Fungal spores

Spores may be of following types

A. Sexual spores
Arise by meiosis
1. Ascospore – Aspergillus, Saccharomyces, Pseudoallescheria
Endogenous 4-8 ascospores in asci in ascocarp
2. Basidiospore – trichosporon, malassezia
Basidiospores in basidia in basidiocarp
3. Zygospore – Zygomycetes
Fusion of hyphae

B. Asexual spores

1. Types
a. Arthrospore – septa of hyphae, cuboidal
Dermatophyte, Geotrichum, Trichosporon, Coccidiodes
b. Blastspore – budding
Candida, Cryptococcus, yeasts
c. Chlamydospore – candida

Yeast and Yeast like organism

Features Candida albicans Crytococcus neoformans

Colony character of culture on nonmucoid colonies mucoid colonies

SDA slope
Gram stain Gram positive oval budding Gram positive spherical
yeast cells budding pleomorphic yeast
cells

India ink Oval budding yeast cells Spherical budding

without capsule pleomorphic yeast cells with
capsule seen as a clear
unstained halo 2-3 times the
cell in dark background

Urease test Negative Positive

Capsule Absent Present

Disease caused oral thrush, cutaneous, Meningitis, pulmonary,
systemic infections cutaneous

1
 Different types of yeasts

Features Candida Cryptococc Rhodotorula Saccharomy Geotricum Trichosporon

us ces
Germ tube C. albicans - - - - -
Capsule - + + - - -
Urease C.krusei + + - - +
Pseudohyphae + - - + - +
True hyphae - - - - + +
Arthrospore - - - - + +
Chlamydospore C.albicans - - - - -

Pathogenic yeast

1. Candida
2. Crytococcus
3. Trichosporon
4. Rhodotorula and Geotrichum in extremely immunocompromised

Candida
163 species

Features albicans tropicalis krusei glabrata parapsilosis dubliensis

chrom agar light blue dry flat smooth pink light cream deep green
green pink
germ tube + - - - - +
blastospore + + + + no + +
pseudohyph
ae
chlamydospore + - - - - +
azole S S R R develop S R
inherent develop
diseases with HIV, haemat, haemat, nail, skin,
diabetes neutropenia, neutropenia, neonatal
transplant, transplant, ICU
commonest sepsis
in sepsis

Albicans – normal flora of skin, GIT, female genital tract

Hence causes endogenous infection
Use of antibacterials, AIDS increase candida infections

2
Pathogenesis
Colonisation
Conversion to hyphal form – invasiveness
Biofilm formation

Clinical features
Carriage – mouth, skin, anorectal, vaginal
Predisposing factors – extremes of age, diabetes, malignancy, HIV, antibiotics,
immunocompromised, radiation, chemotherapy, catheter, surgery, zinc or iron deficiency
1. Cutaneous – intertrigo in skin folds
Paronychia, onychomycosis – nails
Diaper dermatitis
2. Mucocutaneous infections
Oral thrush – buccal mucosa, gum, tongue, palate
Congestive reddening, dry, smooth, shiny varnish-like appearance
Thirst, metallic taste sensation, dryness, burning
Candida albicans – commonest
Angular cheilitis – fissures at angle of mouth
Glossitis, oesophagitis, gastritis
Vulvovaginitis, Balanitis, balanoposthitis
Keratoconjunctivitis
3. Systemic – UTI, endocarditis in prosthetic valve, pericarditis, pulmonary candidiasis
Meningitis, sepsis
Arthritis, osteomyelitis, endophthalmitis

Laboratory diagnosis
1. Direct examination – KOH, gram stain
Yeast cells with budding, pseudohyphae
2. Culture – SDA, SAB
3. Germ tube formation
4. Chlamydospore formation
5. Sugar fermentation and assimilation
6. Chrom agar
7. Antigen detection – mannan, b-1,3 D glucan, enolase, HSP-90

Germ tube formation

Also called Reynold – Braude phenomenon
Use – presumptive diagnosis of candida albicans
Albicans is more susceptible than other candida species
Pooled human serum 0.5ml + candida 2-3 colony incubate at 37C for 3 hours
Include positive and negative control – albicans and tropicalis
Pooled sera – gives uniform environment for test and control strain and 12-18% humans have
antibodies to candida and antifungals may be present in sera

3
Wet preparation made after 3 hrs
Observation – germ tube is an outgrowth from the cell with no constriction at base, length 2-3
times of vegetative cell and breadth half of cell, parallel walls
95% albicans positive
Dubliensis and stellatoidea also positive
Stellatoidea – sucrose negative c.albicans

features germ tube pseudohyphae true hyphae

formation outgrowth from cell budding apical elongation
walls parallel not parallel parallel
septa absent constricted, curved straight,
perpendicular
base no constriction constricted no constriction
branching absent present present
terminal cell only one cell spherical cylindrical

