Cryptococcus neoformans (Cryptococcosis)

MICROBIOLOGY

Cryptococcus spp.is a basidiomycetous yeast. Polysaccharide capsule and melanin are virulence factors. Two Cryptococcus  species are pathogenic in humans: C. neoformans (serogroups A and D) andC. gattii (serogroups B and C). C. neoformans variety grubii corresponds to serogroup A and C. neoformans variety neoformans is serogoup D (Table 1) (24, 36).  These species are characterized by distinct geographic distribution, host preference clinical presentation and therapeutic recommendations. C. gattii can be divised into four genotypes: VGI, VGII, VGIII, VGIV[63]. VGII genotype can be further divided into three subtypes: VGIIa, VGIIb, VGIIc.

EPIDEMIOLOGY

C. neoformans

C. neoformans  exposition is frequent as 80% of adults have antibodies directed against C. neoformans. However, cryptococcosis was rare before 1980’s (36, 132).   With the emergence of AIDS, the yeast gained increasing medical importance. In 1990’s, 80% of cryptococcosis occurred in patients with AIDS (39).   Thanks to the introduction of highly active antiretroviral therapy (HAART), the rate of cryptococcosis has declined significantly in developed countries in the past few years with an increase of cryptococcosis in HIV-negative patients  (131). However, this infection has emerged as a major cause of mortality among persons who do not have access to HAART in developing countries. One million cases are reported each year in Sub-Saharan Africa, with more than 600 000 deaths/year in HIV-infected patients (123).  

Patients with T-cell mediated immunodeficiency are at greatest risk for cryptococcal infections. Nowadays, HIV represents the major risk factor (more than 50% of cases in the Western world while in Sub-Saharan Africa cryptococcosis is almost exclusively observed in HIV-infected patients). Other predisposing factors such as solid organ transplant are listed in Table 2(73).  Use of corticosteroids or others immunosuppressive drugs are frequently reported in association with others risk factors (122).    Some cases occur in otherwise healthy patients with possible single-gene errors of immunity in the IL-12/IFNγ axis. Furthermore, autoantibody against GM-CSF (C. neoformans and C. gattii) or IFNγ (C. neoformans) have been recently described in patients with cryptococcosis without known risk factors (24, 73).

C. gattii

Phylogenic studies showed a divergence of C. gattii and C. neoformans approximately 50 million years ago (112).   First human cryptococcosis caused by C. gattii was recorded from a Congolese Bantu boy with meningitis in 1970 (48).  Gatti et al. isolated a different strain of Cryptococcus with unique elongated cells, which culture did not differ from typical C. neoformans strain. The first environmental isolation of C. gattii(serotype B) was reported from Eucalyptus trees in 1990 by Ellis and Pfeiffer in Australia, it confirmed an unusually high incidence of C. gattii in Oceania in 1980’s (41).

Since the description of an ecological niche from Eucalyptus trees, the fungus has been isolated in more than 50 species of trees and their hollows (159).  The soil can be contaminated near from trees surroundings. C. gattii can be present in export trees and woody products. Some native and domestic animals are also affected (66).

Distribution of  C. gattii was initially confined to tropical and subtropical regions (Australia, Brazil, southern California, Thailand, Vietnam, Hawaii, Central Africa) with sporadic cases of meningoencephalitis among otherwise healthy individuals (70).  Distribution of each genotype was variable between each region. Then an endemic focus was noted in the arboriginal community in North Australia and in Papua New Guinea since 1990 (45, 149, 150).   However, many laboratories did not differentiate routinely the two species until recently. In 1999, C. gattii emerged in British Columbia and caused an unprecedented outbreak in Vancouver island with more than 200 cases in 9 years (47).  Unlike endemic cases in Australia, infected patients presented with respiratory syndrome and seemed to have underlying conditions associated with immunosuppression. A clonal predominant strain (VGIIa) could be responsible, but reasons for its dissemination remain unclear. Following this outbreak, since 2004, it has been observed an increasing incidence of C. gattii infections in the US Pacific Northwest with more than 60 cases between 2004 and 2010 (20).  In parallel to outbreaks, emergence of cases was reported in Europe, Asia, South-America after travel in endemic areas (50).  Since 2009 in USA, some patients infected with VGI or VGIII genotype have been reported in non-endemic areas without history of travel mainly in Washington and Oregon states (54).  There is now a complex distribution of each genotype among the 5 continents, despite recent outbreaks and travel-related spread (Table 3).

