Cryptococcosis

Dr. Basim M. Ibrahim

Department of Microbiology, College of Medicine,
University of Baghdad
2023
Objectives
 With the end of this lecture the students will be able to:
1. Define Cryptococcosis and Candidiasis.
2. Demonstrate some important CCCs. of Cryptococcus spp. and
Candida spp.
3. State main clinical presentations of the two cases.
4. List laboratory diagnostic steps.
Cryptococcus spp.
 Classified into the Division: Basidiomycota.
 Encapsulated yeast (surrounded by a large LPS capsule both
in host and on some culture media).
 First case of cryptococcal meningitis was reported in 1905.
 Include 37 species, only C. neoformans is pathogenic to human.
 Classified into 4 serotypes according to capsular LPS Ags.; A, B,
C and D.
 Capsule importance:
1. Resistance to phagocytosis.
2. Aids in identification.
3. Suppress T-cell function.
 Difference between Yeast and Mold
CCC. Yeast Mold
Definition Microscopic fungus, consisting Growth form of fungus, which grows in
of a single, oval cell the form of multicellular filaments called
hyphae
Habitat Common in environment Found in damp, dark or steam-filled areas

Appearance Oval in shape, and is colorless Fuzzy appearance, and the colors can be
and smooth green, orange, black, brown, purple and
pink
Energy Converts carbohydrates to Secretes hydrolytic enzymes to external
production alcohol during fermentation food sources and absorbs nutrients

Reproduction Budding Sexual or asexual spores

Uses Baking industry and in the Production of cheese
production of ethanol
Examples Saccharomyces cerevisiae (baking Mucor, Penicillium, Rhizopus,
yeast) and C. neoformans and Aspergillus

Center for Food Security and Public Health, Iowa State University, 2012
Cryptococcus serotypes
1. C. neoformans var neoformans
 Two serotypes: A and D.
 Found in soil contaminated with pigeon droppings (birds
excreta).
 Causative agent of most common cases of Cryptococcosis,
especially in immunosuppressed individuals.

2. C. neoformans var gattii

 Two serotypes: B and C.
 Found in bark and plant debris under Eucalyptus trees or
decaying wood, not associated with pigeons.
Source of infection??
 Note:
Host’s immune status seems to be more important
than virulence of organism.
Current model of immune response to Cryptococcus spp. in
health and disease
1. Inhaled cryptococcal spores are recognized by alveolar macrophages
through pattern recognition receptors (PRR).
2. This stimulates macrophages to release C–C motif chemokine ligand 2
(CCL2) to recruit monocytes and dendritic cells (DCs) to lung.
3. Recruited DCs are capable of breaking down cryptococcal lifeforms and
present Ag to CD4+ T-cells.
4. Activated Th1 and Th17 secrete IFN-Ɣ, IL-6, IL-10 and granulocyte-
macrophage colony stimulating factor (GM-CSF) to recruit/differentiate
classical (M1) macrophages.
5. Exact mechanism of fungicidal activity by M1 macrophages against
Cryptococcus spp. is still unclear; however, they known to upregulate iNOS,
CD80 and TLR4 as well as produce IL-1β, IL-8 and TNF-α.
6. Cytokine/chemokine mileu organize leukocytes to encapsulate/eliminate
cryptococcal organisms within granulomas.
7. Most common association with cryptococcal disease is absence/dysfunction
of at least one aspect of healthy immune response, leads to uncontrolled
fungal growth.
8. Some patients develop a skewed hyper-immune response to pathogen,
causing inflammatory damage to host tissue even with fungal clearance.
Cryptococcosis
 Potentially fatal fungal infection, presenting mainly as
pneumonia or meningitis.
 Clinical presentations:
1. CNS.
2. Pulmonary.
3. Visceral.
4. Cutaneous/Mucocutaneous.
1. Cryptococcal meningitis
 Meningitis/Brain abscess are most common presentations.
 Symptoms including headache, fever, nausea, vomiting, neck
stiffness, vision problems, coma, photophobia and changes in
mental status (confusion or change in behavior).
 Untreated infections are often fatal.

Center for Food Security and Public Health, Iowa State University, 2012
2. Pulmonary cryptococcosis
 Either asymptomatic (majority) or symptomatic (self-limiting)
infection.
 After initial infection, spreading to other organs may occur.
 Symptoms include cough, fever, headache, weight loss,
dyspnea, malaise, chest pain and difficulty breathing.
 Section of Rt. lung showing Distended alveoli filled
apical cavity and multiple foci with Cryptococcus spp.
of consolidation with a mucoid (HE stain)
appearance

Center for Food Security and Public Health, Iowa State University, 2012
3. Ocular lesions
 Optic neuritis, chorioretinitis and endophthalmitis.

