crypto cocco sis

Cryptococcosis

John R. Perfect, MDa,*, Arturo Casadevall, MD, PhDb

aDepartment of Medicine, Division of Infectious Diseases,

Duke University Medical Center, PO Box 3353, Durham, NC 27710, USAbDepartment of Medicine, Division of Infectious Diseases, Albert Einstein

College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Epidemiology

Varieties of C. neoformans

Cryptococcus neoformans strains classically have been grouped into two

varieties that included five serotypes known as C. neoformans variety neofor-

mans (serotypes A, D, and AD) and C. neoformans variety gattii (serotypes

B and C). The serotype differences between the different varieties reflect anti-genic differences resulting from differences in the structure of the capsular

polysaccharide. The serotype classification of a strain is determined using

absorbed rabbit sera [1]. Some monoclonal antibodies are now available

that are useful for typing strains [2,3]. C. neoformans var. neoformans is

found throughout the world in association with excreta from certain birds

including pigeons, canaries, and cockatoos. C. neoformans var. gattii is

found primarily in tropical and subtropical regions and has been associated

with several species of eucalyptus trees.After many years of stable taxonomic classification the availability of

DNA typing data has shown major differences between isolates grouped

within a serotype and, as a consequence, there is now considerable uncer-

tainty as to the exact relationship between the varieties and serotypes. The

situation is made more complex by the fact that C. neoformans has a sexual

cycle and that some strains are capable of mating with consequent genetic

recombination. Recently it was proposed to separate serotype A into a

different variety known as grubii on account of significant genetic dif-ferences from serotype D [4]. The varietal status for the serotype AD strains

Infect Dis Clin N Am 16 (2002) 837–874

* Corresponding author.

E-mail address: [email protected] (J.R. Perfect).

The authors are supported by the National Institutes of Health with NIAID grants

AI33774, AI3342, and HL59842 (AC); and AI28388, AI49975, PA 98100, and AI39115 (JRP).

0891-5520/02/$ - see front matter � 2002, Elsevier Science (USA). All rights reserved.

PII: S 0 8 9 1 - 5 5 2 0 ( 0 2 ) 0 0 0 3 6 - 3

is uncertain but there is increasing evidence that these represent diploid

strains, possibly resulting from matings of A and D strains. There is now

a further proposal to abolish the current varietal system and replace it withtwo species that contain groupings of genotypes [5]. Given the impending

completion of the C. neoformans genome and rapid advances in the under-

standing of genetic diversity among fungi it is likely that taxonomic relation-

ship of strains will remain in flux for some time. For clinicians, however, the

standard serotype classification, which has been in use for half a century,

remains a useful method for approaching strain differences.

Most clinical isolates are serotype A (variety grubii) even in geographic

regions where strains of variety gattii are found in the environment [6,7].Serotype D is common in certain European countries but constitutes a

minority of isolates in other parts of the world [6,7]. For example, in New

York City serotype D comprises approximately 13% of isolates [8]. From

a clinical viewpoint there has been little emphasis in determining the sero-

type of clinical strains because all varieties produce similar forms of disease

and there do not seem to be significant differences in drug susceptibility

among the various isolates. Hence, strain serotyping is not routinely per-

formed by microbiology laboratories and serotype information is seldomavailable to clinicians. There is increasing evidence, however, that the differ-

ent varieties result in different clinical manifestations or clinical syndromes.

For example, cryptococcosis caused by C. neoformans var. gattii occurs

primarily in immunologically competent hosts and is associated with the

formation of large granulomas (cryptococcomas) that may require surgical

removal for optimal management [9]. Serotype D strains have been associ-

ated with a propensity to cause cutaneous lesions [10].

Pathogenesis and prevalence of human C. neoformans infection

In the 1950s, Baker et al [11–13] carried out autopsy studies and docu-

mented the existence of a primary infection complex in individuals with and

without a history of cryptococcosis. These studies established that the

pathogenesis of cryptococcosis was similar to that of Mycobacterium tuber-

culosis and involved an initial pulmonary infection that was contained with-

in the lung by granulomatous inflammation. Evidence that latency canfollow an initial infection and reactivate later in life comes from DNA typ-

ing studies of C. neoformans isolates from African expatriates who devel-

oped cryptococcosis while living in Europe [14]. In this study isolates

from African expatriates were more similar to those from Africa than to the

prevailing European strains [14]. Other evidence consistent with the exis-

tence of a latent state is the description of cryptococcosis caused by C. neo-

formans var. gattii strains in individuals living outside tropical areas with a

history of travel [15]. There is also evidence, however, that initial exposuresto the yeast can lead to symptomatic infection. For example, there are anec-

dotal reports of cryptococcosis following direct exposure to pigeons and

838 J.R. Perfect, A. Casadevall / Infect Dis Clin N Am 16 (2002) 837–874

their excreta [16]. Recently, an immunocompromised patient was shown to

have cryptococcosis with a strain that was indistinguishable from an isolate

found in the excreta of a pet bird in the home [17]. Hence, initial acquisitionof C. neoformans from the environment can presumably lead to infection

with at least three outcomes: clearance of infection, development of latent

infection, or acute infection with or without disseminated cryptococcosis.

The outcome of infection is presumably a function of the inoculum, the

immunologic state of the host, and the virulence of the infecting strain.

At this time the interaction of these factors to determine precisely the out-

come of infection remains poorly understood but it is believed that most pri-

mary C. neoformans infections are asymptomatic.The prevalence of infection with C. neoformans is believed to be high even

when symptomatic disease is rare. Several methods have been used to study

the prevalence of infection with C. neoformans among individuals without

evidence of cryptococcosis. Skin testing for delayed hypersensitivity to C.

neoformans antigens has shown positive responses in individuals with a his-

tory of known exposure to the fungus [18]. The prevalence of positive skin

reactions in individuals with presumed high exposure to C. neoformans, such

as pigeon fanciers and laboratory workers in cryptococcal research labora-tories, is high suggesting that asymptomatic infection is common [19]. Sero-

logic studies have consistently provided evidence for widespread infection in

individuals without a history of cryptococcosis. Practically all adults have

serum antibody to C. neoformans antigens [20]. Analysis of sera from chil-

dren of different ages in New York City has shown that the age of serocon-

version for most individuals is before the age of 10 [21]. Interestingly, that

study reported one child who presented to the emergency room with vomit-

ing and fever and was positive for serum cryptococcal antigen, which raisesthe issue that some childhood infections are accompanied by antigenemia

and may be an early indicator of disseminated infection [21].

Although there are reports of occasional isolation of C. neoformans from

individuals with no signs or symptoms of cryptococcosis [22,23], this fungus

is not generally considered to be a normal constituent of the human micro-

bial flora. The likelihood of recovering C. neoformans from asymptomatic

humans increases, however, in individuals with chronic pulmonary diseases

[24,25]. Hence, isolation of C. neoformans from a human specimen shouldprompt consideration of the possibility of chronic infection.

