INFECTIOUS DISEASE OF PATHOLOGY

Fungus

Dr. Naila Awal(Postgraduate student)

Definition

• Fungi are the eukaryotic, saprophytic, parasitic micro organism, unicellular or multicellular having chitin in their cell wall & ergosterol & zymosterol in their cell membrane.

Classification Morphological classification-

1) Mold- fungi which are structurally long filamentous & multicellular & form a network of hyphae/mycelium.

• Ex-

Dermatophytes Septate

Aspergillus

Mucor --Aseptate

2)Yeast- unicellular round/oval like fungi reproduction by budding.

3) Yeast like fungus- Fungus that reproduction by budding, but bud fails to separate from parent cell & ultimately form a structure that looks like a chain of elongated cell. They form pseudohyphae.

• Ex- Cryptococcus neoformans.

• Ex- Candida

• 4) Dimorphic fungus• Fungi that can remain

either mold/ yeast form depending upon the temperature of the environment.

• Ex- Histoplasma capsulatum

• 37C-->change to yeast form

• 25C --> mold form

Depending upon the site of infection

1) Superficial mycoses- Those fungal infection ,caused by the fungus that lies on the surface of the stratum corneum.

2) Cutaneous mycoses- caused by the fungus that liberate keratinase enzyme & therefore can invade deep into keratin layer of skin & also nail &hair.

• Ex-Malassezia furfur -->• Pityriasis versicolor

• Ex-• Dermatophytes-->

dermatophytoses (Ring worm)

3) Sub cutaneous mycoses- caused by the fungus that lies on the surface of the stratum corneum.

4)Deep mycoses- caused by the fungus that infect internal organ

• Ex- • Rhinosporidium seeberi–

Rhinosporidioses.

• Ex- • Candida–Candidiasis.• Histoplasma capsulatum-

Histoplasmosis.

Superficial mycoses(Pityriasis versicolor)

• Disease- Malassezia furfur

• Clinical manifestation-Lesions are macular, non-inflammatory, well

demarcated & hypopigmented.

Lab diagnosis

• Sample- Skin scrapping• Lab procedure-• The scrapping material is placed on glass slide few

drops of KOH is added cover slip wait for 20 mins.• Observation-Cluster of rounded yeast cell & short ,stout,

curve, septate hyphae.

Cutaneous Mycoses(Dermatophytes)

• Genus-1)Trichophyton- Nail & Hair

2)Epidermatophyton- Nail

3)Microsporum- Hair

C/F-Skin-->Ring like lesion. Ring- Scaly, hypopigmented & inflammatory.

Nail yellow, brittle & thick.Hair brittle.

Lab diagnosis

• Sample-Skin- Skin scrapping

Hair- Hair clipping. If infection present in scalp, then skin adjoining scalp is taken.

Nail Nail shaving at the margin between healthy & unhealthy area & from the deeper portion of nail.

Lab procedure

1)The material is placed on glass slide few drops of KOH is added cover slip wait for 30 mins (skin)/10mins (hair)/several hours.

• Observation- long thin, branched & septate hyphae.

2) Culture- Sabouraud's dextrose agar media-

25-28 C for 3-4 weeks.

• Colony morphology- colony is picked up place on glass slide Lacto phenol cotton blue dye is added cover slip examine under microscope.

• Spiral Hyphae

Tinea

mentagrophytes

• Macroconidia

Microsporum

• Microconidia• Trichophyton rubrum

• Anthar hyphae-

Tinea

schoenlenii

Sub cutaneous mycosesMycetoma

• Definition- It is a granulomatous disease of subcutaneous tissue caused by various fungi & bacteria where there is gradual destruction of tissue leading to loss of function of the affected area.

• Disease- Maduromycosis /Madura foot• Actinomycetoma when madura foot is caused by

bacteria.• Eumycetoma when madura foot is caused by

fungus.• Site- 1)Foot most common site 2)Lower extremities 3)Back

• Clinical manifestation-1)Suppuration

2)Abscess formation

3)Granuloma

4)Tumor like swelling containing multiple draining sinus.

Pathogenesis

• Local trauma of skin

Micro organism enters to the body

Inflammatory response

Granulomatous inflammation.