By contrast with C. neoformans, C. gatti can affect apparently healthy individuals. In Australia, 72% to 100% patients had no known risk factors while the others had solid or hematopoietic malignancy, corticosteroid or immunosuppressive therapy, renal transplants or idiopathic CD4 lymphopenia (28). Conversely, in the British Columbia and US Pacific Northwest outbreaks, more than 70% of patients had pre-existing conditions such as lungs disease, smoking and corticosteroid therapy (82).  Other risks factors included renal or cardiac failure, a history of cancer. This link between genotypes and host is not well understood. HIV patients did not seem to be over-represented compared to C. neoformans infections. Furthermore, high level of anti-GM-CSF autoantibodies have been recently described as a risk factor for CNS infection by C.gattii in otherwise immunocompetent patients (143).  C. gattii infections are uncommon in children but are over represented in Brazil possibly related to VGII genotype (144).

CLINICAL MANIFESTATIONS

C. neoformans

Although the lungs are the most frequent portal of entry for C. neoformans, central nervous system involvement is the main clinical manifestation in 90% patients with AIDS and 70% of non-HIV patients  (40).

Meningo-encephalitis

Infection can be asymptomatic or present with asthenia, weight loss, memory lapse. The main presentation is febrile encephalitis with altered mental status and headache. Focal deficit, convulsions and meningism are rare, especially in non HIV patients (122).   Fever is reported in 50 to 70 % of cases. Abnormal mental status at baseline is highly predictive of mortality (141).  Elevated intracranial pressure occurs in 60% to 90% of HIV patients in sub-Saharan Africa (89).  They have more frequently headache, meningismus, hearing loss or papilledema (49).  Several studies thus far have linked increased opening pressure with death rate (137).   CD4 count below 100/mm3 is a major risk factor for cryptococcosis in HIV subgroup. Patients with hematological malignancies have also a higher risk of mortality (40).

Brain MRI is more sensitive than CT scan (abnormal images in 92% vs 53% for AIDS patient with proven meningitis). Related lesions include mass(es), dilated perivascular spaces (Virchow Robin spaces), pseudocysts and basilar meningeal enhancement (3, 22).

Cerebro-spinal fluid (CSF) characteristics typically include elevated cellular counts <200 leucocytes/mm3, median CSF protein <1,2g/L, median CSF:serum glucose ratio <0,5 (40).  A higher leukocyte count or CSF protein level are more frequent in non-HIV and non-transplant patients. On the contrary, a low or even normal cell count could be an unfavourable outcome associated with a high fungal CSF burden. HIV-patients with CSF glucose levels <2mmol/L had also an increased risk of mortality (33).

Pulmonary Involvement

The lung is commonly the portal of entry following pathogen inhalation. Clinical and radiological presentations are non-specific and are more severe in immunocompromised hosts with AIDS. Respiratory symptoms include cough with or without sputum production, dyspnea and pleuritic pain but some patients can quickly developsevere acute respiratory distress syndrome (166, 167).  Incidental finding on radiological imaging can occur up to one third of patients. Fever and hemoptysis are uncommon. In AIDS patients, pulmonary cryptococcosis diagnosis is usually concomitant with extrapulmonary dissemination (14, 86).

CT scan shows mostly solitary nodule (or mass) or multiple nodules. Micronodules are reported, mimicking a pneumocystosis. Air space consolidations are more frequent in non-HIV patients (136). Pleural effusion and thoracic lymphadenopathy rarely appear (86).