Incidence on human cryptococcosis

Despite strong serologic evidence for widespread infection in certain

human populations, cryptococcosis is a rare disease in individuals without

impaired immunity. Before the AIDS epidemic, cryptococcosis was consid-

ered an unusual infection and there was a dearth of robust epidemiologicstudies to provide the true incidence and prevalence of cryptococcosis in the

absence of HIV infection. A retrospective review of the Kaiser-Permanente

839J.R. Perfect, A. Casadevall / Infect Dis Clin N Am 16 (2002) 837–874

records in Northern California for the years 1971 to 1980 in the pre-AIDS

era yielded only 10 cases with an overall incidence of 0.8 cases per million

persons per year [26]. Extrapolation of these numbers to calculate a rate forthe United States per year would have yielded 176 cases [26], which is not far

off from the estimates of 200 to 300 cases per year made in the 1960s [27]. A

retrospective analysis of the data at the Centers for Disease Control and

Prevention, however, revealed a steady rise in the number of identified cases

through serologic testing from 24 cases in 1965 to 336 in 1976 [27]. Whether

these numbers reflected increased awareness of the infection, improvements

in serologic detection, or a true increase in the incidence of cryptococcosis

cannot be discerned from the information available. Nevertheless, it is clearthat in the 1970s there was an increasing recognition of cryptococcal disease,

which may have reflected the beginning of the AIDS epidemic, the increas-

ing use of organ transplantation to treat certain incurable diseases, and

progress in cancer therapy resulting in larger numbers of immune-sup-

pressed survivors. A recent population-based study using active surveillance

has documented that the incidence of cryptococcosis in non–HIV-infected

individuals varies with geographic location and ranges from 0.2 to 0.9 per

100,000 in Atlanta and San Francisco, respectively [28]. An active surveil-lance study in Alabama found an overall annual incidence of 0.84 cases per

100,000 of cryptococcosis in non–HIV-infected individuals [29]. Rates of

cryptococcosis approaching 1 case per 100,000 in patients without HIV

infection are comparable with the incidence rates described for meningococ-

cal meningitis [30].

After the recognition of the AIDS epidemic in 1981 cryptococcosis in

individuals without an obvious cause of immune impairment became an

AIDS-defining diagnosis. Many studies documented a high prevalence ofcryptococcosis in AIDS patients with rates ranging from 2.9% to 13.3%

[31–37]. The annual incidence of cryptococcosis in patients with advanced

HIV infection in 1992 to 1994 was 17 and 66 per 1000 persons living with

AIDS in San Francisco and Atlanta, respectively [28]. These incidence num-

bers predate the introduction of more effective antiretroviral therapy, which

has been associated with a decline in the prevalence of cryptococcosis in this

population (see later).

Two other groups are at significantly higher risk for cryptococcosis: can-cer patients and recipients of organ transplants. Lymphoproliferative dis-

orders were associated with an increased risk for cryptococcosis in the

1950s [38,39]. Subsequent studies have confirmed a continuing association

between cryptococcosis and hematologic malignancies [40–42]. Although

prospective population-based incidence data for cryptococcosis in patients

with cancer are not available a retrospective analysis of case records at the

M.D. Anderson Cancer Center (Houston, TX) for the years 1989 to 1999

revealed an incidence of 18 cases per 100,000 admissions. Cryptococcosisoccurred in 2.8% of organ transplant recipients with an overall associated

mortality of 42% [43]. Kidney and liver transplant recipients seemed to be


at high risk for cryptococcosis and there was a significantly higher preva-

lence of C. neoformans infection among organ transplant recipients in the

Northeastern region of the United States [43]. Furthermore, both in cornealand lung transplantation, the transplanted organ has carried the cryptococ-

cal infection to the host [44,45].

Sources of infection

Cryptococcus neoformans var. gattii (serotype B) is found in several spe-

cies of eucalyptus trees, whereas C. neoformans varieties neoformans (sero-

type D) and grubii (serotype A) have been isolated from various sourcesincluding fruits, trees, and avian excreta [46]. There is general agreement

in the field that most cryptococcal infections are acquired by inhalation of

infectious propagules. The specific source of infection and the form of C.

neoformans responsible for human infection, however, have not been defin-

itively established. Encapsulated yeast cells of the type found in laboratory

cultures and in clinical isolates are thought to be too large to be inhaled effi-

ciently. Dehydrated yeast cells or basidiospores (<5 to 10 lm), however, are

sufficiently small for inhalation and alveolar deposition. Analysis of soilscontaminated with pigeon excreta has shown the presence of large numbers

of propagules with diameters compatible with alveolar deposition [47,48].

Air in sites heavily contaminated with pigeon excreta has also been shown

to contain viable cryptococci. Analysis of the air in a tower in Oklahoma

City contaminated with pigeon excreta revealed an average concentration

of 45 viable cells of C. neoformans cells per 100 L of air with approximately

60% of the cells being less than 5 lm in diameter [49]. Based on these num-

bers it was estimated that a human exposed to this air for 1 hour would have41 C. neoformans cells deposited in the lungs [49]. Several studies have

shown that clinical isolates are indistinguishable by molecular typing from

environmental isolates [50–52].

Direct evidence for human infection from either eucalyptus trees or

pigeon excreta is not yet available. The finding of viable C. neoformans cells

of size compatible with alveolar deposition in air from sites contaminated

with pigeon excreta and the association of DNA types in clinical- and

excreta-derived isolates, however, provides strong circumstantial evidenceimplicating this material as a potential source of infection. There is one

well-documented case that suggests acquisition of C. neoformans infection

from a pet cockatoo [17]. It is advisable that individuals at risk for crypto-

coccosis avoid sites that are contaminated with pigeon excreta and desist

from having certain birds, such as pigeons, canaries, and cockatoos, as pets.

Although there is not a strong seasonal association for the occurrence of

cryptococcosis, some studies have noted a trend toward higher rates in the

fall and winter [37]. A study from Thailand, however, noted no seasonalassociation for C. neoformans infections while demonstrating a strong asso-

ciation for Penicillium marneffei infection in the dry season [53].


Risk factors

Life-threatening cryptococcal disease can occur in apparently normal

individuals but such cases are relatively rare. The likelihood of cryptococco-sis rises dramatically, however, in individuals with impaired immunity. Spe-

cifically, conditions that predispose to impaired cellular immunity have been

associated with a significantly increased risk of cryptococcosis and these

include advanced HIV infection [32], lymphoproliferative disorders [39], ste-

roid therapy [54], and organ transplantation [55]. Cryptococcosis has also

been associated with hyper-IgM syndrome in children [56–58] and HIV-

negative CD4+ T lymphopenia in adults [59]. A smoking habit may predis-

pose to C. neoformans infection in certain individuals [28,60]. Population-based studies have also yielded an association between outdoor activities

and the risk of acquiring cryptococcosis [60].

In patients with HIV infection the incidence of cryptococcosis is inversely

proportional to the CD4 lymphocyte count [61]. In patients with HIV infec-

tion the risk of cryptococcosis rises rapidly when the CD4 lymphocyte count

drops below 100/lL [37]. The average CD4 lymphocyte count of HIV-

infected patients with cryptococcosis has been measured at 73/lL [61].

Current trends in the epidemiology of cryptococcosis

After rising dramatically until the early 1990s, the prevalence of crypto-

coccosis in patients with HIV infection began to decline with the introduc-

tion of more effective antiretroviral therapy and the widespread use of

fluconazole therapy for the treatment of oral candidiasis [28]. Temporal

trends noted a rising incidence of cryptococcosis in HIV-infected patientsfrom 1985 to 1992 [62], but by the mid-1990s there was evidence for fewer

infections in this population [63]. The introduction of highly active anti-

retroviral therapy (HAART) reduces viremia and increases CD4 counts,

resulting in an improved immunologic state that is associated with signifi-

cantly reduced risk for cryptococcosis. In The Netherlands the number of

patients with HIV infection complicated by C. neoformans infection has

declined by as much as 75% with the introduction of HARRT [64]. Another

important contributor is probably the use of fluconazole for the suppressionof oropharyngeal candidiasis in patients with AIDS [65]. In this regard flu-

conazole therapy has been shown to be effective prophylaxis against the

development of cryptococcal meningitis [66,67].

