1. Cryptococcosis Reference:
Monograph on Cryptococcus and Cryptococcosis In man, animals and birds
A guide for postgraduate students in developing countries By
Mohamed Refai, Mahmoud El-Hariri and Randa Alarousy

2. History and nomenclature of the genus Cryptococcus
The name Cryptococcus was created from the Greek kryptus meaning ‘hidden’ for a group of yeasts that lacked the ability to produce endospores by Kützing (1833) .

3. Morphology of Cryptococcus Macro- and microscopic morphology
On solid media most species have a slimy appearance, carotenoid pigments may be produced.
The cells of Cryptococcus species are spheroidal, ovoidal, elongate, amoeboid or polymorphic.
Cells of most strains are covered by a thin layer of glycoprotein capsular material that has a gelatin-like consistency and that, among other functions, serves to help extract nutrients from the soil.

4. Ultra structure
The electron microscopy of Cryptococcus cell reveals the presense of a capsule, cell wall, plasma membrane and nucleus, as well as other intracytoplasmic organelles.
The nucleus shows a clear membrane and generally has disintegrated chromatin material, instead of a definite nucleolus.
Mitochondria are consistent in size and shape.
Lipid granules with glycogen in transition, large numbers of vacuoles, well preserved ribosomes and endoplasmic reticulum are observed.
Bud separation demonstrates a simulated break-off appearance from mother cells.
The electron microscopy of Cryptococcus cell reveals the presense of a capsule, cell wall, plasma membrane and nucleus, as well as other intracytoplasmic organelles. The capsule, sometimes thicker than cells, consists of either coiled, intertwining microfibrils or dots; the dot-type appears with or without radiating cilia-type filaments at the periphery. A clear zone often separats the capsule from the cell wall. The wall appears to be composed of multiple parallel layers of thin membraneous sheaths containing an inner dense dark zone and an outer lighter area. The plasma membrane is similar to that found in other yeasts. The nucleus shows a clear membrane and generally has disintegrated chromatin material, instead of a definite nucleolus. Mitochondria are consistent in size and shape. Lipid granules with glycogen in transition, large numbers of vacuoles, well preserved ribosomes, few plasmalemmosomes and endoplasmic reticulum are observed. Bud separation demonstrats a simulated break-off appearance from mother cells.

5. Biochemical characteristics of Cryptococcus species
Biochemically, the members of the genus Cryptococcus are unable to ferment sugars, but do assimilate different sugars, inositol and produce urease. Carotenoid pigment production is extremely variable. Cryptococcus differs from Rhodotorula* by assimilating inositol, and from Candida glabrata by assimilating inositol and being urease positive.
* Both Cryptococcus and Rhodotorula species produce carotenoid pigments and capsule and both are urease positive.

6. Pathogenicity Cryptococcus neoformans is an opportunistic fungi.
Virulent isolates of Cryptococcus must be able to:
produce small particles that can get into the alveolar spaces,
Grow at 37oC at a pH of 7.3 to 7.4 in an atmosphere of approximately 5% CO2
Have the ability to produce a large capsule and shed great amounts of capsular material into the body fluids makes the organism highly virulent.
Other factors, such as melanin, mannitol, superoxide dismutase, protease, and phospholipase production, may enhance the pathogenicity of C. neoformans.
The effectiveness of many of these cryptococcal virulence factors depends on the status of the host's defensive mechanisms.
Virulence factors increase the degree of pathogenicity of a microbe. Cryptococcus has a number of virulence factors; however, the severity of the host's disease results from a combination of several virulence factors superimposed on the host's innate and immune resistance status

7. Epidemiology of Cryptococcosis
Emmons (1952) was the first one who
isolated C. neoformans from the pigeon droppings after an outbreak occurred in new York squares.
Inhalation is the most common mode of transmission.
Incubation period is unknown.
Person-to-person transmission has not been documented other than through transplanted organs.
Host range: humans and various domestic and wild animals.
C. neoformans serotypes A and D are distributed worldwide and cause the vast majority of cryptococcal infections, predominately in immunocompromised individuals. Serotype A is responsible for over 95% of cryptococcosis cases worldwide.

8. Cryptococcosis in man Pulmonary Cryptococcosis :
Radiographic features. In general
several CT patterns that can be seen:
clustered nodular
pattern - most prevalent
solitary pulmonary nodular
scattered nodular
bronchopneumonic
The most common CT findings in immunocompetent patients with pulmonary cryptococcosis are pulmonary nodules. The nodules are most often multiple, smaller than 10 mm in diameter, and well defined with smooth margins.
Associated cavitation may be seen in up to 40% of cases

9. Central Nervous System: Dissemination to the brain and meninges is the most common clinical manifestation of cryptococcosis and includes meningitis, meningoencephalitis or expanding cryptococcoma.
i. Meningitis is the most common clinical form, accounting for up to 85% of the total number of cases, however the clinical signs are rarely dramatic.
Symptoms usually develop slowly over several months, and initially include headache, followed by drowsiness, dizziness, irritability, confusion, nausea, vomiting, neck stiffness and focal neurological defects, such as ataxia. Diminishing visual acuity and coma may also occur in later stages of the infection. Acute onset cases may also occur, especially in patients with widespread disease, and these patients may deteriorate rapidly and die in a matter of weeks.

