Cryptococcosis main doc

8/7/2019 Cryptococcosis main doc

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Cell structure of Cryptococcus fungi


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What is Cryptococcosis ?

Cryptococcosis is infection with Cryptococcus neoformans fungus .

Etiology and incidence

Cryptococcosis is nearly always caused by Cryptococcus neoformans, an encapsulated

yeast (Division Basidiomycota). Unlike most pathogenic fungi, this organism

occurs in the yeast form both in the host and in the environment. The perfect (mycelial)

stage of this fungus is called Filobasidiella neoformans or Filobasidiella bacillisporus.

This stage has never been isolated from patients or found in nature; it is only found in

the laboratory under certain conditions.

C. neoformans is surrounded by a large capsule within its hosts and on some culture

media. This capsule is important in its resistance to phagocytosis and in the identification

of the organism. Strains differ in their virulence for animals and possibly humans,

but the immune status of the host seems to be more important than the virulence of the

strain. There are four serotypes - A, B, C and D - based on capsular antigens . There are

three varieties:

Serotype A now classified as variety grubii

Serotypes B + C variety gattii

Serotype D variety neoformans


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Cryptococcus neoformans var. neoformans comprises serotypes A and D . This

organism is ubiquitous and causes most cases of cryptococcosis. In humans, it is an

opportunistic pathogen that mainly affects immunocompromised hosts.

Cryptococcus neoformans var. gattii comprises serotypes B and C . This variety is

less common in the environment than C. neoformans var neoformans. In humans, it is

mainly found in immunocompetent hosts. It has also been isolated from some cases of

cryptococcosis in animals including cats, dogs, porpoises and llamas.

There are two perfect states of C. neoformans: Filobasidiella neoformans var neoformans

is the result of mating between C. neoformans var neoformans serotypes A and D.

Filobasidiella bacillisporus is the result of mating between C. neoformans var gattii

serotypes B and C.

Some strains of serotypes A and D can mate with strains of serotypes B and C.

Cryptococcus species other than C. neoformans are, with rare exceptions, considered to

be saprophytic and nonpathogenic.

Cryptococcus laurentii has been associated with 15

cases of human disease. These cases mainly occurred in hosts with diseases or conditions

that predisposed them to fungal infections .


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Cryptococcus lifecycle

E xists in asexual and sexual forms, with the asexual form existing as a yeast, which

reproduces by budding. This is the only form associated with human infection.

P roduces white mucoid colonies in vitro which become visible within 48 hours

Thick capsule visible in India ink suspension .

Capsule has important antiphagocytic properties

Cryptococcus epidemiology


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Increasing proportions of patients have an underlying immune deficiency virtually all Var

neoformans or Var grubii

HIV/AIDS

Accounts for up to 50% cryptococcal infections

CD 4 less than 200

Incidence has declined in Australia since advent HAART

P rolonged steroid therapy

Organ transplantation

Malignancy

Sarcoidosis

In contrast to Var neoformans, Var gattii is geographically restricted to:

Australia, PNG

N . Africa and Mediterranean

India, S E Asia

Mexico, Brazil, P araguay, S California

Commonly non-immunocompromised hosts

L arge mass lesions (cryptococcomas) common, resulting in significant morbidity.

Transmission

C. neoformans grows naturally in the environment. C. neoformans Var neoformans

is ubiquitous in the soil, where it grows as a saprophyte. It is common in old pigeon nests

and around pigeon droppings; the bird droppings appear to create a favorable environment


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for its growth. It can also be isolated from numerous environmental sources including

vegetables and fruit, house dust, air conditioners, air and sawdust. It can survive for

months to years outside the host.

C. neoformans Var gattii is found in bark and plant debris under eucalyptus trees

(the river red gum tree E ucalyptus camaldulensis and the forest red gum tree, E . tereticornis).

It is also found in the air around these trees, particularly when they bloom in late

spring. It is not associated with pigeon droppings. Recently, C. neoformans Var gattii

was isolated from trees and soil on Vancouver Island in British Columbia.