Because HIV infection is currently the major risk factor for cryptococco-

sis in countries affected by the AIDS epidemic the use of HAART has been

associated with a reduced incidence of cryptococcosis in areas where it is

available. The high cost of HAART and the complicated logistical infra-

structure that is necessary for its administration, however, have precludedits use in less-developed countries of Africa and Asia where the prevalence

of HIV infection and AIDS increased dramatically in the 1990s and so has


cryptococcosis. For example, in Asia a prospective study of bloodstream

infections in patients hospitalized at a Bangkok hospital revealed that C.

neoformans was the most common pathogen recovered in culture [68]. Thenet result of these epidemiologic trends has been an explosion in the inci-

dence of cryptococcosis in Africa, Thailand [68], and India [69], whereas the

number of cases in Europe and the United States has declined.

Several reports early in the AIDS epidemic suggested that cryptococcosis

was more prevalent in Africa than in other geographic regions [70,71]. Cur-

rently, cryptococcal meningitis is the leading cause of culture-positive men-

ingitis in Zimbabwe, constituting 45% of all cases and easily outnumbering

the cases of pyogenic (16%) or tuberculous (12%) meningitis [72]. A similarsituation exists in Malawi where C. neoformans is the most common cause of

meningitis having surpassed both Neisseria meningitidis and Streptococcus

pneumoniae in this role [69]. The mortality in African patients is high, aver-

aging over 40%, and the mean duration of symptoms before hospitalization

is only 4.8 days [69]. The exact reason for the apparently higher prevalence

of cryptococcosis in African patients with HIV infection is not known.

There is some evidence that the incidence of cryptococcosis in Africa has

been increasing for decades possibly as a consequence of the developingHIV epidemic in that continent [73]. Interestingly, a population analysis

of AIDS-defining illnesses in Australia revealed that birth in Africa was

associated with an odds ratio for developing cryptococcal meningitis that

was 2.43 times that observed for individuals born in Australia or other

industrialized nations [74]. The authors speculate that the higher likelihood

of cryptococcal disease in African expatriates may be a result of greater

exposure and lower rates of antifungal prophylaxis [74]. It is also possible,

however, that host genetic and nutritional differences, possibly combinedwith C. neoformans strain differences, are responsible for the greater preva-

lence of cryptococcosis in sub-Saharan patients with AIDS.

Host defense and immune response

There is evidence that C. neoformans infections are common in the gen-

eral population based on serologic [21] and skin hypersensitivity [75,76]

studies. Because the prevalence of infection in the general population seemsto be very high yet the incidence of cryptococcosis is relatively low, one must

conclude that normal host defense mechanisms are highly effective at con-

taining and preventing disease. Nevertheless, the reality of reactivation dis-

ease in many cases of human cryptococcosis [14] strongly suggests that the

establishment of latent infection in granulomas may be a relatively common

event following acute infection in normal individuals. C. neoformans

presents the paradox that normal immune mechanisms must be highly effec-

tive at containing infection but may be unable to eradicate the organismonce established in tissue in many immunocompetent hosts. The immune

response to C. neoformans has been studied extensively in humans and


experimental animals and is relatively well understood. The mechanisms by

which C. neoformans subverts the immune system to establish chronic infec-

tions are only beginning to be unraveled.

Effective immune response

Most cryptococcal infections are associated with conditions whereby cell-

mediated immunity is compromised [77]. This observation strongly suggests

an important requirement of cell-mediated immunity in host defense against

C. neoformans and there is general agreement that a strong cellular immune

response producing granulomatous inflammation is essential for contain-ment of C. neoformans infection [78–81]. In humans, containment of crypto-

coccal infection in the lung is associated with the formation of a primary

complex, which consists of a granulomatous subpleural nodule [11]. In brain

tissue a granulomatous response is associated with control of infection in

both humans [82] and rats [82,83]. Experimental cryptococcal infection in

immunocompetent rats reveals that control of infection with diminution

of fungal burden is temporally associated with a granulomatous tissue reac-

tion [78]. Similarly, normal rabbits infected with C. neoformans mount astrong cellular immune response in the meninges that is associated with con-

tainment of an intracisternal infection [84]. Because granuloma formation is

a result of a Th1-polarized response, there is a requirement for the produc-

tion of Th1-associated cytokines, such as interferon-c and interleukin (IL)-2,

and effective host responses to C. neoformans infection [85,86].

The macrophage is generally considered to be the central effector cell

against C. neoformans [87–89]. Alveolar macrophages are probably the first

immune effector cell encountered by inhaled cryptococcal cells [90,91]. Themacrophage functions in defense against cryptococcosis by ingesting and

killing yeast cells and by producing proinflammatory cytokines, such as

IL-12. Chemokines, such as MCP-1 and MIP-1a, are important for recruit-

ment of inflammatory cells to sites of cryptococcal infection [92]. Histologic

examination of infected tissue reveals that macrophages are closely associ-

ated with cryptococci [87]. T cells are believed to contribute to host defense

by providing cytokines that activate macrophage fungicidal activity, such as

interferon-c [93], and by promoting the transformation of alveolar macro-phages into giant cells capable of ingesting large encapsulated yeast cells

[94]. Natural killer cells, neutrophils, eosinophils, and certain types of lym-

phocytes have each been reported to mediate direct antifungal effects in vitro

suggesting that they contribute to host defense [46].

There is evidence that humoral immunity contributes to protection

against C. neoformans [95]. Four independent research groups have shown

that passive administration of certain monoclonal antibodies to the polysac-

charide capsule can prolong survival or reduce fungal burden [96–99]. A pol-ysaccharide-tetanus toxoid conjugate vaccine was shown to elicit high titers

of antibody to the capsule that protected against an intravenous infection in


mice [100]. The availability of specific antibody can help the host response

by providing opsonins for efficient phagocytosis, enhancing natural killer

cell function, and clearing capsular polysaccharide [101]. Antibodies to glu-cosylceramide [102] and melanin [103] in the cell wall have been shown to

modify the course of infection suggesting the existence of other targets for

humoral immunity in addition to the polysaccharide capsule. Serologic stud-

ies in patients at risk for infection suggest qualitative and quantitative differ-

ences in the types of immunoglobulins present that may predispose to

disseminated disease [104].

Intracellular pathogenesis

Histologic examination of C. neoformans–infected tissue can reveal

diverse inflammatory responses ranging from the virtual absence of tissue

reaction to intense granulomatous inflammation resembling caseous

necrosis [79]. The budding index of C. neoformans in tissue is inversely pro-

portional to the intensity of the granulomatous reaction which is consistent

with the view that strong cell-mediated tissue responses are required to con-

tain infection [79]. The protean nature of tissue responses to this fungusseems to be a function of both the immunologic status of the host and cer-

tain yet undefined characteristics of C. neoformans. For example, switch var-

iants of a single C. neoformans strain can elicit diverse inflammatory

responses in rats ranging from minimal inflammation to caseous necrosis

[105]. There is evidence that the size of the capsule affects the type of

response. In this regard, infection with small capsule variants often elicits

intense inflammatory responses that have been confused with Histoplasma

capsulatum [106]. This phenomenon is believed to represent interference ofcapsular polysaccharide during infection with these well-encapsulated

strains and the inflammatory response (see later).

The location of C. neoformans vis-a-vis inflammatory cells is also a func-

tion of the inflammatory response. Although the capsular polysaccharide

is antiphagocytic in vitro in conditions where there are no complement or

antibody opsonins, the fungus is rapidly ingested by alveolar macrophages

after experimental infection [107]. Granulomatous responses are associated

with a predominance of intracellular forms, whereas in the absence of in-flammation cryptococci can grow as extracellular fungal masses, which are

grossly visible in tissue and can give a ‘‘soap suds’’ appearance [79]. There is

increasing evidence, however, that C. neoformans can survive in macro-

phages for prolonged periods of time [89]. Studies on the course of C. neo-

formans infection in immunocompetent mice have shown that C. neoformans

can replicate inside alveolar macrophages resulting in host cell lysis [107].