Treatment
Cutaneous and mucocutaneous – ketoconazole topically
Systemic – amphotericin B + flucytocine, azoles

CRYPTOCOCCUS
It is an encapsulated heterobasidiomycetous fungus

A common worldwide opportunistic pathogen

It is true yeast – both sexual and asexual stage

Cryptococcus neoformans:

C. neoformans strains have five serotypes based on capsule structure.

The serotype classification (A to D) describes antigenic differences in the structure of the
polysaccharide capsule; these differences can be detected by antibodies from rabbit sera or by
specific monoclonal antibodies
Serotyping done by:
A) Inhouse or commercial antisera
B) Monoclonal antibodies (E1)
C) Immunochromatographic test
D) Molecular methods: RFLP

It is divided into varieties based on biochemical, morphological and DNA typing methods.

4
Features Crytococcus grubii Cryptococcus Cryptococcus gattii
neoformans
Serotype A D B, C
Epidemiology Europe, USA Europe, USA Tropics, Subtropics
Telemorph Filobasidiella Filobasidiella Filobasidiella
neoformans neoformans basilispora
Risk group HIV On steroids Immunocompetent
Ecology Pigeon droppings Pigeon droppings Eucalyptus
camaldulensis
Virulence Highly virulent Highly virulent Low virulence
Onset of d/s Acute Acute Insidious
D/s spectrum Cryptococcemia Cryptococcemia Focal CNS lesions
Neurological sequelae Less Less More
Blastospores Rounded Rounded Elliptical
CGB Negative Negative Cobalt blue colour
GCP Negative Negative Positive
CDBT Negative Positive (orange) Blue
D-tryptophan Negative Negative Utilised
D-proline Negative Negative Utilised
Malate Negative Negative Utilised
Mortality High High Low
Drug susceptibility Susceptible Susceptible Resistant
Treatment Shorter Shorter Longer, Relapse

Ecology

C. neoformans
Soil contaminated by bird droppings- pigeons (Columba livia), turkeys, and chickens
Association with pigeon droppings may be due to nitrogenous compounds particularly
creatinine that favours the growth of this organism.
Birds resistant to disease by the yeast may result from their very high body temperature (41-
43ºC), which is not conducive to growth of C. neoformans. However, the yeast may
transiently colonize the gastrointestinal tract of the birds. The birds may simply represent
vectors.

C. gattii
Australian tree Eucalyptus camaldulensis (red gum) is the natural reservoir.
Trees such as firs, maples, and oaks may also be an ecologic niche for specific strains of C.
gattii.
it has been found in soil associated with a variety of bacteria,amebae, mites, worms, and sow
bugs.

Other trees associated with Cryptococcus:

Serotype C: tropical almond trees (Terminalia cattapa)
C.neoformans var grubii – rotting wood debris of Cassia grandis (pink shower tree)

5
 Mycology and life cycle

It is a gram positive, 4-20 µm, spherical, budding (narrow based) yeast cell. It has a capsule
which is 2-3 times the size if the yeast cell.
On modified SDA it forms cream to white opaque mucoid colonies,
brown colonies on Bird seed agar due to production of the enzyme laccase which converts
caffeic acid in the medium to melanin.
The life cycle of C. neoformans involves two distinct forms, asexual and sexual.
The asexual stage exists as encapsulated yeast cells that reproduce by simple, narrow-based
budding.
The sexual form is a laboratory observation

Virulence factors
I. Capsule
glucuronoxymannan + galactoxylomannan
Polysaccharide capsule
II. Melanin
C. neoformans possesses laccase(phenol oxidase)

Pathogenesis:

Mode of infection: Inhalation of infectious propagules

The pathogenesis of cryptococcosis is determined by three broad factors:
(1) the status of the host defenses;
(2) the virulence of the strain of C. neoformans; and
(3) the size of the inoculums

Yeast survives in the environment for 2 years or more, loses its capsule and is inhaled.

In the alveoli, the yeasts contact the alveolar macrophages, which recruit other inflammatory
cells through cytokines or chemokines, and a proper Th1 response and granulomatous
inflammation is elicited.