Skin involvement

Skin involvement is the third localization. Cutaneous lesions are usually polymorphic. The classical presentation is molluscum contagiosum-like manifestations commonly seen during disseminated disease. However, an isolated primary cutaneous cryptococcosis can occur in patients reporting some risk factors: direct inoculation in a preexisting skin lesion, outdoor activities, living in rural areas, exposition to contaminated source such as avian excreta, wood debris and soil and more frequently ascribed to serotype D at least in Western Europe (110).

Fungemia

It generally precedes the occurrence of meningoencephalitis by dissemination. One positive blood culture should lead to exhaustive work-up for spread of C. neoformans and specific therapy. HIV patients have more frequently blood infection compared to non-HIV patients (46% vs 12% respectively) (40).

Others Involvement

C. neoformans can also affect the genitourinary tract. Relapse may result from reservoir in the prostate (74).  Other rare localizations include lymph-nodes, muscle, bones and joints, heart valve and peritoneum (105).

C. gattii

C. gatii involves mainly the lungs or central nervous system or both. Primary cutaneous infections are also reported (78, 108).  Because of the non-specific symptoms and the rarity of the infection, a delayed diagnosis is frequent (mean 50 days). Incubation period is shorter than C. neoformans, with a median time of 6 months (extremes 2 weeks-2 years) (27).   Obtaining history of travel in endemic or outbreak regions may help clinicians.Summarily, CNS infections are more common in non-outbreak infections, while lung disease predominates in the North American outbreak with at least one genotype. Both CNS and pulmonary involvement are reported in Australia (80% of cases). Others involvement such as skin, bones, joints, lymph nodes disease rarely occur.

Pulmonary involvement

In US Outbreaks, 75% case-patients had respiratory syndrome including cough, dyspnea, and chest pain while 20-50% case-patients had CNS disease (20).  Fever, chills are uncommon (<50%). Hemoptysis is reported. Some patients are asymptomatic. Chest imaging (X-ray, CT-scan) often shows large unique or multiples mass lesions called « cryptococcomas » (1 to 10 cm diameter). They are found mainly in immunocompetent patients. Pulmonary infiltrates (alveolar, interstitial) are reported in immunocompromised patients.

Central nervous system involvement

Symptoms are non-specific. The diagnosis delay from the onset of CNS symptoms was 45 days (interquartile range 21 to 120 days) for endemic cases in Australia. Headache and neck stiffness are common. Fever, chills occur sometimes (<50%)  (28).  Neurological presentation can include cranial nerve palsy (most VI and VII nerve), vision loss, seizures, focal signs, impaired consciousness with coma (28). Measure of opening pressure should be practised in every case when a lumbar puncture was performed. Increased intracranial pressure (>20cm H20) often occur (>50% of cases) (28).  Hydrocephalus is more common than in C. neoformans infection (30% of cases in Australia). Ophtalmoscopy can show papilledema and haemorrhages which are responsible for visual loss.  CT-scan and MRI of the brain can show cryptococcomas near from the basal ganglia, hydrocephalus, basilar meningeal enhancement. The cryptococcoma lesion is similar like an abscess. MRI is more sensitive to diagnose small lesions and detect dilatations of perivascular Virchow-Robin space, which are typical of cryptococcal infections. Despite lower mortality than C. neoformans infections, C. gattii is associated with more neurological sequelea such as loss of visual acuity, cranial nerve palsy, memory impairment or epilepsy (28, 104).

LABORATORY DIAGNOSIS

The diagnosis of cryptococcal infection can be done by several means: 1) direct microscopic examination of infected body fluids; 2) histological examination of tissues samples; 3) detection of cryptococcal polysaccharide antigen in body fluids; and 4) culture.

Direct Microscopic Examination

India ink examination is a useful and rapid diagnostic test that relies upon direct visualization of the distinctive cryptococcal capsule within body fluids, especially CSF. India ink examination can be performed on all liquid samples (urine, sputum, ascite). The sensitivity of india ink examination of CSF from patients with cryptococcal meningitis is approximately 80% in HIV patients and 30% to 50% in non-HIV patients (12, 24, 40).  The presence of yeast cells of 4-6 μm with a clear halo around (capsule thickness: 1 to 30 μm) indicates presence of cryptococcus. False negative results are possible in case of thin capsule and lead to incorrect diagnosis (Candida spp, Histoplasma spp.).