At the initial stages of infection, before the immune response is established,

most of the replication occurs in the intracellular compartment. C. neofor-mans has a unique strategy of intracellular replication in macrophages

whereby capsular polysaccharide is secreted into vesicles resulting in


disruption of the vesicular network, which presumably leads to cyto-

toxicity [108].

From a clinical viewpoint the occurrence of intracellular parasitism in C.

neoformans infection is significant because it suggests that efficient eradica-

tion of infection requires antifungal agents capable of host cell penetration

and that are active in phagolysosomes.

Immunomodulation by C. neoformans and its products

There is considerable evidence that C. neoformans infection is able to

modulate the host immune response through various fungal products. C.

neoformans infection elicits suppressor responses in experimental animals,which presumably down-regulate the cellular immune response to infection

[109,110]. In rats, the maintenance of chronic infection is associated with

down-regulation of both humoral and cellular immune responses [89]. The

best characterized immunomodulator is the capsular polysaccharide, which

has been shown to interfere with a variety of immunologic functions [111].

C. neoformans sheds capsular polysaccharide into tissue during infection,

which presumably results in both local and global inhibition of the immune

response [111]. Locally, shed polysaccharide may interfere with leukocytemigration, cytokine production, and phagocytosis, whereas globally this

antigen is believed to elicit a state of immunologic unresponsiveness such

that affected hosts seldom mount significant antibody responses [112].

In addition to exopolysaccharides, C. neoformans synthesizes three other

products that can affect the immune response: (1) mannitol, (2) melanin, and

(3) prostaglandins. C. neoformans produces mannitol in tissue [113], which

can interfere with oxidative killing by phagocytic cells [114]. C. neoformans

has a laccase that can synthesize melanin from phenolic precursors [115].Melanin production in tissue has been documented during both human and

rodent experimental infection [116,117]. Melanin is believed to contribute to

virulence by protecting the fungal cell against oxidative and nonoxidative

fungicidal mechanisms of host immune effector cells [118]. Melanin may also

act as an immunomodulator by down-regulating the cellular immune

response in the lung [119]. C. neoformans has recently been shown to pro-

duce prostaglandins in vitro that could modulate the immune response if

they are produced in vivo [120].

Clinical manifestations

There are two primary sites of infection with C. neoformans: the lung and

the central nervous system (CNS). In a recent review of non–HIV-infected

patients, there were 109 (36%) with only pulmonary involvement and 157

(51%) with CNS disease and the rest with other or multiple sites [121]. Threeother sites of infection (the skin, prostate, and eye) have particular clini-

cal features that require some specific clinical attention. It should be


emphasized, however, that C. neoformans can infect any organ in the body

[122]. There are also a few differences that distinguish cryptococcosis in

patients with or without HIV infection, such as more CNS and extrapulmo-nary involvement, higher rate of positive India ink examinations, positive

blood cultures, and fewer cerebrospinal fluid (CSF) inflammatory cells. This

is primarily a distinction of severity of immunosuppression and burden of

yeast and not a specific feature or tropism between HIV and the fungus.

Lung

This organ is the most common portal of entry for infection and symp-toms range from asymptomatic colonization [123] to life-threatening pneu-

monias [124]. Although at least a third of normal hosts are asymptomatic

and infection is found by an abnormal chest radiograph, patients may

present with evidence of acute infection, such as fever, chest pain, cough,

weight loss, and sputum production [125,126]. There are also a series of

unusual presentations of pulmonary cryptococcus, such as ‘‘allergic’’ cryp-

tococcal pneumonia [127], pseudo-Pancoast’s tumor [128], bronchiolitis

obliterans with organizing pneumonia [129], and superior vena cava syn-drome [130,131]. It is also possible to have simultaneous infection with

other pathogens, such as tuberculosis, Nocardia, and Echinococcus [132–135].

The chest radiographs in apparently normal hosts range from well-defined,

noncalcified, single or multiple lung nodules; indistinct mass-like infiltrates;

hilar lymphadenopathy; pleural effusions; and lung cavitation [136]. There is

no characteristic radiograph, although single or multiple nodules are most

common and may radiographically mimic a malignant tumor. If the infec-

tion is limited to the lung, the serum cryptococcal antigen is generally neg-ative and in the authors’ opinion a positive cryptococcal serum antigen is a

sign that the yeast may have disseminated from the lung to other body sites.

Patients with C. neoformans isolated from the lung and with an underlying

immunosuppressive risk factor for invasive infection should probably be

considered for lumbar puncture to rule out simultaneous CNS infection

with or without symptoms. In the apparently normal, asymptomatic patient

with C. neoformans isolated from the lung, however, the yield of positive

findings with lumbar puncture is so low that it does not need to be per-formed routinely.

In the severely immunocompromised host, cryptococcal pneumonia can

progress more rapidly; the ability to disseminate outside the primary lung

focus to the CNS allows patients to present with a meningeal rather than

a pulmonary syndrome [137]. Patients can, however, develop an over

whelming cryptococcal pneumonia with features of adult respiratory distress

syndrome [138–140] without CNS involvement. Unlike immunocom-

petent patients, most immunosuppressed patients have constitutionalsymptoms, such as fever, malaise, chest pain, cough, dyspnea, weight

loss, or headaches. Chest radiographs are similar in spectrum to


nonimmunocompromised hosts except that alveolar and interstitial infil-

trates may be more common and can potentially be confused with other

pathogens, such as Pneumocystis carinii. In AIDS patients, cryptococcalpneumonia is primarily symptomatic and over 90% already have concomi-

tant CNS infection at clinical presentation [124]. This observation reflects

the extreme nature of the immunodeficiency in these patients at the time

of cryptococcosis in which CD4 counts have generally fallen under 100

cells/lL. It should also be noted that all severely immunocompromised

patients with pulmonary cryptococcosis may present with other concomi-

tant opportunistic pulmonary and disseminated infections, such as

typical or atypical mycobacteria, cytomegalovirus, Nocardia, and P. carinii

infections.

Central nervous system

Most patients with cryptococcal meningitis present with signs and symp-

toms of subacute meningitis or meningoencephalitis, such as headaches,

fever, lethargy, coma, or memory loss over 2 to 4 weeks. Patients may not

have classic symptoms, however, and present with acute (several days)symptoms of severe headaches, intermittent headaches, or no headaches

with altered mental status. It is difficult to separate AIDS patients from

others in their presentation except to point out that symptoms may be

shorter; there is a greater chance of finding a secondary site of infection; a

greater likelihood of having a second CNS event with either infections, such

as Toxoplasma gondii, or lymphoma; and a higher burden of yeasts. A recent

new cryptococcal syndrome associated with HAART and partial immune

reconstitution has been described during HIV infection. Several patientsafter starting or changing HAART developed acute symptoms of cryp-

tococcal meningitis or pain and swelling in tissues [141]. It is hypothesized

that with an improved immune system produced by HAART, a silent or

latent cryptococcal infection was then clinically made apparent as an inflam-

matory reaction was mobilized to the clinically silent yeast cells or polysac-

charide antigen [141].

There are little data that relate the severity of CNS disease to the infecting

strain of C. neoformans and it is generally considered that the state ofhost defenses primarily determines clinical manifestations. There are some

suggestions, however, that certain strains can influence clinical presen-

tation. For instance, in Australia, which attends to infections with both

C. neoformans var. neoformans-grubii and C. neoformans var. gattii, cerebral

cryptococcomas or hydrocephalus with pulmonary mass lesions in immuno-

competent host, were generally found with the variety gattii infections [142].

In general, infection with this variety has longer survival rates than those

with variety neoformans-grubii. In a subgroup of patients with variety gattii

infection, however, there are more brain-enhancing lesions by scan, compli-

cations of hydrocephalus, cranial nerve palsies, and elevated intracranial


pressures, and a poor prognosis is noted [142,143]. Although these findings

may relate to the distribution of infection in normal hosts and their degree

of immune suppression [9], it is possible that there may be an intrinsic path-obiologic varietal difference, particularly in the propensity to invade the

brain parenchyma.