Immunocompetent Immunocompromised

Yeast continues to multiply and disseminates

Effective immune Yeast produces
causing clinical disease
Response eliminates yeast lung/lymphnode

complex and

Loss of immunity
Asymptomatic until 6
loss of immunity
Clinical manifestations:

Predisposing factors
 HIV infection
 Immunosuppressive treatment
 Organ transplantation
 Lymphoma and leukaemia (CLL)
 Diabetes mellitus
 Chronic corticosteroid therapy
 Chronic renal failure

1. Pulmonary
Range from asymptomatic infections to life threatening pneumonia.
Symptoms are fever, chest pain, cough, weight loss, sputum production

2. CNS cryptococcosis
Cryptococcal meningitis -most common
CD4 <200cells/cumm
7-10% AIDS patients have cryptococcal meningitis
Signs and symptoms of subacute meningitis or meningoencephalitis: headache, fever, cranial
nerve palsies, lethargy, coma, or memory loss over several weeks.

3. Cutaneous:
Skin is third most common site of infection (scalp, face, neck)
Lesion is a papule or maculopapule, with a soft orulcerated center, in severely
immunosuppressed patients, skin infections may present as a cellulitis or an abscess that
mimics a bacterial skin infection

4. Osseous: Osteolytic lesions, arthritis

5. Genitourinary tract – Renal absess, Prosatitis

6. Others: lymph nodes, liver, spleen, adrenals,thyroid, eyes, muscles can also be affected.

Laboratory diagnosis
Samples received: CSF, Serum, Sputum, Skin biopsies, blood, Urine

I. Direct microscopy:
a. India ink preparation: (10% India ink/nigrosin) ;
4-20 µm, spherical, budding capsulated yeast cells.
Can be done on CSF and urine samples
Approximately 50% of non-AIDS patients with cryptococcal meningitis and over
80% of patients with AIDS have a positive India ink examination of the CSF.

b. Gram’s stain :
Gram positive, 4-20 µm, spherical, budding (narrow based) yeast cell,
pleomorphic due to precocious budding.

7
 c. Calcoflour white: yeasts can be detected in a specimen when numbers are reduced

d. Histopathology:
Histopathologic stains such as hematoxylin and eosin, the yeasts are
surrounded by empty spaces, which reflect the capsule.
The polysaccharide capsule can be identified with stains such as mucicarmine
and Alcian blue
ability to produce melanin allows it to be stained with theFontana-Masson
stain.
Gomori’s methenamine silver (GMS) fungal stain identifies the narrow-based
budding yeast in tissue.

II. Culture and biochemicals:

SDA, SAB (Cryptococcus is inhibited by cycloheximide)

Staib’s bird seed agar / Pal’s sunflower seed agar: brown colony
Urease positive
Carbohydrate assimilation and nitrate reduction tests can be used
C. gatti – cobalt blue color
C. neoformans and C.grubei – no color change

III. Serology:
ii. Antigen detection: Serum, CSF, BAL, urine

a. Latex agglutination: Reverse passive agglutination

Can be done on CSF and serum
b. Enzyme immune assay
c. Lateral flow assay:
PCR

Treatment

Immunocompetent patients

Pulmonary disease only: No therapy

Extrapulmonarydisease: Induction: Amphotericin B + flucytosine for 2 wks
0.7mg/kg/day 100-150mg/kg/day
Consolidation: Fluconazole 400mg/kg/day
Criteria for stopping therapy: Resoution of symptoms, 2 CSF cultures
are negative and CSF glucose is normal.

Immunocompromised patients

Induction (2 weeks): Amphotericin B +flucytosine

Consolidation: (8-10 wks) Fluconazole
Suppressive (lifelong?): Fluconazole

8
Malassezia infection
Lipophilic fungus
11 species - Malassezia furfur most important
Pathogenesis – interfere with melanin production
Clinical features
Pityriasis versicolor - Patchy discolouration – hypo/ hyper
Neck, trunk, face, arms
Multiple, well-defined, non-inflammatory, macular, fine scaling, non-pruritic
D/D – vitiligo, PKDL, tinea, leprosy, erythrasma, pityriasis alba, rosea
Wood’s lamp – golden yellow fluorescence
Laboratory diagnosis
Sample – skin cleaned with 70% alcohol, allowed to dry
Edge of lesion is scraped
Microscopy – KOH, CFW
2-7um round yeast cell with occasional budding
Hyphae – blunt, short, stout, infrequent branching
Appearance - Banana and grapes, spaghetti and meat balls
Culture – lipid containing media – oleic acid, olive oil, tween in SDA
Identification – non fermentative
Urease positive
Treatment
Topical application
25% sodium thiosulphate, 10% sulphur ointment, Ketoconazole 2%
Oral – itraconazole 200mg daily for 7 days, Fluconazole 400 mg daily for 3 days