Transmission seems to be mainly by inhalation, but C. neoformans can also enter

the body through the skin. Infections seem to be acquired mainly from the environment.

Cryptococcosis can also result from the reactivation of a latent infection.

Cryptococcal mastitis in cattle is usually associated with the treatment of the mammary gland

for another condition. The organism may be introduced into the teat in contaminated

syringes, cannulas or antibiotic preparations. It can also enter the mammary gland if the teat

ends are not adequately cleaned before treatment.

Cryptococcosis does not seem to be very contagious. There are no reports of transmission

from mammalian animals to other animals or to humans. However, in one recent

case, an immunosuppressed human probably acquired C.neoformans from the feces of an

asymptomatic pet bird.


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Human-to-human transmission is extremely rare and has mainly occurred under unusual

circumstances. Vertical transmission was recently described, when a HIV-positive

mother with peri-partum cryptococcal meningitis infected her newborn.

S ymptoms

y

Blurred vision or double vision (diplopia)

y

Bone pain or tenderness of the breastbone (sternum)

y

Chest pain

y

Confusion

y

Cough -- dry

y

Fatigue

y

Fever

y

Headache

y

N ausea

y

Skin rash, including pinpoint red spots (petechiae), ulcers, or other skin lesions

y

Sweating -- unusual, excessive at night

y

Swollen glands

y

Unintentional weight loss

N ote: P eople with a normal immune system may have no symptoms at all.

Diagnosis

P hysical examination may reveal:


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y

Abnormal breath sounds

y

Fast heart rate

y

Fever

y

Mental status changes

y

Stiff neck

Tests that may be done include:

y

Blood culture

y

CT scan of the heady

Sputum culture and stain

y

L ung biopsy

y

Bronchoscopy

y

Spinal tap to obtain a sample of cerebrospinal fluid (CSF)

y

Cerebrospinal fluid (CSF) culture and other tests to check for signs of infection

y

Chest xray

y

L umbar pucture

y

Measure and record opening pressure


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y

Repeat at least fortnightly during therapy and daily if pressure is more than 25 cmH 2O

I ndia ink examination

y

CSF WCC (usually mononuclears) typically low (< 50) in those with advanced

immunosuppression

y

CSF glucose + protein often only minimally abnormal

India Ink preparation taken from SAB isolate showing numerous Cryptococcal cells

surrounded by clear capsule (negative staining)

(Inset: E nlarged photo of Cryptococcal cell & budding daughter cell surrounded by clear

capsule )

Cryptococcal antigen assay

Cryptococcal antigen test (looks for a certain molecule that the Cryptococcus fungus can shed

into the blood.)


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y

Rapid diagnostic test

y

Rare false positives

y

Titre generally correlates to organism burden

y

Serum assay useful screen in AIDS patients

y

Extraneural cultures

y

Occasionally positive from another site

y

Full evaluation needed to exclude disseminated disease, or C N S disease

y

R adiology - Detection of cryptococcomas,may detect hydrocephalus ( need for shunt)

Cryptococcus neoformans in sputum Alcian blue- P AS stain ..

Clinical Manifestations

P ulmonary cryptococcosis


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Asymptomatic carriage may occur in healthy people as well as those with

chronic lung disease

May experience a self limited pneumonia

Invasive chronic pulmonary disease may occur and may disseminate to the

CN S

CN S disease

Meningitis (85%), meningoencephalitis, cryptococcoma

G enerally symptoms more insidious and of longer duration in the non-

immunosuppressed

Higher burden of organisms in AIDS, with variable inflammatory response,

which parallels degree of immunosuppression.

Cutaneous cryptococcosis

Ulcerated or nodular lesions usually portend poor prognosis in disseminated

disease

Cellulitis

Cryptococcus, cutaneous on the hand Cryptococcosis on the forehead

Bone and joint disease


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L ytic lesions in up to 10% with disseminated disease

O cular cryptococcosis

Rare, other than pressure effects

G enitourinary disease

P rostate acts as sanctuary site in immunosuppressed

What Makes Cryptococcus neoformans a P athogen?