Histological Examination

Non-specific histologic stains (Periodic acid–Schiff, May-Grünwald-Giemsa) are more sensitive than India ink stain to detect the yeast in tissue specimens. Hematoxylin-eosin stain reveals the clear halo. Some stains target specifically the polysaccharide capsule (mucicarmine, alcian blue). Inflammatory response in tissue appears to be minimal, particularly in HIV-infected patients with a moderate polymorphic infiltrate (mainly macrophages and T-lymphocytes).

Cryptococcal Polysaccharide Antigen in Body Fluids

Detection of cryptococcal polysaccharide capsular antigen (CRAG) is useful in both the diagnosis of infection and the prediction of prognosis and response to therapy. Several latex, enzyme-linked immunoassays and more recently immunochromatographic cryptococcal antigen tests are available. CRAG assays are based on detection of cryptococcal capsule polysaccharide glucuronoxylomannan. Sensibility and specificity in serum and CSF are excellent especially in AIDS patients(over 95%) (67). Clinicians should be informed that CRAG in serum is positive in only 85% of non-HIV patients with disseminated and/or meningitis  (40).

Unexpected positivity of a cryptococcal antigen in body fluids should lead to a control and when appropriate to exhaustive work-up. An isolated positive antigen can precede an authentic cryptococcosis from more than several months  (46).  High titres of CRAG in serum (>1/512) or CSF at baseline predict high fungal burden in CSF and thus higher mortality (59).  Kinetics of disappearance is not used in routine practice and previous studies have reported no correlation between decrease of serum but not CSF CRAG and the outcome of cryptococcal meningitis (1, 4).

Both false positive (due rheumatoid factor, hydroxyethyl starch fluid, cross-reaction with Trichosporon spp antigen and non-pathogenic Cryptococcus species), and false negative tests (due to prozone phenomenon, absence of pronase use, early infection) have been reported (52, 55, 97).

In 2011, the United States FDA approved a lateral flow assay (LFA) for the rapid (≤15 mins) semi-quantitative detection of polysaccharide antigen in serum or CSF (53).  The test consists in dipstick-like strips optimized to detect all four major cryptococcal serotypes. Recently a prospective multisite study has shown the highest sensibility and specificity for CSF (>99%) compared to others diagnostic methods such as India Ink staining, CSF cultures or CRAG (12).

Culture

Culture is the gold standard for cryptoccocosis diagnosis. C. neoformans can grow on most standard media used for isolating bacteria and fungi except media with cycloheximide. C. neoformans can grow within 48 to 72 hours (up to 7 days in case of anti-fungal therapy), at 30°C rather than 37°C on Sabouraud dextrose agar. C.neoformans colonies are translucent, smooth and gelatinous (71).

Niger seed agar supplemented with antibiotics is a selective medium for C. neoformans, which possess phenyloxidase that oxidizes caffeic acid (in the niger seed extract) to produce melanin. C. neoformans are visualized as brownish colonies on this medium, contrary to Candida sp. growing as white colonies. This characteristic is useful for culture of C.neoformans from contamined or colonized specimens by Candida spp. However some Cryptococcus neoformans and non-neoformans strains lack phenoloxidase and do not produce brown colonies (71).

Culture performance can be improved by high volume of body fluids like 3 to 5 mL for CSF (125).  Furthermore, the lysis-centrifugation method may be more sensitive in detecting low fungal inocula in blood culture. Negative cultures with positive India ink examinations can be explained by the presence of non viable yeast after therapy (37).

Biochemical profiles show no fermentation, urease production and no assimilation of nitrate. The full physiological profile provides definite identification after primary culture.

Distinction between Cryptococcus spp.

Laboratory diagnosis of C. gatti does not differ from C. neoformans. Diagnosis of Cryptococcus spp. is based upon: direct visualization of an encapsulated yeast, positive culture or CRAG (cryptococcal antigen) positive test. The work-up strategy should be similar than in C. neoformans infection (Table 2).