Piedra
Definition – superficial infection of hair shaft characterised by development of irregular, firm
nodules composed of fungal elements
White piedra Black piedra
Trichosporon species – T.beigelli Piedraia hortae
yeast like fungus phaeoid haphae
soft, whit, grey light brown nodules on hair discrete, hard brown black nodules
shaft 1mm diameter, firmly attached
facial, axillary, beard, moustache, pubic scalp hair most
less on scalp, eyebrows may be moustache, beard, pubic hair
grow as width of hair grow outside cuticle
easily detached from hair -less organised not detached – more organised
hair is brittle hair may break at site on infection
relapse more less

Treatment – shaving hair, Topical – ketoconazole

9
Dermatophytes
Definition – cutaneous fungal infection affecting skin, hair and nails
Also called tinea or ringworm

Features Microsporum Trichophyton Epidermophyton

Sites affected Skin, hair Skin, hair, nail Skin, nail

Macroscopy White White Khaki green

Hyphae Hyaline septate Hyaline septate Hyaline septate

branched hyphae branched hyphae branched hyphae

Macroconidia
Number Few Few to many Abundant
Shape Barrel/spindle Pencil or cigar shaped Club shaped
Size 7-16u 20-50u 20-40u
Arrangement Singly Singly Singly or in clusters
Wall Thick rough spiny Smooth, thin, wall Thin smooth wall
wall
Cells 2-8 cells 3-6 cells 2-5 cells

Microconidia Absent
Shape Ovoid Pyriform or globose
Number Few Many
Arrangement Sessile along sides Grape like clusters
of hyphae along the side of
hyphae
Species 16 24 2 (1 in human)

Epidemiology
Based on habitat dermatophytes are of three types-
Anthropophilic Zoophilic Geophilic
only humans animals, birds soil
T.concentricum T.equinum T.ajelloi
T.mentagrophytes T.mentagrophytes var menta T.terrestre
T.rubrum T.simii M.gypseum
T.schoenleinii T.verrucosum M.nanum
M.audouinii M.canis E.stockdaleae
M.ferrugineum M.equinum
E.floccosum
inflammation less inflammation severe inflammation severe
disease chronic and self-healing self-healing
intractable
healing difficult quick quick

10
Spread – direct inoculation
Arthrospores help in spread

Pathogenesis – grow in dead keratinised tissue

Clinical features
Serpentine, circular, annular ring like lesions
Central clearing
Depending on the site affected it has different types

A. Tinea capitis – hair affected

B. Tinea corporis – non hairy glabrous skin
Erythematous scaly lesion, annular, sharp margin, plaques, raised border
Single, multiple, confluent
Partial central clearing
T.rubrum

C. Tinea incognito – application of topical steroids

D. Tinea faciei – non bearded area of face

E. Tinea barbae – beard and moustache

Barber’s itch

F. Tinea cruris – infection of groin

G. Tinea manuum – palmar aspect of hand

H. Tinea pedis – plantar aspect of foot, toes, interdigital web space

I. Tinea unguium – infection of nail plate

J. Dermatophytid reaction – secondary eruption

Delayed reaction to dermatophyte antigen

Laboratory diagnosis
Wood’s lamp examination
Consists of barium silicate with 9% nickel oxide
UV light 365 nm - Characteristic fluorescence in infected hair
Bright green M.audouinii, M.canis, M.ferrugineum
Dull green T.schoenleinii
Golden yellow Malassezia furfur
Coral red Corynebacterium minutissimum
Dull yellow M. gypseum

11
Samples
Skin – cleaned with 70% alcohol, allowed to dry
Scraping from the margin
Nails – clipped and sent
Hair – plucked, basal root portion needed
Cut hair or clipping not useful
Hair brush sampling may be done – brush pressed on agar

Transport - Dry condition in black paper, envelope at room temperature

Direct microscopy- with KOH - Branching hyaline hyphae, arthrospores are seen
Culture - SDA with antibiotics and cycloheximide, 10 days to 3 week time
Dermatophyte test medium – identification
Identification - Morphology in LPCB preparation
Hair perforation test
Urease test
Hair perforation test
Differentiate between T.mentagrophytes and T. rubrum / M.canis and M.equinum

Treatment
Oral griseofulvin – 10mg/kg
Itraconazole
Terbinafine
6 weeks in skin, 12 weeks in hair, 6-12 months in nail

Mycetoma
Definition – slowly, progressive, chronic granulomatous infection of skin and subcutaneous
tissue
Triad of –
a. Tumefaction of affected tissue
b. Multiple draining sinuses
c. Presence of oozing granules
Madura foot, maduramycosis
Padavalmikam – anthill foot
Types of mycetoma based on agents-
1. Eumycetoma – caused by fungi - 40%
2. Actinomycetoma – caused by Nocardia, Streptomyces, Actinomadura – 60%
3. Botryomycosis / schizomycosis – S.aureus, Pseudomonas, CONS, E.coli, Proteus