Conjunictival Cryptococcosis
4. Skin lesions
 Formation of granulomatous reaction with giant cells.
 Presentations: Papules, vesicles, ulcers, purpura,
subcutaneous tumor-like masses and abscesses.
 Lesions can mimic: Acne, Lipomas and Basal cell carcinoma.

Annular, confluent,
erythematous plaques on
posterior Rt. calf 5
months after onset of
primary cutaneous
cryptococcosis
A. Initial presentation of erythematous and indurated skin
B. Skin biopsy sample showing yeast forms with mucoid capsules
C. Progressive ulceration of skin despite antifungal therapy
D. Slow re-epithelization of skin after surgical debridement and 6 months
of antifungal therapy Center for Food Security and Public Health, Iowa State University, 2012
5. Cellulitis
 Common in organ transplant recipients.

Cryptococcal cellulitis on Rt. forearm

Lab. Diagnosis
1. Specimens: CSF, biopsy, blood, sputum and urine.
2. Slide: Encapsulated (clear halo), round to oval, single cell.

Capsule in tissue section

stained with Meyers-
Mucicarmine stain
India ink preparation of CSF
3. Culture: At 25-37°C for 48-72 hrs. on:
 SDA without cycloheximide: White-creamy to yellowish-
brown, soft, fast growing, glistening, smooth and mucoid.
 Bird seed agar OR Caffeic acid agar: Brown-black colonies
(due to melanin produced).
 Urea agar: +ve. Bird seed agar

-ve / +ve
 SDA medium

Note:
C. neoformans differs from Candida spp. by hydrolyzing urea
and not forming pseudohyphae.
4. Biochemical examination of CSF in addition to numerous
organisms.
5. Serology:
• Not useful in humans as Abs are often found in healthy
people.
• Serological tests involve:
1. Latex agglutination test:
• Detect cryptococcal Ags.
• Sensitive and specific.
• In serum, CSF and broncho-alveolar lavage, false-ve results
may exist.
• Patient improves = ↓ titer ; No respond to therapy = ↑ titer.
2. Indirect fluorescent Ab test:
Identifies organism in culture/tissue section, by staining yeast
cell wall.
3. Tube agglutination test.
4. ELISA.
Center for Food Security and Public Health, Iowa State University, 2012
Treatment
 Long-term therapy is required in HIV patients after initial
therapy, as infection respond slowly to treatment.
 Prophylaxis: Fluconazole or itraconazole.
 Rx.: Amphotericin B, 5-Fluorocytosine (both are synergistic),
Fluconazole, Itraconazole, Ketoconazole and Flucytosine.
 Treatment consists of 3 phases:
1. Induction (at least 2 weeks plus clinical improvement).
2. Consolidation (8 weeks or until CSF cultures are sterile).
3. Maintenance therapy (lifelong).
 Prevention and Control: Environmental exposures are
difficult to prevent; avoid pigeon droppings and remove them
from the environment, carefully cleaning of birds
cages/handling of infected animals and avoid eucalyptus
trees.
Candida albicans
Objectives:
 With the end of this lecture, the students will be able to:
 State main CCC. features of the yeast C. albicans.
 Define Candidiasis and list main clinical presentations.
 Identify main lab. Dx. steps.
Introduction:
 Genus Candida includes approximately 154 species, of which
C. albicans is most frequently isolated in human infs.
 C. albicans is single-celled dimorphic (have 2 forms; yeast in
host and mold in environment).
 Endogenous; N.F of human oral cavity, gut and vagina.
 Frequently recovered from hospitals, medical equipment and
food.
 Able to produce blastoconidia, pseudo- and true-hyphae,
germ-tube and chlamydospore by conversion of its hyphal
elements.
 Plasma membrane similar to that found in mammalian.
 Cell wall contains 30-60% glucan, 25-50% mannan, 1-2%
chitin, 2-14% lipid, and 5-15% protein.
Virulence Factors:
 Adhesins (MP66, MP-hemed and Ala1p).
 Enzymes: Proteinase, phospholipase and lipase.
 Toxins: Gliotoxin and acetaldehyde.
 Interference with phagocytosis, immune defenses and
complement.
 Mannan: Major Ag of Candida species.
 Glucan: Impede amphotericin B from gaining access to
plasma membrane.
 Morphogenesis (yeast to filamentous hyphae).
 Germ tube.
 Synergism with certain bacteria.
 Acidic metabolites.
 Growth rate and undemanding nutrient requirements.
Predisposing factors:
 Overuse of antibiotics, oral contraceptives, steroid hormone
medications and corticosteroids.
 Suppressed immune system (D.M, AIDS, radiation or
chemotherapy).
 General and invasive surgical procedures, including catheters.
 Prolonged hospitalization
 Bone-marrow and organ transplantation.
 Premature low-weight births.
 Minimal vegetable consumption (alter pH to suit fungal
growth).
 Sexual intercourse.
 Trauma.
 Pregnancy.
Pathogenesis:
 Most common fungal pathogen worldwide.
 Disease caused via invading human tissue by pseudohyphae.
 Impair immune function by negatively affecting T helper: T
suppresser, resulting in unregulated Ig production.
 Produce disguising Ags deterring immune system from
recognizing it as foreign body (non-responsive condition).
 Infs. usually occur when a patient has:
1. Alteration in cellular immunity (resulted in decreased
resistance to infection) or intestinal N.F balance (allowing
yeast to overcome host).
2. Invasive procedures, such as cardiac surgery or indwelling
catheters (producing alterations in host physiology).
3. Minimal vegetable consumption (leading in pH altering to
suit fungal growth).
Clinical presentations:
1. Oropharyngeal candidiasis
 Incidence: Healthy newborns, infants during 1st year, elderly
persons and D.M pts.
 Candidal esophagitis is most frequent inf. among HIV pts.
with an incident rate of 50-90%.
 Areas affected: Moist surfaces around lips, inside cheeks,
tongue and palate.
 Prevention:
1. Treatment of vaginal yeast inf. during last 3 months of
pregnancy.
2. Wash bottles and pacifiers.
3. Do not reuse bottles more than an hour after a baby has
drank from it.
4. Practicing good oral hygiene.
Pseudomembranous:
White thick plaques in mouth, tongue,
gums, palate and/or pharynx.