Skin

Cryptococcus neoformans has been reported to produce almost any

type of human skin lesion [144–147]. Skin manifestations include acneiform

lesions, purpura, papules, vesicles, nodules, tumors, abscesses, ulcers,

superficial granulomas, plaques, sinus tracts, and herpetiformis- [148] and

molluscum contagiosum–like lesions [149,150]. Even cellulitis around an

intravenous catheter, which resembles a bacterial infection, may be caused

by C. neoformans in a high-risk patient [151,152]. The variety of skin presen-

tations make it essential that a biopsy with culture is obtained and histopa-thology is performed on any new skin lesions in immunocompromised hosts.

These skin lesions can be a sentinel finding for disseminated cryptococcosis.

In fact, except for occasional clinical, laboratory accidents [153,154] and

direct trauma, cutaneous cryptococcosis generally represents disseminated

disease and it may occur simultaneously on the skin with other systemic

mycoses in severely immunocompromised hosts [155]. A recent observation

in a cohort of solid organ transplant recipients suggests that the type of pri-

mary immunosuppressive agent used for rejection of transplanted organscan influence clinical manifestations of cryptococcosis. In this report, trans-

plant patients with cryptococcosis receiving tacrolimus were significantly

less likely to develop CNS disease and more likely to have skin, soft tissue,

and osteoarticular involvement compared with patients receiving a nonta-

crolimus-based immunosuppressive regimen [43]. Tacrolimus has anticryp-

tococcal activity at high temperatures (37� to 39�C) but loses activity as the

environmental temperatures decrease [156]. This observation might explain

the more frequent skin involvement with its lower body temperatures.

Prostate

The prostate gland has been recognized as a potential clinical site forsanctuary of this yeast from antifungal treatment [157]. In fact, a silent C.

neoformans infection has been spread into the blood during urologic surgery

on the prostate [158]. It has been shown during careful follow-up of patients

with cryptococcosis and HIV infection that cultures of urine with or without

prostatic massage and seminal fluid were frequently positive at the end of

initial therapy [159,160]. This site may represent a major reservoir for

relapse of infection in all patients, although this site is less well-studied in

non-AIDS patients with cryptococcosis. Despite reports of a penile ulcer[161] and a vulvar lesion with C. neoformans [162], there have been no

reports of conjugal spread of this yeast.


Eye

Before the HIV pandemic, ocular signs and symptoms could be found in

45% of patients with cryptococcal meningitis [163]. Manifestations canrange from ocular palsies to retinal involvement. Ocular infections can occur

simultaneously with other pathogens, such as HIV or cytomegalovirus [164–

166]. In one fourth of cases, eye involvement may occur before the diagnosis

of meningoencephalitis [165]. Ocular cryptococcosis may lead to visual loss.

In fact, most cases of cryptococcal endophthalmitis lead to blindness with

only an occasional case successfully managed [167]. There have also been

reports of catastrophic loss of vision in patients without evidence of endoph-

thalmitis [168,169]. Two pathogenic processes have been hypothesized.First, rapid visual loss associated with optic neuritis caused by infiltration

of the optic nerve with yeasts. There are few therapeutic options for success-

ful therapy of this form of visual loss. On the other hand, a second group of

patients presents with slower visual loss and later in therapy or during the

follow-up period. In this group, symptoms may be related to increased intra-

cranial pressure and treatment with ventricular shunts or optic nerve fenes-

trations may halt progression of visual loss. Most eye infections are from an

endogenous source, such as the CNS and blood, but occasionally the yeastcan be introduced directly into the eye from outside sources, including

trauma and transplantation of human tissue [44,170].

Other Body Sites

Cryptococcus neoformans has been described to infect most areas of the

body. The previous five sites and their clinical manifestations are the mostcommon. Cryptococcemia, bone and joint, and cryptococcal peritonitis,

however, have been reported enough not to be considered rare infections.

Cryptococcemia occurs in advanced HIV infection or during periods of high

amounts of immunosuppression from either the underlying disease or drug

treatments, such as corticosteroids. It generally reflects a high burden of

yeasts in tissue but it can occur without an apparent secondary site of

involvement. Cryptococcemia rarely causes vascular instability (ie, shock

or endocarditis) but these conditions have been reported. Bone and jointinvolvement is an occasional extraneural site. Before the HIV epidemic, it

had been reported that up to 5% of disseminated cases of cryptococcosis

showed bone and joint involvement [171]. Osteoarticular cryptococcosis can

occur in both immunocompromised and nonimmunocompromised hosts

and the only clinical link with infection is the suggestion that sarcoidosis

is a common underlying disease with this site of infection. In AIDS patients,

it might be found in a random bone marrow biopsy without any radio-

graphic evidence of disease. Cryptococcal peritonitis is an occasional clinicaloccurrence and generally presents in two groups of patients: patients on

chronic ambulatory peritoneal dialysis; and patients with severe underlying


illnesses, such as cirrhosis [172]. The following body sites are considered rare

manifestations of cryptococcosis because less than a dozen cases per site

have been presented in the literature: genitourinary tract (pyelonephritis andgenital lesions); muscle (myositis); heart (native and prosthetic endocarditis,

mycotic aortitis or aneurysm, myocarditis, pericarditis, and vascular foreign

body); thyroid (thyroiditis and mass); adrenal gland (adrenal insufficiency

and excess); head and neck (gingivitis, sinusitis, salivary gland involvement,

larynx, and neck mass); gastrointestinal (esophagitis, biliary tract, enteritis,

and hepatitis); breast (mastitis and mass); and lymph node (lymphadenop-

athy) [46].

Laboratory diagnosis

Direct microscopic examination

Clinical specimens from the CSF to aspirates of cutaneous lesions can be

examined by India ink examination for the 5 to 10 lm in diameter yeast cells

with capsules. Approximately 80% of AIDS patients and 50% of non-AIDS

patients with cryptococcal meningitis reveal the yeast with this simple and

immediate test. For a routine positive India ink test from CSF, it is esti-mated that the specimen needs to contain greater than 103 CFU/mL of CSF

(personal observations). Although calcofluor white is not specific for C. neo-

formans, this solution can be used on tissue aspirates with a fluorescent

microscope to find these yeasts quickly when they are in a reduced number

within a specimen. In tissue sections, the encapsulated yeasts may be sur-

rounded by empty spaces because the capsule does not routinely stain with

many of the standard histologic stains, such as hematoxylin and eosin. The

appearance of the polysaccharide capsule can be identified with specificstains, such as mucicarmine and alcian blue. The Gomori methenamine sil-

ver stain for fungi identifies C. neoformans as a yeast but it is not specific for

this yeast and the Fontana-Masson stain, which identifies melanin, selec-

tively stains these yeasts in tissue. Most clinical isolates in both tissue and

fluids have evidence of a capsule, although occasionally there are reported

hypocapsular strains in which the capsule is difficult to observe histologi-

cally [173].

Cultures

Cryptococcus neoformans can be isolated on most routine mycologic or

bacteriologic media. Standard blood culture methods, including radiometric

methods, now routinely detect cryptococcemia. In fact, one study using a

lysis-centrifugation procedure showed over 70% sensitivity of detecting

cryptococcemia in AIDS patients with cryptococcosis [174]. Most C. neofor-mans isolates can be detected in culture between 3 and 7 days after the speci-

men has been placed into or on media.