10. (a) Gross photograph of the brain showing opaque meninges in cryptococcal meningitis; (b) dense lymphomononuclear inflammation over meninges with scattered yeast like fungal organisms. (H and E; ×400); (c) yeast like forms of Cryptococcus highlighted by mucicarmine stain (×400).

11. Cryptococcosis in man
ii. Meningoencephalitis due to invasion of the cerebral cortex, brain stem and cerebellum is an uncommon, rapid infection, often leading to coma and death within a short time. Symptoms include slow response to treatment and signs of cerebral edema or hydrocephalitis.

12. iii. Cryptococcoma
Cryptococcoma is a rare entity, characterized by localized, solid, tumor-like masses, usually found in the cerebral hemispheres or cerebellum, or more rarely in the spinal cord. Symptoms are consistent with an expanding intracranial mass and include headache, drowsiness, nausea, vomiting, mental changes, slurred speech, double vision, unsteadiness of gait, coma, paralysis and hemiparalysis. These symptoms may mimic cerebral neoplasm which may delay a true diagnosis

13. Cutaneous Cryptococcosis:
In patients with AIDS, skin manifestations represent the second most common site of disseminated cryptococcosis.
Lesions often occur on the head and neck and may present as papules, nodules, plaques, ulcers, abscesses, cutaneous ulcerated plaques. Anal ulceration may also occur.

14. Cutaneous Cryptococcosis

15. Cryptococcal peritonitis:
Cryptococcal peritonitis has been well- described. It may present in the ascites of chronic liver disease or in the peritoneal dialysate of patients with chronic renal disease.
Cryptococcal pyelonephritis:
Cryptococcuria occurs and even pyelonephritis is described but the urinary tract site is less common than one would expect with severely disseminated disease and may simply reflect the length of routine culture methods.
Prostatic cryptococcosis:
Prostatic cryptococcosis is generally asymptomatic and has been considered a site for occult or sequestered infection that is protected from antifungal treatments.

16. Ocular cryptococcosis:
It is reported in up to 45% of patients with cryptococcosis and thus becomes a significant site for disease .
The most common manifestations are ocular palsies and blindness but in severely immunosuppressed patients retinal lesions may appear.
Loss of vision without evidence of endophthalmitis also occurs.
Cryptococcal endocarditis
Despite frequent cryptococcemia, the reported cases of cryptococcal endocarditis are less than a dozen.

17. Diagnosis of cryptococcosis
The workup in patients with suspected cryptococcosis includes the following:
Cutaneous lesions: biopsy with fungal stains and cultures
Blood: fungal culture, cryptococcal serology, cryptococcal antigen testing and molecular biological assays.
Cerebrospinal fluid: India ink smear, fungal culture, cryptococcal antigen testing and molecular biological assays .
Urine and sputum: cultures, even if renal or pulmonary disease is not clinically evident.
Cryptococcal pneumonia: culture of bronchoalveolar lavage washings.

18. Direct microscopic examination of India ink preparation
Preliminary diagnosis of cryptococcal infection is made by direct microscopic examination of India ink preparations of samples.

19. Cryptococcus colonies on Sabroud's dextrose agar
Isolation and Identification:
Definitive diagnosis is confirmed by the culture of specimens, often the cerebrospinal fluid (CSF) or blood, and sometimes in respiratory secretions.
Cryptococcus neoformans and C. gattii grow well at 37oC.
On Sabouraud dextrose agar colonies appear soft, creamy, opaque in 3-5 days, then colonies become mucoid and creamy to tan.

20. Demonstration of brown colour effect
Cryptococcus colonies are brown on bird seed agar, modified tobacco and Eucalyptus leave extract agar as well as on Pal ׳s medium.
Other yeasts develop white to creamy colonies.
Colonies on bird seed agar

21. Differentiation of C. gattii and C
Differentiation of C. gattii and C. neoformans on canavanine glycine bromothymol blue (CGB)
On Canavanine Glycin Bromthymol blue (CGB) medium, Cryptococcus neoformans develop non-coloured colonies and , while C. gattii develops blue colonies.

22. Biochemical identification
Cryptococcus neoformans and C. gattii do not ferment sugars, but assimilate several sugars such as glucose, galactose, sucrose, maltose and inositol, but not lactose or nitrate and hydrolyses urea.