To be classified as a pathogen, an organism must be able to cause infection under certain

conditions. By this definition, C. neoformans can certainly be classified as a pathogen. Because

the immunodeficient are more susceptible than the immunocompetent to infection with this

yeast-like organism, C. neoformans is frequently referred to as an opportunistic pathogen.

The factors that make C. neoformans a pathogen can be divided into two major groups:

The first comprises the basic characteristics needed to establish an infection and survive in the

human host

The second comprises the virulence factors that affect the degree of pathogenicity.

Basic Characteristics for P athogenicity


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T he I nfectious P article

Cryptococcus neoforma ns is an encapsulated fungal organism ( F igure 1) that can cause disease

in apparently immunocompetent, as well as immunocompromised, hosts. To enter the alveolar

spaces of the lungs and establish pulmonary infection, an organism must produce viable forms

smaller than 4 m in diameter. The typical vegetative form of C. neoformans is the yeast form

with a cell diameter of 2.5 m to 10 m.

The organism can also undergo sexual reproduction, and since it is a basidiomycete

(Filobasidiella neoformans), it forms basidiospores. Sexual reproduction appears to occur much

less frequently in nature than asexual or vegetative reproduction. The sexual spores

(basidiospores) are approximately 1.8 m to 3 m in diameter .Although the exact nature of the

infectious particles of C. neoformans is not known, they are presumed to be the dehydrated yeast

cells or basidiospores of the appropriate size range to get into the lungs. Once inside the lungs,

the yeast cells become rehydrated and acquire the characteristic polysaccharide capsule (F igure

2)In the case of basidiospores, they would convert to encapsulated blastoconidia.

F igure 1. T ransmission electron micrograph C. neoformans showing the characteristic

polysaccharide capsule.


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F igure 2. P roposed means of infection by C. neoformans .

G rowth I n Vivo

To cause infection in humans, a C. neoformans isolate must grow at 37 oC in an atmosphere of

approximately 5% CO 2 and at a pH of 7.3 to 7.4. To survive at 37 oC, the organism must have an

intact gene that encodes the C. neoformans calcineurin A catalytic subunit Calcineurin is a

serine-threonine specific phosphatase that is activated by Ca 2+-calmodulin and is involved in

stress responses in yeasts . Although calcineurin A mutant strains of C. neoformans can grow at

24oC, they cannot survive in vitro at 37 oC, in 5% CO 2, or at alkaline pH Since these are similar

to conditions in the host, one would predict that the calcineurin A mutant would not survive in

the human host. In support of that prediction, Odom et al. have shown that such mutants are not

pathogenic for immunosuppressed rabbits . Calcineurin A appears to be a basic requirement


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for C. neoformans survival in the host and consequently is a necessary factor for the

pathogenicity of the organism.

Virulence F actors

Virulence factors increase the degree of pathogenicity of a microbe. C. neoforma ns has a number of

virulence factors; generally, the virulence of an isolate cannot be attributed to any single factor, but rather

it is attributed to many working in unison to cause progressive disease. As virulence factors go, those

of C. neoforma ns would be considered low-grade. 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.

T he fungus has the following essential virulence factors:

S ynthesis of the pigment melanin. Melanin protects the fungal cells against oxidative stress,

phagocytosis, and antifungal drugs, and also can modify host immune responses.

Development of polysaccaride capsule, which helps the fungus cells to withstand phagocytosis

by alveolar macrophages.

Ability to grow at body temperature (37-39C). The vast majority of fungal species grow

optimally between 25 and 35C and there are only a few fungal species that appear

thermotolerant and this physical characteristic is a pre-requisite phenotype for invasive mycoses

in a mammalian host. Furthermore, C. neoformans cells can survive in the gastrointestinal tract

of birds (~40C), which in many instance responsible for environmental spread of the pathogen.


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Neurotropism

T here are three possible hypotheses that can explain the pathogen's predilection for the

Central Nervous S ystem (CN S ):

1. Specific neuronal substrates such as dopamine and epinephrine can facilitate fungal growth;

2. Being a privileged tissue site in terms of vigorousness of immune responses, the C N S might

provide safer environments for the fungus then other body sites;

3. Specific receptors present on neuronal cells could be more attractive for Cryptococcus cells

than other organs during systemic infection.