C. gatti specificities:

- India ink staining is more sensitive than for C. neoformans in non-HIV patients (25, 28, 72)

- cryptococcal antigen sensitivity in serum is 90% for lung disease

- cryptococcal antigen sensitivity in CSF is 87 to 100% for meningitis

- LFA is not evaluated but could be effective since one study enrolling 4 patients did not show false-negative results

- CRAG serum titers of ≥256 at baseline predicted death and/or neurologic sequelae at 12 months

- colonies grown on agar media are more mucoid and stickier in texture than those of C. neoformans.

Culture on CGB (selective medium L-canavanine glycine bromothymol blue agar) is the gold standard to differentiate the two species. C. gattii turns the medium blue by assimilation of glycine as the sole carbon and nitrogen source, which raises the pH whereas C. neoformans does not change the color (68).  C.gatti grows in the presence of D-proline whereas C. neoformans does not. Cryptococci other than C. neoformans or C. gattii do not produce the brown pigment on Niger seed agar.

In combination with diagnostic media, serotyping of Cryptococcus spp with a direct immunofluorescence assay with one monoclonal antibody (El) specific for cryptococcal polysaccharide allows a rapid and reliable method to serotype clinical isolates in four categories (A or D for C. neoformans, B or C for C. gattii). However as serotyping is not routinely used, some clinical laboratories recommend to combine molecular method such as D2 large ribosomal subunit DNA sequencing (D2 LSU) in addition to culture on CGB. In fact, some non-cryptococcal yeasts can grow on CGB medium (68).

For some authors, clinicians do not need to know in routine practice the species causing infection to well manage a patient with cryptococcosis especially in resource-limited countries (138). Some promising molecular or proteomic methods would be able to distinguish the two species and genotypes, especially in particular situations: failing of culture methods, histologic specimens without culture or differential diagnosis with others fungal agents. Accessibility to these methods depends on experience of certain laboratories and these are sometimes unable to distinguish the two species. Multilocus sequence typing (MLST) seem to be currently the molecular reference method resulting from high discriminatory power and reproducibility between different laboratories (103). It allows genotype identification and can further divide VGII isolates in subtypes. A consensus MLST typing scheme for the C. neoformans-C. gattiispecies complex has been published in 2009 using seven variable genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region (103).

PATHOGENESIS

C. neoformans

The most common reservoir of C. neoformans is bird droppings. The infection starts following inhalation of the pathogen from the environment (86).  Spores migrate in pulmonary alveolar regions, germinate into mature yeast and acquire their capsule. Thereafter a complex interaction between the yeast and alveolar macrophages starts. Capsule confers protection against phagocytosis. Macrophages may not recognize the yeast with their innate surface receptors, therefore, C. neoformans may survive extracellularly or in resident macrophages. This phase can be asymptomatic and this dormant latent infection may persist for a prolonged period (109).  When a significant immunosuppression appears, mature yeast can spread by lymphatic and haematogenous dissemination. Thus, most cases of cryptococcosis seem to be due to reactivation. Reason for targeting of central nervous system by C. neoformans is unknown. Three specific mechanisms determine yeast entry into the brain to cross the blood-brain barrier (142).    A transcellular passage can occur through endothelial cells with a specific ligand-receptor, a paracellular route between the endothelial cells is possible or a Trojan Horse mechanism, in which yeast cross inside a host monocyte (23).   Direct inoculation by traumatism and by transplant infected organs have been described as different pathways (162). Nevertheless there were no human-to-human transmissions, except for one case described in literature (169).  Three main virulence factors are well known: a complex capsule, melanin production and growth at human body temperature (57). The most virulent factor is the capsule (165).   C. neoformans is the only eucaryotic pathogen to possess it. Composition is about 88% glucuronoxylomannan and 10% galactoxylomannan, it has an extreme complexity and adaptation with the host. The capsule interferes with phagocytosis and decreases complement activity. The capsule activity can also modulate intracellular survival of the yeast to allow long-term latency. Melanin is a pigment, which protects the yeast from ultraviolets in environment. During pathogenesis, melanin is produced by oxidation of host catecholamines helping it to resist to free radicals (117).  The ability to grow at body temperature is provided by the RAS1 signalling pathway. Mutated  RAS1  strains cannot grow at 37-39°C and are also avirulent in animal models of cryptococcosis (69).