All Cryptococcus species are nonfermentative and produce extracellular

starch and urease. C. neoformans is unique among Cryptococcal species in

its ability routinely to grow at 37�C. It also produces a phenoloxidase. Thesefeatures have allowed for the rapid identification of C. neoformans by the

use of a rapid urease test or the use of Staib’s birdseed agar, DOPA, or caf-

feic acid media to detect the brown or black colonies as the phenoloxidase

breaks the diphenolic compounds in the media down into melanin [175–

177]. Isolates can also be identified by several commercial systems based

on biochemical reactions [178,179] or the use of DNA-based methods

[180,181].

The two varieties of C. neoformans can be differentiated by a color reac-tion when grown on concanavine-glycine thymol blue agar [182] and a sero-

type commercial kit using monoclonal antibodies is available [183,184].

Finally, specific strains can be fingerprinted or biotyped using DNA-based

or enzymatic methods, respectively [185–188], and these DNA methods can

identify specific varietal genotypes.

Serology

The serologic test for the detection of cryptococcal polysaccharide and

the diagnosis of invasive cryptococcosis is both specific and sensitive

(�90%). The most commonly used method of antigen detection is the latex

agglutination test, but the enzyme immunoassay is occasionally used in

some medical centers. A positive test at titers of greater than or equal to

1:4 in a biologic fluid strongly suggests infection. In fact, every positive test,

no matter what the titer, should be considered carefully within the clinical

context of the patient. Many kits can now detect as little as 10 ng/mL of poly-saccharide in specimens. With proper treatment of specimens, such as elim-

ination of rheumatoid factor by boiling and pronase or 2-mercaptoethanol

treatment, there are few false-positives. One rare false-positive occurs when

there is a cross-reactive antigen, such as the polysaccharide of Trichosporon

beigelii, in the specimen or another microorganism [189–191]. False-negative

results, however, are also rare. These false-negative tests may be caused by

low titers, early infection, presence of immune complexes, prozone effect of

high titers, or poorly encapsulated strains with low production of polysac-charide. The antigen detection system has its most clinical experience with

CSF and serum specimens, but it has been used to detect polysaccharide

antigen in urine or bronchoalveolar lavage fluid [192].

In diagnosis, cryptococcal antigen tests should be part of the standard

analysis on CSF specimens in immunocompromised hosts with meningitis.

They may also be used to screen serum in febrile, very high-risk patients,

such as those with HIV infection in areas where there is a known high rate

of cryptococcal infection [193]. The detection of cryptococcal polysacchar-ide is the leading example of a useful test for serologic diagnosis of an inva-

sive fungal infection and it may even be positive before detection of the


organism by culture. Despite its excellence as a diagnostic test, it is more dif-

ficult to use as a treatment barometer except as a general prognostic feature.

High titers (�1:1024) generally reflect high burden of yeasts, a poor hostimmune response, and a greater likelihood of therapeutic failure. Crypto-

coccal antigen titers have been less useful to make specific clinical treatment

decisions. For instance, the use of serial antigen titers to develop a treatment

algorithm remains imprecise and not validated by clinical trials.

Antibodies to C. neoformans may not be detected or are at low levels dur-

ing active infection because many patients are severely immunosuppressed,

but they may rise during clinical recovery. They are not useful, however, for

diagnosis or treatment decisions.

Radiology

Chest radiograph

The chest radiograph of pulmonary cryptococcosis in the immunocompe-

tent host can show a variety of features including nodules, infiltrates, hilar

lymphadenopathy, or pleural effusions [136,194–198]. In AIDS patients the

radiograph may reveal either diffuse or focal interstitial infiltrates with orwithout lymphadenopathy [124,199,200]. This radiograph may be confused

with P. carinii infection or represent co-infection with it or another

pathogen.

CT and MRI

CT and MRI are frequently used in the diagnosis and management of

cryptococcal meningitis. CT scan findings in non-AIDS patients with men-

ingitis can reveal hydrocephalus; gyral enhancement; or multiple nodules,which may be enhancing or nonenhancing [201]. Cryptococcomas can be

either single or multiple and occur in up to 25% of patients [202,203].

Approximately one half of CT scans, however, are normal in cryptococcal

meningitis. In patients with HIV infection, the scans are similar to non-

AIDS patients except approximately one third of patients have cortical atro-

phy from their underlying HIV infection [204].

The MRIs are more sensitive than CT scan for detecting abnormalities.

MRI findings include numerous clustered foci that are hyperintense onT2-weighted images and nonenhancing on postcontrast T1-weighted images

within basal ganglia and mid-brain, which represent Virchow-Robin spaces.

There may also be the multiple miliary enhancing parenchymal and lepto-

meningeal nodules with gadopentetate dimeglumine [205].

It should be emphasized that there is no pathognomonic radiographic

picture for cryptococcal meningitis. It may present simply as an idiopathic

hydrocephalus identified by CT or MRI [206]. In AIDS patients, however,

a scan may identify a parenchymal lesion, which represents a second CNSdisease process, such as toxoplasmosis or lymphoma. Finally, the use of

CT or MRI in follow-up of cryptococcal meningitis may show worsening


(enlargement or new lesions) or persistence of the cryptococcomas [207]

while the patient continues to improve clinically or is stable. This finding

may simply represent enhancement by inflammatory cells within granulo-mas as microscopic infectious foci are being eliminated; it is not necessarily

a criteria for treatment failure.

Management

General

The management of cryptococcosis has been extremely well-studied and

although all aspects of treatment are not consistently successful or uni-

formly agreed on, there is substantial evidence-based data to make recom-

mendations. A group of experts have attempted to collate the therapeutic

evidence into a series of guidelines for therapy of cryptococcosis [208]; these

guidelines are excellent starting points for therapeutic decisions, but this dis-

cussion adds the authors’ own insights and opinions. The breadth of deci-

sions to be made in cases of cryptococcosis can be quite diverse. Forinstance, it is clear that some individuals with asymptomatic isolation of

C. neoformans from respiratory secretions have been untreated without clin-

ical compromise. A recent review of this issue suggested that approximately

20% of patients with positive pulmonary cultures received no treatment

[121]. Cryptococcal meningitis, however, is uniformly fatal without antifun-

gal treatment. Before availability of antifungal agents, there were cases that

survived for years with this chronic CNS condition before succumbing. The

rapidity of progression to death for the natural history of this infection withsevere immune suppression, however, such as HIV infection, is illustrated

in the comprehensive report on meningitis in HIV-infected patients in

Zimbabwe [72]. Of 406 patients, 45% had cryptococcal meningitis and

almost 40% of those with this infection died during the initial hospitalization

without treatment. Therapeutic decisions do have a wide range of options

and consequences in the management of cryptococcosis.

In vitro susceptibility testing

Methods for testing C. neoformans isolates in vitro for minimum

inhibitory concentrations (MICs) have been standardized and modified for

media, inoculum, and end point determinations [209,210]. Most initial

isolates of C. neoformans have lowMICs to amphotericin B, flucytosine, and

azoles [211,212]. Initial resistance to flucytosine is low [213] but rising MICs

occur during therapy with this agent and correlate with clinical relapse

[137,214,215]. There has been identified, however, only an occasional

C. neoformans isolate resistant to in vitro amphotericin B [216], andmost relapse isolates remain fully susceptible to this polyene. During the

HIV epidemic and before HAART the widespread use of azoles in severely


immunocompromised host showed an increase in lumenal infections with

azole-resistant Candida albicans. Although there have been some apparently

azole-resistant C. neoformans strains isolated from patients, azole resistanceprobably runs less than 5% of initial isolates [217]. In fact, when secondary

isolates are examined, it seems through molecular typing methods that these

are relapse isolates rather than reinfection [218] and most have a similar (sus-

ceptible) MIC as the primary isolates [219]. There are cases of increasing flu-

conazole MICs during treatment [220], however, and present animal data

suggest a correlation between in vitro susceptibility testing and in vivo out-

come [221]. It is not certain what the precise breakpoint is for fluconazole sus-

ceptibility in cryptococcosis treatment. MICs, however, of greater than orequal to 16 lg/mL from both pharmacodynamic studies in vitro [222] and

some experience with isolates from relapsed patients with prior azole expo-

sure [223] should make the clinician consider very high dosing of drug or the

possibility of using another agent. It is possible that more azole-resistant C.

neoformans with its heteroresistant phenotypes [224] may be observed in the

future. At this time with the low incidence of primary azole-resistant isolates

it is probably not necessary to check MICs before treatment if patients had

not received prior azole therapy. All initial isolates should be saved and com-pared with relapse isolates, however, to determine if drug resistance is a pos-

sible factor for treatment failure. A general overview of in vitro and in vivo

reports suggests that there is a positive interaction against C. neoformans for

the following drug combinations: amphotericin B and flucytosine; amphoter-

icin B and azole; amphotericin B and rifampin; flucytosine and azole; and tri-

ple combination of amphotericin B, flucytosine, and azole [225–227].