P athophysiology

Of the 19 species that comprise the genus Cryptococcus, human disease is associated with only C

neoformans. Animal models provide much of the understanding of the pathogenesis and the host

defense mechanisms involved in C neoformans infections. The organism is primarily transmitted

via the respiratory route and not directly from human to human.

Following inhalation, the yeast are deposited into the pulmonary alveoli, where they must

survive the neutral-to-alkaline pH and physiologic concentrations of carbon dioxide before they

are phagocytized by alveolar macrophages. G lucosylceramide synthase ( G CS) has recently been

identified as an essential factor in the survival of C neoformans in this extracellular environment.

Although G CS is a critical factor in extracellular survival of the yeast, the yeast no longer

requires G CS to survive the intracellular, more acidic, environment of within the macrophage

once it is phagocytized by alveolar macrophages.


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Unencapsulated yeast are readily phagocytosed and destroyed, whereas encapsulated organisms

are more resistant to phagocytosis. A cryptococcal polysaccharide capsule has antiphagocytic

properties and may be immunosuppressive. The antiphagocytic properties of the capsule block

recognition of the yeast by phagocytes and inhibit leukocyte migration into the area of fungal

replication.

The host response to cryptococcal infection includes both cellular and humoral components.

Animal models demonstrate that natural killer cells participate in the early killing of cryptococciand, possibly, antibody-dependent cell-mediated killing. In vitro monocyte-derived

macrophages, natural killer cells, and T lymphocytes can inhibit or kill cryptococci. A successful

host response includes an increase in helper T-cell activity, skin test conversion, and a reduction

in the number of viable organisms in the tissues. In addition to cellular mechanisms,

anticryptococcal antibodies and soluble anticryptococcal factors have been described. Antibodies

to a cryptococcal antigen and its complement play a critical role in enhancing the macrophage-

and lymphocyte-mediated immune response to the organism. Researchers use monoclonal

antibodies to capsular polysaccharide to passively immunize mice against C neoformans.

C neoformans infection is usually characterized by little or no necrosis or organ dysfunction until

late in the disease. Organ damage may accelerate in persons with heavy infections. The lack of

identifiable endotoxins or exotoxins partly causes the absence of extensive necrosis early in

cryptococcal infections. Organ damage is primarily due to tissue distortion secondary to the

expanding fungal burden. E xtensive inflammation or fibrosis is rare. The characteristic lesion of


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C neoformans consists of a cystic cluster of yeast with no well-defined inflammatory response.

Well-formed granulomas are generally absent.

C neoformans can cause an asymptomatic pulmonary infection followed later by the

development of meningitis, which is often the first indication of disease. If limited to the lungs,

C neoformans infection may cause pneumonia, poorly defined mass lesions, pulmonary nodules,

and, rarely, pleural effusion. Although immune defects are common in patients with meningitis

or disseminated infection, patients with disease that is confined to the lungs are usually

immunocompetent.

T reatment

CN S disease uniformly fatal without prescription.

Immunocompromised patients need longterm suppressive therapy, unless immune status

substantially recovers

Aim for complete eradication of organism in the nonimmunosuppressed:

Amphotericin B 0.5-0.7 mg/kg/d + flucytosine 100-150 mg/kg/d for 6 weeks followed by

fluconazole 400 mg/d for 3-6 months+

Debate re switch to fluconazole after 2 weeks if favourable clinical(including L P )

response

In HIV/AIDS most switch early to oral therapy, or use high dose oral fluconazole from the

outset if mild disease

L iposomal amphotericin, if develop toxicity.


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N ew azoles

E chinocandins have no anticryptococcal activity

Management of raised intracranial pressure often the most problematic issue

L arge volume (30-50 m L ) CSF removal up to daily

Shunt or drain placement (does not prevent clearance of infection)

Steroids generally of no use in management of pressure, except where oedema

associated with cryptococcomas

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Cryptococcosis main doc