Lab diagnosis

• Sample- muco pus

• Lab procedure-• Muco pus is taken in 3 separate glass slides.• 11stst slide- slide- 1 drop of normal saline is added cover slip

viewed by hand glass• Observation-• If granules are black- Madurella grisea

red-Madurella mycetomatis

yellow white-Pseudallescheria boydii

22ndnd glass slide glass slide

• Add few drops of 20% KOH in muco pus wait for 10 mins

• Observation- • If fine fillamentous branch with no chlamydospore

Bacteria• If thick septate branched hyphae with chlamydospore

Fungus

33rdrd glass slide- glass slide-• Granules are crushed on a slide Z-N stain

decolorized with 1% H2SO4 Red branching filament Nocardia

• Histopathhology-Suppurative granuloma-• Center abundant polymorphonuclear cells & granules• Surrounded by --lymphocytes, plasma cell, histiocytes &

fibrosis.

Rhinosporidiosis

• Definition- It is a chronic granulomatous disease characterized by production of large polyp / wart like lesion.

• Causative organism- Rhinosporidium seeberi

• Site- Common site –Nose-78% Nasophaynx-68% Tonsil-3% Eye-1%.

Pathogenesis

Local trauma

Fungus is inoculated into the mucosal epithelium of nasal cavity

Replication of fungus

Hyperplasic growth of host tissue & immune response

Granulomatous disease

Morphology

• The lesions are polypoid, reddish & granular.• May be multiple & pedunculated.• Highly vascular & bleed on touch.

M/E-• Papillomatous hyperplesia of nasal mucosa.• Multiple mature & immature cyst (sporangia) are packed

with spores.• Infiltration of chronic inflammatory cells into normal

tissue.

Deep Mycoses

• Classification- Deep mycoses

Primary deep mycoses Opportunistic mycoses(Fungal infection that (Fungal infection that infect healthy Individual) infect immunosuppressive individual)Ex Ex-Coccidioidomycosis CandidiasisParacoccidioidomycosis CryptococcosisHistoplasmosis AspergillosisBlastomycosis Mucormycosis

Histoplasmosis

• Causative organism- Histoplasma capsulatum.• Pathogenesis-• Source of infection- Soil• Mode of transmission- Inhalation.

Through inhalation

macro & microconidia enters into lung

engulf by alveolar macrophage

through macrophage they spread to the various tissue. (Liver, spleen, lymph node)

• C/F-1)Asymptomatic

2)Pulmonary histoplasmosis- fever, dry cough.

More severe case- granulomatous lesion in lung.

3)Localized lesion in extrapulmonary site- mediastinum, adrenal,liver, meninges.

4) Disseminated histoplasmosis- Immunosuppressed person.

Lab diagnosis

• Specimen-1)Pulmonary histoplasmosis- Sputum.

2)Buffy coat of blood.

3)Bone marrow aspirate.

4)Biopsy specimen from different internal organ.

• Lab procedure-• 1) Direct microscopy- Specimen is taken on the glass

slide Giemsa staining Microscopic examination.• Findings- Yeast like fungus within the cytoplasm of

macrophage.

2)Culture-Sabouraud’s dextrose agar media- incubate at 37 C for 4 weeks.

Observation of colony morphology- hyphae with macro & micro conidia.

3) Serology-Patient’s serum-Anti histoplasma capsulatum .

4)Skin-Histoplasmin skin test.

5) Histopathology-

Pulmonary histoplasmosis-Epithelioid granuloma with

central caseous necrosis,

which coalesce

to form

areas of consolidation.

Disseminated histoplasmosis--->Mononuclear phagocytes filled with fungal yeast.

-->Epithelioid granuloma are not formed.

Cryptococcosis

• Definition- It is a true yeast surrounded by thick polysaccharide capsule.

• Causative organism- Cryptococcus neoformans.

• Nice to know- Capsulated bacteria-

Streptococcus pneumoniae

Klebsiella

Haemophillus influenzae

Neisseria meningitidis

Mode of transmission- Inhalation of yeast .• Pathogenesis-

• It is asymptomatic / produce influenza like symptoms which resolves automatically in healthy person.

But in immunocompromised person -Through inhalation enters into lung via

blood meninges cryptococcal meningitis.

