Ajello, CDC Public Health Image Library.)
Figure 25 Close-up of a dematiaceous fungus growing on Sabouraud dextrose agar.
Figure 26 Image showing phialides with terminal conidia of Madurella mycetomatis. (Lactophenol cotton blue stain) (Courtesy of Dr Lucille K. George, CDC Public Health Image Library)
Figure 27 A fluffy gray colony of Rhizopus species fills the space within the Petri dish (lid lifter). (Sabouraud dextrose agar)