12
Epidemiology
Tropical and subtropical counties
More in developing countries, rural parts
20-40 years
Men: women = 3.5:1
Men more prone to injury, female hormones protective
Pathogenesis – introduction by prick
Microabscess formation, inflammation, neutrophil
Host immunity and evade host defence by the organism

Agents
agent colour texture size shape
Madurella mycetomatis black hard 0.5-5mm oval, lobed
Madurella grisea black soft 0.3-0.6mm oval, lobed
Exophiala jeanselmei black soft 0.2-0.3mm irregular
curvularia geniculata black hard 0.5-1mm oval
pseudoallescheria boydii white soft 0.5-1mm oval, lobed
aspergillus nidulans white soft 1-2mm oval
acremonium falciforme white soft 0.2-0.5mm oval
fusarium sp white soft 0.2-0.6 oval

Clinical features
Progress in years
Lesions are firm, painless, subcutaneous nodules
Spread direct
Hand, feet, head, back
Diagnosis – Xray, CTscan, MRI
Determine bone involvement

features Actinomycetoma Eumycetoma

agents nocardia, Streptomyces, actinomadura fungi
tumor mass multiple, diffuse, ill-defined margin single, well defined
sinus more less
opening raised, inflamed, flared up flat, not flared
flap opening easily removed not so
discharge purulent serous/ serosanguinous
grain white usually black or white
lesions extensive less extensive

Laboratory diagnosis
Samples – grain, pus
Grains collected on sterile gauze
Grains – gross- colour, shape, size, texture
Washed in saline
Direct microscopy – crushed between two slides

13
Fungus – gram stain, KOH
Bacteria – gram stain, Kinyoun’s acid fast stain
Fungus – 2-6um wide septate hyphae, chlamydospores seen
Bacteria – 0.5-1um filamentous bacteria, coccoid, bacillary forms
Culture – washed in antibiotics like penicillin
Inoculated in SAB, SDA

Treatment
Eumycetoma – ketoconazole 200mg twice daily or itraconazole 100mg twice daily for 8-24
months
Amphotericin B – madurella, fusarium
Actinomycetoma – antibiotics
Surgery

Dimorphic fungus
Definition
Grow at 250C, in soil as hyphae – saprophytic form
Grow at 370C, in body as yeast or spherule – parasitic form
Thermal dimorphism is necessary
1. Histoplasma capsulatum
2. Blastomyces dermatidis
3. Coccidiodes imitis
4. Paracoccidiodes braziliensis
5. Sporothrix schenckii
6. Penicillium marneffei

Comparison of different yeast phase of dimorphic fungus

Features Histoplasma Penicillium Sporothrix Blastomyces Coccidio Paracocci

Shape oval round – cigar spherical spherules spherical
oval shape
Size 2-5um 3-4um,8- 3-5 um 8-20um 10-80um 15-30um
13um
Budding single absent single non multiple
narrow-neck broad neck budding thin neck
attached to endospore
mother cell 2-4um
Wall cross-walls thick thick thick
Appearance intracellular sausage asteroid figure of 8 spherule mariner’s
in like bodies hour glass wheel,
macrophage pilot
wheel

14
Comparison of different mycelial phase of dimorphic fungus

Features Histoplasma Penicillium Sporothrix Blastomyces Coccidio Paracocci

hyphae thin septate thin septate thin thin septate thin septate thin 1-2u
hyaline twisted branched septate
conidia tuberculate phialide, flower 2-10 um arthroconidia single
macroconidia metullae, like spherical, cell
and conidia in pattern smooth direct
microconidia chains palm tree conidia conidia
like
pattern

Sporotrichosis
Definition – It is a chronic pyogranulomatous fungal infection of cutaneous and subcutaneous
tissue
Agent – Sporothrix schenckii
Also called Rose gardener’s disease
Epidemiology - Introduced in skin following minor trauma
Clinical features
Lymphocutaneous –
Upper and lower extremity
IP 8-30 days
Firm, non-tender, mobile subcutaneous nodule
Involvement of lymphatics draining the site
Nodulo-ulcerative secondary lesions along the couse of lymphatics
80% cases
Laboratory diagnosis
Sample – pus, tissue
Microscopy – KOH, gram stain – yeast cell very scanty
HP- HE, PAS, GMS- asteroid body
Splendore-Hoeppli phenomenon- immune complex deposition around the organism
Culture gold standard - SDA, BHIA, 25C and 37C
LPCB of culture at 25C shows hyphae
37C shows round, oval, ellipsoidal, fusiform, budding yeast cells
Slide culture
Thermal dimorphism