Lab Diagnosis

• Specimen-1)CSF2)Sputum3)Tissue sample

Lab procedure-1)Wet film microscopy- circular/oval yeast cell.2)India ink preparation- CSF is centrifuged deposit is

taken on glass slide1 drop of india ink is added cover slip examine under microscope.

Observation- against dark background darkly illuminated single circular/oval yeast with budding.

3) Culture-

Blood agar media- 37 C for 24 hours

Saborauds dextrose agar media-37 C for 2 days.

• 2 days after, colonies are picked up on glass slide examine under the microscope

• Observation-

Spherical yeast cell

with budding.

4)Serological test- Anti cryptococcal Ab +

Histopathological findings

• CNS-• In immunosuppressed person-Soap bubble lesion- gelatinous masses of fungi in

meninges or may expand the perivascular Virchow- Robin space within the grey matter.

• In non immunosuppressed person-Chronic granulomatous lesion composed of macrophage,

lymphocyte & foreign body giant cell. Suppuration may also occur.

• Lung- Solitary pulmonary granuloma.

Aspergillosis

• Causative agent- Aspergillus fumigatus.• Mode of transmission- By the inhalation of

Aspergillus spore.• Spectrum of disease-1) No infection- b/c alveolar macrophage engulf & destroy

the conidia.2)Person who have allergic to Aspergillus antigen, manifest

as Allergic bronchopulmonary Aspergillosis (ABPA).3)Person who have cavity to lung (due to TB, sarcoidosis),

conidia after reaching the cavity they germinate & produce abundant hyphae in lung cavity. This clinical condition is known as Aspergilloma.

• They manifest as-Haemoptysis

Dyspnoea

Asthma

4)Person who are in steroid therapy (leukemia, bone marrow transplantation) inhale conidia produce severe manifestation of disease Invasive Aspergillosis which is clinically manifest as pneumonia.

• After lung infection hyphae invade blood vessels haematogenous spread to different internal organ Abscess (brain, liver, kidney).

Lab diagnosis

• Sample-1)Sputum

2)Blood

3)Lung biopsy.

• Lab procedure-• 1) Gram staining- Septate hyphae

with dichotomous branching.

• 2)Culture-Blood agar

Sabouraud's dextrose agar media

• Observation of colony- Aspergillus colonies are usually fast growing, white, yellow, yellow-brown, brown to black or shades of green.

• 3) Serological test- Immunodiffusion tests for the detection of antibodies to Aspergillus species .

• 4) Histopathology-• Aspergilloma-

Proliferative mass of hyphae form fungal ball which lies freely within the cavities.

Surrounding inflammatory reaction may be sparse/chronic inflammation/ fibrosis.

Invassive aspergillosis

• 1) lung- Aspergillus form fruiting bodies (usually in lung cavities) &

Septate filaments, branching at right angles (40 degrees)

Mucormycosis

Definition- It is an opportunistic infection caused by ‘bread mould fungi’ including- Mucor, Rhizopus, Abscidia & Cunninghamella.

Predisposing factors-• Neutropenia• DM• Corticosteroid use• Iron overload• Breakdown of cutaneous barrier as a result of burning,

surgical wound/ trauma.

Major route of infection- 1) Inhalation 2) Ingestion 3) Traumatic inoculation.Clinical presentation-

• 5 clinical form of Mucormycosis- Rhino cerebral, pulmonary, gastrointestinal, primary cutaneous & Disseminated.

• Rhino cerebral Mucormycosis-

Initial symptoms- acute sinusitis, congestion, purulent nasal discharge, fever, unilateral headache, peri orbital edema, proptosis, facial numbness, cranial nerve palsy.

• Pulmonary Mucormycosis-

Fever, haemoptysis • Gastrointestinal Mucormycosis-• Bowel perforation, peritonitis, GIT hemorrhage.• Severe immunocompromised person manifest as

primary cutaneous lesion.

• They do not harm in immunocompetent individual but infect immunosuppressed person.

Pathogenesis

• It is transmitted by air borne asexual spore.• After inhalation of spore

Colonize into nasal sinus

Orbit Lung

Brain Engulf by alveolar macrophage & oxidative killingRhino cerebral Mucormycosis

Lab diagnosis

• Specimen- Biopsy from suspicious areas.

• Lab procedure-1) Staining-• H&E stain- often hard to see

– GMS, PAS stains better

Observation-Non septate,

irregularly wide fungal

hyphae with frequent

right angle branching.