24. Serological diagnosis of cryptococcosis
Cryptococcal antigen from cerebrospinal fluid is the best test for diagnosis of cryptococcal meningitis in terms of sensitivity. Rapid diagnostic methods to detect cryptococcal antigen by:
Cryptococcal antigen (CRAG) detection:
i. Rapid latex agglutination: either by polyclonal Ab as: Crypto-La; Myco-immune; CALAS or IgM based assay as: Murex Cryptococcus and
ii. Cryptococcal antigen lateral flow assay (LFA):uses gold-conjugated, monoclonal antibodies impregnated onto an immunochromatographic test strip to detect cryptococcal capsular polysaccharide glucuronoxylomannan antigen (CRAG) for all 4 C. neoformans serotypes (A–D).
The LFA is a point-of-care dipstick test that uses gold-conjugated, monoclonal antibodies impregnated onto an immunochromatographic test strip to detect cryptococcal capsular polysaccharide glucuronoxylomannan antigen (CRAG) for all 4 C. neoformans serotypes (A–D)  If cryptococcal antigen is present in a specimen, suspended, gold-conjugated antibodies bind to the antigen.

25. Serological diagnosis (contin’d)
iii. Cryptococcal Antigen enzyme immunoassay (CrAg EIA) :The detection of Cryptococcus antigen by enzyme immunoassay is a highly sensitive and specific test. It is extremely useful in cases where microscopic techniques are inconclusive (when?).
Detection of Cryptococcus antibodies Cryptococcal antibodies are not helpful in diagnosis because of their poor sensitivity and specificity.

26. 135
135. Iatron serotyping kit can be used to serotype isolates of Cryptococcus neoformans.

27. 136
136. A positive cryptococcal antigen latex test. It should be noted that the detection of cryptococcal capsular polysaccharide antigen in spinal fluid is now the method of choice for diagnosing patients with cryptococcal meningitis. In AIDS patients, cryptococcal antigen can be detected in the serum in nearly 100% of cases. However, in non-AIDS patients antigen detection in serum is less sensitive with only about 60% of patients with cryptococcosis reported as being positive. Note, serum specimens should be pretreated with pronase to enhance detection of antigen and avoid false negative results.

28. 137
137. Negative cryptococcal antigen latex test.

29. Diagnosis of cryptococcosis (contin’d)
Histopathology
i) Hematoxylin and eosin stain shows lightly basophilic cell wall surrounded by a clear zone.
ii) Cryptococcus neoformans will stain with Periodic acid–Schiff or silver methenamine.
iii) Mucicarmine stains the capsule - shows clear zone containing carminophilic material. Alcian blue also stains the capsule
iv) A combined Periodic acid–Schiff - alcian blue stain contrasts the cell wall and capsule.
Capsule-deficient variety is demonstrated by:
(a) Immunofluorescent antibody technique.
(b) Fontana-Masson silver staining

30. Cryptococcus in the lung. Methenamine silver stain (MSS)
Cryptococcal prostatitis. Left:Fontana-Masson stain demonstrates melanin in the cell wall. Right: The mucoid capsule of the pathogen is stained with mucicarmine.
Cryptococcus in the lung. Methenamine silver stain (MSS)

31. Diagnosis of cryptococcosis “Radiology”
Upper photos: MRI scans showing multiple cryptococcomas [white masses] in the brain. (Courtesy Prof. Tania Sorrell, Sydney NSW).
X-ray showing pulmonary cryptococcal infection [right upper lobe]. (Courtesy Prof. Tania Sorrell, Sydney NSW).

32. Molecular Diagnosis of Cryptococcus
Molecular typing techniques:
1. Multilocus enzyme electrophoresis (MLEE)
2. DNA fingerprinting
3. Random amplification of polymorphic DNA (RAPD)
4. PCR fingerprinting amplified fragment length polymorphism (AFLP)
5. Restriction fragment length polymorphism (RFLP) of PLB1 gene
6. Restriction fragment length polymorphism (RFLP) of GEF1 gene
7. Restriction fragment length polymorphism (RFLP) of URA5 gene
8. Sequencing of ITS1-5.8S-ITS2 rDNA region
9. Sequencing of intergenic spacer region (IGS)
10. Sequencing of multilocus sequence typing (MLST)
11. Sequencing of multilocus microsatellite typing (MLMT)
12. Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method (MALDI-TOF) analysis
Notice the differences between eukaryotic and prokaryotic cells as well as the capsule during DNA extraction.

33. Treatment and Prognosis
TREATMENT: Give appropriate antifungal therapy .
IMMUNIZATION: No immunization is currently available; however, some vaccines are currently in clinical trials, including GXM conjugated to tetanus toxoid vaccine, which has been shown to be effective in immunocompetent individuals in clinical trials.
PROPHYLAXIS: HIV patients may receive antifungal therapy such as fluconazole when no symptoms of infections are present