Treatment strategies

It should be emphasized that success rates from any study depend pri-

marily on study definitions and the patient’s prognostic factors. These fac-

tors always need to be taken into account when reviewing the published

data and treating the individual patient.

Amphotericin B therapy converted cryptococcal meningitis from a uni-

formly fatal infection to one that is curable. Amphotericin B successes

before the AIDS epidemic were reported at 60% to 70% [146,228]. A 10-week course of amphotericin B at 0.4 mg/kg/d for 10 weeks recorded a suc-

cess rate of 68% in non-AIDS patients [228]. In AIDS patients at 0.5 mg/kg/

d of amphotericin B, the success rate dropped to 40% [229]. Recent studies

have suggested that a higher daily dose of amphotericin B may be more

effective [230,231] and this strategy has been more commonly adopted. Fur-

thermore, amphotericin B can now be given in higher doses with reduced

toxicity in lipid preparations if toxicities develop with standard amphoteri-

cin B. Ambisome at 4 mg/kg/d has the most clinical experience [3] andincludes a comparative trial in which it performed similar to amphotericin

B [232]. It is probably the best alternative polyene preparation for treatment


after standard amphotericin B deoxycholate, although ABLC has shown

some treatment success in cryptococcal meningitis [233].

Flucytosine was initially used alone in the treatment of cryptococcal men-ingitis [214,234], but development of resistance on therapy reduced enthusi-

asm for monotherapy. Several studies used the combination of amphotericin

B (0.3 mg/kg/d) and flucytosine (150 mg/kg/d) to reduce the course of treat-

ment to 6 weeks and the toxicity of amphotericin B [228,235,236]. In some

cases, with good prognostic signs there was success with 4 weeks of treat-

ment [236]. Furthermore, in a small number of AIDS patients, the combina-

tion therapy was found to be superior to fluconazole [237] and itraconazole

alone [238]. The combination regimen reproducibly sterilizes CSF at 2 weeksin most patients including those with AIDS and there are several studies that

support less relapses with an amphotericin B plus flucytosine treatment reg-

imen [239,240]. This led to a definitive study using higher doses of ampho-

tericin B (0.7 mg/kg/d) and lower doses of flucytosine (100 mg/kg/d) for a

2-week induction period of treatment and then switching to fluconazole

(400 mg/d) alone for 8 weeks and completion of initial therapy [241]. This

strategy has become a primary modality in AIDS patients and frequently

chosen now for non-AIDS patients with cryptococcal meningitis [121] andin the authors’ opinion represents the initial regimen of choice for treatment

of cryptococcal meningitis.

Fluconazole has been studied in cryptococcal meningitis because of its

excellent pharmacokinetics within the CNS. In a comparative trial with

amphotericin B in AIDS patients, the success rate was similar to amphoter-

icin B alone but sterilization of the CSF took longer with the azole therapy

[229]. Fluconazole may have its most difficult time in controlling infection

when the burden of yeasts is high and infection is well-established[242,243]. In initial therapy of cryptococcal meningitis it may be less useful.

Fluconazole has also been used as salvage therapy with higher doses [244].

Furthermore, it has been used successfully with flucytosine in treatment of

an animal model [245] and humans [246]. It has also been used in successful

suppressive therapy for AIDS patients [247,248] and may be useful for pro-

longed initial therapy in non-AIDS patients. Although optimal dosing for

initial stages of meningitis treatment is not precise, doses of 400 to 800

mg/d are likely to give the best results.Itraconazole, despite its poor penetration into CSF, has been used in the

successful management of cryptococcal meningitis [249–251]. Direct com-

parison for initial treatment of cryptococcal meningitis with fluconazole has

not been made. For suppressive therapy in AIDS patients, however, fluco-

nazole has been shown to have a distinct advantage over itraconazole [239].

Miconazole has rarely been used in cryptococcal infection and with

mixed results [252–254]. Ketoconazole has not been successful in treatment

of cryptococcal meningitis [255], but it is likely to be effective in non-CNSinfections [256]. Voriconazole and posaconazole have excellent in vitro

activity against C. neoformans and animal studies support their potential


in this infection [257,258]. Present trials for these drugs in cryptococcosis

have generally used them as therapy for patients who failed standard regi-

mens. Successes in this unique population of patients run from 30% to70% [259,260]. At present, the new b-glucan synthase inhibitors, such as cas-

pofungin, micafungin, and anidulafungin, do not have clinical activity for

treatment of C. neoformans. There are a series of concepts in treatment of

cryptococcosis that require further explanation.

Site of infection

In pulmonary cryptococcosis, there are few comparative studies to deter-

mine which drugs are the best when treatment is indicated. Azoles, however,

such as fluconazole, at 200 to 400 mg/d for 3 to 6 months, are safe and easy

to administer and generally form the primary treatment strategy [261,262],

unless the patient is severely ill, in which case amphotericin B should beused. In the authors’ opinion, it is clear that both symptomatic and asymp-

tomatic immunosuppressed patients with C. neoformans isolated from lung

must be treated because dissemination from this site is a distinct possibility

[137]. In fact, the authors recommend that most patients irrespective of

immune status should probably be treated when C. neoformans is isolated

from nonsterile respiratory secretions with or without symptoms. Most CNS

cryptococcomas can be treated and although length of therapy is not

defined, the authors treat patients sometimes with 2 years of fluconazole.As previously mentioned, serial scans may not be helpful and complete res-

olution radiographically may take years. Surgery has been recommended for

large CNS lesions (>3 cm) [263], but in the authors’ opinion its use needs to

be individualized, with it rarely needed with present medical management.

Immune status of host

Every attempt to improve immunity of the host during treatment needs to

be made. This may mean reducing corticosteroid treatment and it is recom-

mended that a dose of prednisone less than or equal to 20 mg/d be achieved

during therapy. More studies on cytokines as adjunct therapy are needed(see later). The published results of these studies are awaited to attempt to

put cytokine treatment in proper perspective. Unfortunately, in AIDS

patients who generally develop C. neoformans with less than 100 CD4

cells/lL, immune augmentation had been difficult until the age of HAART

and continuous azole suppressive therapy had been required to prevent a

high rate of relapse (50% to 60%) if antifungals were stopped, but this strat-

egy may be changing (see later). In HIV-infected patients with cryptococcal

meningitis, it may be reasonable first to treat the fungal infection aggres-sively and attempt to sterilize CSF with antifungal drugs before starting

HAART to avoid a rapid inflammatory process return and development


of new symptoms. HAART can probably be started at 2 to 3 months after

initiation of specific anticryptococcal therapy.

Suppressive therapy

Relapse rates in AIDS patients with cryptococcal meningitis before

HAART approached 50% to 60% when antifungal therapy was stopped.