2) Histopathology- Common site-

• nasal sinus, lung &GITRhino cerebral Mucormycosis-

• Local tissue necrosis• Invade arterial wall• Penetrate peri orbital tissue.• Pulmonary Mucormycosis- • Areas of hemorrhagic pneumonia with• Vascular thrombi &• Distal infraction.

Candidiasis

• Species-1)Candida albicans.2)Candida tropicalis.

3)Candida parapsilosis.• Features-1) Dimorphic fungus-• 25 C-True hyphae.• 37C-Pseudohyphae with yeast.

2) May produce chlamydospore- Sexual spore.

3) Causes opportunistic infection.

• Predisposing factor of candidiasis-1) Immunosuppression due to prolong use of – Anti cancer

drug, steroid.

2)AIDS

3)Prolong use of antibiotic

4)DM

5)Pregnancy

6)Bone marrow transplant recipient

7)Leukaemia, lymphoma

8)Very young & very old age.

Clinical spectrum of candidiasis-

• 1) Mucosal candidiasis-• Oral thrush• Candida esophagitis• Vulvo vaginal candidiasis

• 2) Cutaneous Candidiasis-• Nail proper- Onychomycosis• Nail fold- Paronychia• Armpit/ web of the fingers & toes- Intertrigo• Hair follicle- Folliculitis• Penile skin- Balanitis• Perineum of infant- Diaper rash

3) Chronic muco cutaneous candidiasis-• It involves superficial skin & mucous membrane.

4)Invasive candidiasis-

Oral thrush

• Definition- It is the superficial fungal infection on the mucous membrane of oral cavity.

• Seen in-Newborn Debilitated peopleChildren receiving oral steroid HIV positive patientfor asthma & following a course ofbroad spectrum antibiotics that destroy normal bacterial flora.

• Morphology- They form grey-white dirty looking pseudo membrane, composed of matted organism & inflammatory debris.

• Deep to the surface there is mucosal hyperemia & inflammation.

Candida esophagitis

• Commonly seen in-

AIDS patient

Hematolymphoid malignancy.• Clinical presentation-

Dysphagia

Retrosternal pain.

• Endoscopic findings-

Esophageal mucosa-- plaque & pseudo membrane.

Vaginal candidiasis

• Common in- • Women with-

DM

Pregnancy

Taking OCP

• Clinical manifestation-Intense itching

Thick curd like discharge

Invasive candidiasis

• Definition- It is a candidiasis which is caused by blood borne dissemination of organisms into various tissue / organs.

• Persons risk for developing candidiasis-1) ICU patient

2) Surgical patient

3)Patient with central venous catheter

4) Immunocompromised person.

5) Very LBW infant

• Common pattern/ Effect-1) Kidney- Renal abscess

2) Heart- Myocardial abscess & endocarditis.3)CNS- Brain micro abscess & meningitis

4) Liver- Hepatic abscess

5) Eye- Endopthalmitis

Findings- Inflammatory response

Occasionally granuloma.

Lab diagnosis of candidiasis

• Sample-

-->Swab

-->Scrapping from superficial lesion

-->Tissue biopsy

-->Exudates

-->Blood, CSF, Urine

-->Material from I/V catheter

Lab procedure

1) Wet film preparation- From

Swab, exudates, CSF (centrifuge), urine (centrifuge)

• Findings-Spherical/ oval yeast cell with budding.

2)Staining-

A) Gram staining- Gram + round/oval yeast cell with budding.

B) Immuno fluorescence staining- calcoflor white stain.3) Culture-A) Sabouraud's dextrose agar mediaB) Blood agar media• 37 C for 48 hours in aerobic condition.

Colony morphology-

• 2-4 mm in diameter, circular, white/creamy, soft with mucor odor.

4)Confirmation – Gram staining from the culture plate.• Gram + yeast with budding.

5)Germ tube test (Confirmatory test for Candida albicans)

• 0.5ml human serum is taken in a test tube

• Test fungal colony is added

• Incubate at 37C for 4 hours

• Then 1 drop of serum is collected & place on glass slide

• Cover slip

• Microscopic examination

• Observation- Yeast like cell with finger like projection.• Comment- + for Candida albicans

7) Sugar fermentation test-• It differs for different species.