Treatment
Potassium iodide – for 6-12 weeks
Oral itraconazole 100-200 mg/day

15
Histoplasmosis
Definition – systemic granulomatous disease caused by dimorphic fungus
Agent – Histoplasma capsulatum
Epidemiology
Mode of infection – inhalation of microconidia
Adult people, no sex difference
Patients with AIDS
Clinical features
Asymptomatic in 90-95% cases – positive skin test
1. Pulmonary infection
Acute pulmonary –IP 10-16 days
Fever, headache, sore throat, cough, dyspnoea
X-ray small scattered pulmonary infiltrate, hilar lymphadenopathy
Later – calcification of lungs
Chronic pulmonary – haemoptysis, weight loss
Apical subapical cavities – central necrotic area
10-15 years
2. Disseminated infection
Individuals with HIV, less than 2yrs age, organ transplant
Fever, anorexia, weight loss, anaemia, hepatosplenomegaly, lymphadenopathy
Adrenal histoplasmosis – may occur in immunocompetent
More in diabetes
Fever, weight loss, abdominal pain
Ocular histoplasmosis – sequelae POHS
Primary ocular histoplasmosis syndrome
Atrophic histo spots, peripapillary atrophy, choroidal atrophy
3. Cutaneous and mucocutaneous infection
Mucous membrane – oral may be seen in bettle leaf chewing
West Bengal, Bihar
Laboratory diagnosis
Specimen – sputum, bone marrow, tissue, blood, adrenal
Direct microscopy – KOH, gram stain
2-5um oval yeast cell, narrow neck budding, intracellular
HP – PAS stain
Culture – SDA, BHIA 25C and 37C for 4-6 weeks
Immunodiagnosis – 2 antigens species specific protein - H antigen and M antigen
Histoplasmin skin test
PCR
Treatment
Amphotericin B
Oral itraconazole 400mg daily for 6-12 weeks

16
Penicillium
Penicillium marneffi – dimorphic
Soil, saprophytes, contaminant
Endemic SE Asia
Common in AIDS patients
AIDS defining illness – third common in AIDS after Tb and crytococcus, fourth PCP
Yeast form differ from other dimorphic – division by schizogony and not by budding
In India – common in north east, Manipur
Inhalation of conidia
Reservoir – bamboo rat
More in rainy season

Clinical features
IP 4weeks
Low grade fever, chill, malaise, weight loss, anaemia, cough, weakness, lymphadenopathy,
hepatosplenomegaly, skin lesions
Arthritis, osteomyelitis, peritonitis, pericarditis
Xray, CT scan, MRI
D/D – tuberculosis, histoplasmosis, pneumocystis, crytococcosis
CD4 <50

Laboratory diagnosis
Samples- sputum, skin lesion, bone marrow, lymph node, blood
Direct – H&E, PAS, GMS – yeast cells
CFW, papanicolaou stain
Conidiophores are located laterally and terminally
3-5 metullae arise – produce flask shaped phialides
Globose chains of conidia, basipetal 2-3um
Antigen detection - urine

Treatment
Amphotericin B 0.6mg/kg/day for 2 weeks followed by itraconazole 400mg/kg/day in two
divided doses for next 10 weeks
Prophylaxis – itraconazole 200 mg/day
HAART given

17
Aspergillus
Systemic fungal infection
185 species
Saprophytic fungus - Soil, food, water, decaying vegetation
Inhalation of spores
Both in immunocompromised and immunocompetent state
AIDS, malignancy, transplant, cytotoxic therapy, corticosteroid
Nosocomial outbreaks
After inhalation conidia reach alveoli
Colonisation
When host defence is lowered, conidia inhaled, transform into hyphae
Vascular invasion – disseminated infection

Clinical features
1. Allergic manifestations
a. Asthma – type 1
b. ABPA – allergic bronchopulmonary aspergillosis – type 1&3
c. Allergic rhinitis, sinusitis
2. Localised infections
a. Aspergilloma – fungus colonises in pre-existing cavities like tubercular
Compact mass of fungal mycelia surrounded by dense fibrous walls
Also called fungal ball 8-10cm diameter
X-ray – opacity in cavity with crescent of air at upper margin
Treatment - surgery
b. Otomycosis
c. Keratitis, corneal ulcer
3. Disseminated infections – mainly in immunocompromised
a. Invasive pulmonary aspergillosis
Dry cough, dyspnoea, chest pain, fever
b. Cerebral – abscess, granuloma, meningitis
c. Cutaneous – papule, plaque, ulcer
d. Others – endocarditis,
4. Mycotoxicosis - aflatoxicosis

Laboratory diagnosis
Samples – sputum, BAL, biopsy
10% KOH – thin septate hyphae 3-6um with dichotomous branching
CFW, HP stain
Culture – SDA, SAB, Czapek dox agar, 2% malt extract agar
Antigen detection – galactomannan by ELISA
PCR