Alveolar macrophages and T cell Th1 responses are key components of anti cryptococcal immunity.

Macrophages have been well studied in murine models. Activation of macrophages is critical to confer protection against C. neoformans. Depletion of host macrophages leads to decrease survival after challenge. The yeast can take advantage of macrophage metabolism or manipulates the cell cycle. Intramacrophagic residence of C. neoformans allows immune escape and contributes to virulence. The antioxidant properties of melanin and the capsule protect the yeast from killing by macrophages, and C. neoformans can survive through an exocytosis mechanism without killing the macrophages (31). Macrophages can also be derived as Trojan Horse to cross the blood brain barrier. Explanation of latency in phagocytes can be supplied by the recent postulate of « tissue-damage control » (101). Latency might be a tolerance mechanism, which could minimize yeast damage and its exposition to host immune system, allowing advantages to both host and yeast. Moreover, some patients otherwise healthy develop C. neoformans infection with detection of GMCSF antibodies, suggesting that the GMCSF-STAT5 pathway could be critical for immune defenses against the yeast (143).

T cells are important effectors of anti-cryptococcal immunity (30, 163).  A protective response to C. neoformans is related to strong CD4 T cells. In HIV patients, CD4 cell count below 100 cells/mm3 is a major risk factor of cryptococcosis. In mice models, depletion of specific cytokines has shown that IL-12 and IFNγ were critical for survival after challenge with the yeast whereas decrease of IL-13 and IL-4 cytokines allowed more survival (31). The capsule can counteract the production of TNF alpha and IFNγ whereas urease and prostaglandins production by C. neoformans induce synthesis of IL-10 or IL-4 (117).   In accordance with these results, cryptococcosis has been described in patients with antibody against IFNγ or IL12Rβ1-deficiency (7, 64) (Lanternier et al, unpublished data).  These findings support a crucial role for the IL-12/IFNγ axis in anti-cryptococcal immunity (17). Although IL 17 producing T cells have been described as critical in superficial anti-Candida immunity, it is uncertain if they play a specific role during cryptococcosis (171).

C. gattii

The global pathogenesis of C. gattii is close with C. neoformans infections. Main virulence factors are analogous: a complex capsule, melanin production and ability to grow at 37°C (93).  However, phenotype is correlated to genotype, host immune system and the endemic/sporadic or outbreak context. VGI is associated with CNS disease whereas VGII with pulmonary disease. Concerning host, VGIV for example is related to HIV/AIDS patients in Africa whereas VGI strain to non-immunocompromised patients in Australia (81).  These findings are confirmed by mice models in which some genotypes are more virulent than others (VGIIa for example) (114).  Anyway, molecular genotype is not the only difference that explains virulence: inoculum dose, hyper-virulence factors and transcriptional circuits can be very different among one genotype explaining virulence of certain clones (113,168).  Specific interactions of C. gattii with immune system have not been well studied and did not provide relevant differences with C. neoformans. However, the cytokine pattern involves in response to C. gattii could be different when compared to C. neoformans. In vitro studies show higher concentrations of the pro-inflammatory cytokines IL-1β, TNFα and IL-6 in response to C. gattii, using heatkilled isolates incubated with human peripheral blood mononuclear cells (PBMCs)   (146).   This particular pattern of cytokine secretion could explain the frequent occurrence of IRIS in patients infected by C. gattii (138).   Anti-GM-CSF autoantibodies have been described as a risk factor for CNS infection by C. gattii in otherwise immunocompetent patients (143).  The impaired signalling in GM-CSF pathway could lead to a decrease of Th1-type cytokine production and a reduction of T-cell mediated protective immunity.  The ability of C. gattii to infect non-immunocompromised hosts, despite a strong cytokine response, has led some authors to hypothesize that certain inborn errors of immune function-related genes might account for a predisposition to infection.