Studies showed that daily fluconazole can reduce relapse rates to less than5% [248,264]. Fluconazole has been shown to be more effective than weekly

intravenous amphotericin B dosing [248] and itraconazole [239] and it has

become standard clinical practice to prescribe indefinite fluconazole sup-

pressive therapy in AIDS patients. Recent data in several small studies,

however, suggest that with HAART and its immune reconstitution (rising

CD4 counts and lowering HIV loads), antifungal suppressive therapy can

be stopped at 1 year with little chance for relapse in the next year [265–

267]. A large study of stopping the suppressive azole regimen is beginningin Europe and it is hoped this study will bring more certainty to this strat-

egy. There are no guidelines for suppressive therapy in non-AIDS patients

with cryptococcal meningitis who can relapse in the first year at a 10% to

15% rate with standard regimens. With the use of safe and oral azoles, a

common practice has been to extend therapy with fluconazole at 200 mg/d

for 6 months to a year to cover this highest relapse period.

Role of intracranial pressure

Patients with high burden of yeasts and antigen can develop increased

intracranial pressure both before treatment, during early treatment, and

with the chronic phase of treatment and monitoring. With high intracranialpressures during early management of infection, these patients develop

symptoms, such as systemic hypertension, decreased sensorium, cranial neu-

ropathies, visual loss, or increased headaches. It has been postulated that the

elevated subarachnoid pressures are caused by reduced CSF resorption

because of increased outflow resistance possibly from yeasts and polysac-

charide plugging the arachnoid villi and the antigen contributing to brain

edema [268]. Clinical response to therapies is better in those patients with

pretreatment CSF opening pressures less than or equal to 250 mm H2Oor those with stable or decreased (>10 mm H2O) lumbar CSF pressures

at 2 weeks [269]. Control of increased intracranial pressures with techniques

of external drainage, such as repeated lumbar punctures or lumbar drains,

may be necessary during the early treatment phase. Unfortunately, the gen-

eral preciseness of methods and goals remains elusive and generally is based

on individual patient responses. In contrast to early treatment pressure

problems, ventricular shunts are successful for long-term pressure control

in those with classic obstructive hydrocephalus, which may occur monthsafter start of treatment and must be watched for during follow-up visits.

A shunt can be placed successfully while cryptococcal meningitis is being


effectively treated. In cases of cryptococcal meningitis in which the shunt has

already been in place for treatment of hydrocephalus before anticryptococ-

cal treatment, however, it needs to be removed for cure [206]. The patientcan then be started on adequate treatment and when CSF is sterile another

shunt can be placed if clinically indicated.

Antigen titers

Cerebrospinal fluid polysaccharide antigen titers generally reflect the bur-

den of organisms at the initial diagnosis and high antigen titers predict apoor prognosis. The use of specific antigen titers to predict individual out-

come and direct therapy, however, is generally not helpful [270]. Polysac-

charide antigen may persist for long periods of time despite adequate

therapy. Changing or fixed titers are not reliable indicators of treatment out-

come in individual patients. During the AIDS epidemic, isolated cases of

serum cryptococcal polysaccharidemia in HIV-infected patients with nega-

tive cultures have occurred [271]. Management of these cases is difficult but

empiric therapy may be wise in these high-risk patients because many even-tually develop cryptococcosis.

Cytokines and monoclonal antibodies

Disseminated cryptococcosis generally occurs during an episode of

immunosuppression. There are in vitro studies and animal models that indi-cate that biologic response modifiers or antibodies can have a positive effect

on outcome of cryptococcosis (see host factors). Granulocyte-macrophage

colony–stimulating factor, macrophage colony–stimulating factor, IL-2,

IL-12, and interferon-c can positively interact with host cells to inhibit or

kill C. neoformans. Granulocyte-macrophage colony–stimulating factor has

been used in an open trial [272] and results of an adjunct trial of interferon-

c, which was supported by encouraging results in animals, are awaited [273].

It is not yet clear whether these cytokines help or exactly how to optimizetheir use. Specific monoclonal antibodies and serum therapy have a positive

effect on the prevention and treatment of experimental cryptococcosis [274–

276]. A monoclonal antibody is now being studied in early phase I trials in

HIV-infected patients with stable cryptococcal disease but persistent poly-

saccharide antigenemia. There remain little definitive human clinical trials,

however, into immune modulation therapy. It is clear that future studies

with these biologic enhancers are needed as further strategies are developed

for management in an infection, which can still have 10% to 25% acute mor-tality rates during early treatment in medically advanced countries.

Prognosis

The most important prognostic factor for the success and treatment

of cryptococcal meningitis is the ability to control the patient’s underlying


disease. It has been shown that cancer victims have a shorter survival than

AIDS patients because of their underlying disease state [277]. Another major

risk group is solid organ transplant recipients and recent data and prognosisin this group are conflicting. In one study the prognosis of cryptococcosis in

solid organ transplant was similar to patients without an underlying disease

[121] but in a second study there was a death rate of 42% [43]. More studies

are needed in this area. Two other prognostic findings are burden of organ-

isms at presentation and the level of patient’s sensorium. A poor prognosis is

found with a heavily positive India ink examination; high polysaccharide

antigen titers (�1:1024); and a poor inflammatory response in the CSF

(<20 cells/ll). Patients on presentation who have a lucid sensorium have abetter prognosis than those stuporous or in coma [236].

Specific factors have predicted outcome and generally relate to the three

factors previously described. On amphotericin B therapy, Diamond and

Bennett [278] found more than three decades ago that patients who died

during therapy were more likely to have: (1) an initial positive India ink

examination, (2) high CSF opening pressure, (3) low CSF glucose, (4) low

CSF leukocytes, (5) cryptococci isolated from extraneural site, (6) absence of

anticryptococcal antibody, (7) initial CSF or serum antigen titer of greaterthan 32, and (8) corticosteroid therapy or lymphoreticular malignancy.

Patients who relapsed after treatment were characterized by: (1) abnormal

CSF glucose for greater than or equal to 4 weeks of therapy, (2) low initial

CSF leukocytes, (3) cryptococci isolated from extraneural sites, (4) absence

of anticryptococcal antibody, (5) posttreatment CSF or serum cryptococcal

antigen titer greater than or equal to 8, (6) no significant decrease in CSF

and serum antigen titer during therapy, and (7) daily dose of corticosteroid

therapy equivalent to 20 mg of prednisone or more after therapy. Theseinsights may still help to predict failures today. Recent studies with ampho-

tericin B and flucytosine treatment in non-AIDS patients indicated a better

prognosis if there was a normal mental status, headache on presentation,

and a CSF leukocyte count greater than 20 WBCs/lL [236]. In AIDS

patients during treatment with amphotericin B or fluconazole, important

positive pretreatment predictors for death during treatment were abnormal

mental status, a CSF antigen titer greater than 1:1024, and CSF leukocyte

count less than 20 WBC/lL [241]. Identification of these high-risk patientsfor failure and relapse should allow the clinician to design specific antifungal

regimens for these subsets of patients.

Prevention

There are at least four methods for approaching high-risk patients to pre-

vent cryptococcosis. First, antifungal prophylaxis for AIDS patients has

been effective in reducing the incidence of cryptococcosis with the use of flu-conazole [67,279]. Both HAART and concerns about widespread exposure

to azoles worked together, however, to reduce enthusiasm for its general


use. Second, active immunization with a vaccine for high-risk patients is

possible. A cryptococcal GXM-tetanus toxoid conjugate vaccine has been

developed which elicits protective antibodies in mice [280] and an initialparadigm has been completed. It or other vaccine candidates, however,

await clinical trials in humans. Third, the ability to produce protective

humanized or murine monoclonal antibodies could be given to high-risk

patients as passive prophylactic therapy, but protection requires repeated

continuous administration over the risk period. Finally, high-risk patients

can attempt to avoid high-risk environments, such as bird droppings, which

contain yeasts to be aerosolized and inhaled.

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