18
 Treatment
Amphotericin B, Voriconazole

Features Aspergillus Aspergillus flavus Aspergillus Aspergillus tereus

fumigatus niger

Macroscopy
Colour Bluish green Yellowish green Brown to black Light brown
Texture Powdery Velvety Cottony Velvety
Topography Flat heaped up Flat Coarse pepper Flat with heaped up
in centre appearance centre
Reverse Cream Cream Cream Tan

Hyphae Septate hyaline Septate hyaline Septate hyaline Septate hyaline

Width 3-8u 5-6u 6u 5u

Conidiophore
Length 150-300u 400-850u 400-3000u 100-250u
Wall Smooth Rough parallel Braoad brown Smooth parallel
parallel tint

Vesicle Flask shaped Globose/ Spherical Dome shaped

subglobose
Phialides Uniseriate Uniseriate/biseriate Biseriate Biseriate
Surface of Upper half Most of vesicle 3/4 Entire Upper half
vesicle covered

Conidia
Shape Round Round Round Ellipsoidal
Size 2-5u 4u 4-5u 2u
Arrangement Compact Loose divided Ecchinulate in Long straight chains
chains chains compact chains

Amphotericin B susceptible susceptible susceptible resistant

growth at 45-50C + - - -
diseases caused invasive rhinosinusitis, skin otitis externa immunocompromised
disease, infection, diabetes ICU infection
pulmonary endophthalmitis,
infection aflatoxin,
prevalence 66% 14%, more in India 5% 5%

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Zygomycosis
Emerging, opportunistic pathogen
Broad aseptate fungi
Invade blood vessels
2 groups – mucormycosis and entomophthoromycosis

Mucormycosis
Asexual reproduction – sporangium formation
Sexual reproduction – zygospore formation
Found in environment, food, soil, air, laboratory contaminant
Rapid growth, prolific spore-forming capacity
Inhalation of spores
Predisposing factors – diabetes, malignancy, immunosuppressive therapy
Vascular invasion prominent
HIV less common
Pathogenesis
Spores inhaled into lungs, ingested by alveolar macrophages
Diabetic ketoacidosis
Virulent factors – angioinvasive, growth above body temperature, destructive enzymes,
resistant spores
Clinical features
1. Rhinocerebral - Most common, fulminating, acute onset
Spreads from nasal mucosa to turbinate bones, paranasal sinus, orbit, palate, brain
Destruction of bone and infarction – CT, MRI
Most common – rhizopus
Facial pain, headache, loss of vision, proptosis, blood stained nasal discharge
Spread to frontal lobe – abscess formation
2. Pulmonary
3. Cutaneous
4. Disseminated
Laboratory diagnosis
Repeated isolation necessary
Samples – necrotic material
KOH – broad, aseptate, ribbon like, thick walled hyphae with branching at right angles
CFW, H&E, GMS stain
Culture SDA, SAB
LPCB preparation
Treatment
1. Surgery
2. Correction of ketoacidosis
3. Antifungal – amphotericin B and oral posaconazole
4. Hyperbaric oxygen

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Features Mucor Rhizopus Rhizomucor Absidia Apophysomyces
Macroscopy
Colour White white with white – grey pale grey cream, grey
Texture Woolly black spore floccose floccose floccose
Nature Growth in entire tube
Reverse entire tube salt & pepper
with dark appearance
spot cream
Cream
Hyphae Braoad Braoad Braoad Braoad Braoad aseptate
Width aseptate aseptate aseptate aseptate hyaline
hyaline hyaline hyaline hyaline
10u
Rhizoid - nodal internodal internodal +
Sporangiophore Long straight long straight long short straight
arrangement single 2-3 3-7 -corymb single
unbranched branched branched unbranched
septations coenocytic
faint pigment
Columella Round to oval pyriform pear-shaped hemispherical
oval
Apopyses - - - + flask shape + champagne
glass
Collarette Present at the
base of
columella
Sporangia Globose globose spherical globose
Sporangiospores Ovoid striated ellipsoidal spherical cylindrical
3-5u
Stolon - + + +
species racemosus arrhizus pusillus corymbifera elegans

Entomophthoromycosis

Features Mucorales Entomophthorales

patients immunocompromised immunocompettent
infection acute chronic
angioinvasion present absent
lesion destructive cutaneous, mucocutaneous,
visceral
mortality high low
Splendore-Hoeppli phenomenon absent present
culture floccose glabrous
treatment surgery potassium iodide
distribution worldwide subtropical areas
Conidiobolus and
Basidiobolus