Atrophic:
Diffuse erythema affects mainly palate
and tongue, resulting in soreness.

Angular cheilitis:
Signs of cracking/inflammation seen at
corners of mouth; painful, burning and
soreness.
2. Onychomycosis (nail infection):
 Characterized by red swelling around nails, destruction of
nail tissue and loss of nail.
 Difficult to treat.
 Oral antifungal medications work best.
3. Ocular candidiasis:
 Characterized by cloudy vision and lesions within eyes.
 Caused through spreading of C. albicans via bloodstream.
 Treatment: Amphotericin B.
Other presentations:
1. Genital infections.
2. Nosocomial bloodstream infections.
3. STD.
4. Sepsis.
5. Cutaneous infections.
6. Diaper rash (often in newborn infants).
7. Deep (Invasive).

Note:
C. albicans account for 80% of nosocomial fungal infections
and 30% of deaths from nosocomial infections.
Blood, swabs, vaginal discharge, urine, feces, nail
clippings, hair, skin scales, respiratory secretions or
material from cutaneous or muco-cutaneous lesions

(Hyphae, spores or yeast cells)

Biochemical identification tests: API 20C or API 32C.
Serology / Molecular detection methods.
Antibiotic sensitivity test.
Slide:
 Production of Germ-tube and Chlamydoconidium is a key to
identifying C. albicans.
 Stains used: India ink, Periodic acid schiff, Silver stain and
Lactophenol cotton blue.

Culture:
 Facultatively anaerobic; colonies usually visible on most
bacterial and fungal agar media within 24-48 hrs., resembling
bacterial cultures rather than molds.

Serology/Molecular:
 Fluorescent in situ hybridization (FISH).
 Amplified TefI gene from C. albicans.
 Serum concentrations of mannan is useful for determining pts.
with disseminated Candidiasis.
 Skin smear C. albicans
Germ tubes
Antibiotic sensitivity testing, Treatment and
Prevention:
 Rise in incidence and over prescription of antifungals have
contributed to an increasing resistance against C. albicans.
 Recombinant DNA to create a live vaccine, composed of
conjugated fungal Ags with diphtheria toxoid.
 Suggested cure is limiting internal adherence of fungus.
 T-cell efficiency can be influenced to a useful extent by
nutrition.
 Prevention procedures: Good personal hygiene; keeping skin
clean, dry and free and taking sufficient amounts of probiotics
to repopulate N.F.
 Vaginal candidiasis: Monistat, Vagistat, Gyne-Lotrimin,
Diflucan, and Amphotericin B.
 Systemic infs.: Itraconazole and Fluconazole.
 Oropharyngeal: Topical (Nystatin and Clotrimazole) and/or
Oral (Ketoconazole or Fluconazole).
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