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Rhinosporidiosis
Definition – chronic granulomatous disease of mucous membrane characterised by polyposis
of nasal cavity, conjunctiva and other body sites
Agent – Rhinosporidium seeberi
Hyperendemic in Indian subcontinent, Srilanka
More in south India – AP, TN,Kerala, Pondy, WB, Chattisgarh
Hydrophilic organism – fresh stagnant water
Sporangium – contain 12000- 16000 spores, size 200-300um diameter
Wall 5um thick, has two layers – outer chitinous and inner cellulose, at pore cellulose thinned
Spores 6-9um
Seen by PAS, GMS, Mayer’s mucicarmine stain
Life cycle – 3stages – trophocyte, sporangia, endospores
Clinical features
IP – 1-10 months
1. Nasal – commonest, friable polypoid lesion, vascular bleed easily, raspberry like
appearance, anterior nare, septum, nasophaynx, white spot – sporangia, mucoid
discharge, epistaxis, obstruction unilateral, foreign body sensation, painless
2. Ocular – 15% infections, bulbar and palpebral conjunctiva, oculosporidiosis, single
eye common, fleshy polyp, lachrymal sac affected
3. Cutaneous – wart-like papillomatous sessile mass
4. Miscellaneous – vulva, vagina, penis, urethra due to taking bath
Laboratory diagnosis
Excision biopsy, scrape cytology, nasal washing
HP, KOH – sporangia and endospores
Not cultured
Treatment
Surgery, dapsone 100mg daily for 1yr
High recurrence

Pneumocystis
Pyneumocystis pneumonia
Interstitial plasma cell pneumonia
AIDS defining disease
Pneumocystis carinii – infect rats
Pneumocystis jirovecii – infect humans
Life cycle
1. Trophozoites fill up alveoli of infected lungs
They are pleomorphic, 2-5um bodies, exist in clusters
2. Precyst – also called sporocyst

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 Intermediate stage of sexual reproduction leading to cyst formation
Zygote undergoes mitosis and meiosis within sporocyst
3. Cyst – seen in clinical specimen
4. Sporozoites – also called intracystic bodies
Oval, amoeboid, 1-2um, better seen in giemsa stain
Converted into trophozoites
Epidemiology
Acquired by droplet inhalation
Predisposing factors – AIDS, corticosteroid, malignancy, immunosuppression
Clinical features – pulmonary and extrapulmonary
IP 4-8 weeks
Insidious onset, non-productive cough, dyspnoea, fever, tachynoea, tachycardia, occasionally
haemoptysis and sputum production
Extrapulmonary – lymph node, bone marrow, spleen, liver, CNS- late, thyroid
Laboratory diagnosis
CD4 count less than 200/ cu mm
BAL, induced sputum, body fluids on site, bronchial wash, biopsy
Pulmonary function test
DFA – BAL sample
Stain – GMS, toluidine blue O, gram – weigert stain cyst wall
HP – H&E honeycomb appearance
Culture –does not grow in culture media
PCR
Treatment
Trimethoprim – sulfamethoxazole
15-20 mg/kg/day + 75-100 mg/kg/day
Pentamidine – 4mg / kg/ day IV second line

Pigment producing fungi

Category Fungus Colour
Yeast Rhodotorula Coral red
Black yeast Phaeoanellomyces werneckii, Black
Dermatophytes Microspotum canis Yellow
Trichophyton rubrum Red
trichophyton violaceum Violet
Epidermophyton floccosum khaki green
Dimorphic Penicillium marneffii Red

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Mycotoxicosis
Definition – an illness caused due to ingestion of preformed substances produced by fungus
in the food

The different toxins are

Toxin Fungi producing Food
Aflatoxin Aspergillus flavus nut, maize
Fumonisin Fusarium moniliforme maize
Trichothecene Fusarium graminearum maize, sorghum
Ochratoxin Aspergillus ochraceus, A.niger cereals
Cyclopiazonic acid A.flavus, Penicillium corn, groundnut

Mycetismus
Mycetism/ muscariism
Definition – consumption of poisonous fungi
Different agents are
Poisoning Fungi food
Ergot Claviceps purpurea, rye, millet
C.fusiformis
Coprine Coprine atrementarius cream butter sauce
Muscarine Inocybe
Ibotenic acid Amanita mushroom
Cyclopeptide Amanita toadstool

Culture of Fungi
Fungi that do not grow in artificial culture media
• Rhinosporidium siberi
• Pneumocystis jiroveci
Difficult to grow:
• Malassezia furfur (Lipophilic, requires Olive oil on SDA media)
Other organisms (bacteria) that doesn’t grow on artificial media:
• Klebsiella granulomatis (previously Calymmatobacterium: causing
Donovanosis / Granuloma Inguinale)
• Treponema palidum
• Mycobacterium leprae

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