SUSCEPTIBILITY IN VITRO AND IN VIVO

C. neoformans

Interpretive breakpoint values have not been proposed for any antifungal agents against C. neoformans. No solid data are available on PK/PD, clinical outcome compared with MICs. Some studies with heterogeneity and small strain numbers showed a correlation between in vitro susceptibility and clinical outcome but they use different methods, incubation time, media and end-point determination (2, 5, 134).   Nevertheless, absence of correlation is more frequently observed and susceptibility testing should be interpreted with caution, at least for incident isolates in the context of no prior azole exposure (32).  Therefore, epidemiological cut-off values (ECVs) and wild-type susceptibility endpoint MIC distributions help to characterize susceptibility and emergence of resistant strains. ECVs are based on MIC distributions that comprise the wild type (WT) and non-WT populations. ECV is the highest MIC that belongs to the WT population and allows identifying non-WT strains in order to detect emerging resistance.  Table 5 summarizes modal MIC and ECV obtained in an international study of WT strains (more than 3000 strains of C. neoformans) (42, 43).

CLSI/EUCAST broth microdilution methods and E-test are two techniques available for susceptibility testing with good correlation. Modal MICs and ECVs are established with EUCAST/CLSI methods. Exception is 5-FC, for which E-test shows very high-MIC compared to EUCAST technique (32).  Testing susceptibility of 5-FC needs to use different agar-media in this situation. Unlike C. neoformans, C. gattii has not been included in Clinical and Laboratory Standards Institute (CLSI) guidelines for testing of yeasts.

Results from time-kill curves could be an option but are not achievable in practice (135). To further compound the problem, 20% of cryptoccocal infections are a mix of different strains and additionally susceptibility depends with the used media RPMI or YNB (Yeast Nitrogen Base) for which MICs are frequently much higher (36). Susceptibility testing should be considered for azoles if the patient was preexposed to azoles or if the patient had an unexplained relapse.

C. gattii

Unlike C. neoformans, C. gattii has not been included in Clinical and Laboratory Standards Institute (CLSI) guidelines for testing of yeasts. Susceptibility differs among regions of the world and genotypes. For example, VGII genotype exhibits higher ECVs for 5-FC (ECV 95% 16μg/ml, ECV 99% 16μg/ml) and for FCZ (ECV 95% 32μg/ml, ECV 99% 64μg/ml) compared to C. neoformans and others C. gattii genotypes (42). Overall, C. gattii strains show higher MICs for fluconazole than C. neoformans. In agreement, some studies have shown same results for other triazoles agents and propose slightly different ECVs values suggesting that genotype should be taking in consideration (82). It is important to note that clinical relevance of ECVs is not established and should be considered with caution.

ANTIFUNGAL AGENTS AND CRYPTOCOCCOSIS

Drug of Choice

Amphotericin-B

Amphotericin B (AmB) is fungicidal on Cryptococcus. Primary resistance to AmB is extremely rare.  Major limitations of this drug are its renal toxicity, which is limited by the use of lipid formulations when available. Together with 5-FC, it still constitutes the gold standard therapy for induction in cryptococcosis in 2015.

Flucytosine (5-FC)

5-FC has good in vitro activity against C. neoformans and penetrates well into the CSF. The major limitations of this drug are its toxicity and the development of flucytosine-resistant fungal isolates when the drug is used as monotherapy. Therefore, it has to be used only in combination with Amphotericin B (AmB) or high dose fluconazole. The association with AmB is often synergistic in vitro and depends on the pathway in cause in case of 5-FC resistance (147).  5-FC uptake by C. neoformans occurs via cytosine permease. Then 5-FC is deaminated into 5-FU by cytosine deaminase (164).  If defect appears for cytosine permease or cytosine deaminase, the yeast will be 5-FU susceptible but 5-FC resistant. AmB can restore 5-FC activity if there is a defect for cytosine permease, because AmB permits 5-FC uptake into the cell and then its conversion in 5-FU. 5-FC and AmB seems to be never antagonistic in vitro and in vivo. Actual techniques cannot distinguish theses mechanisms of resistance if a strain was resistant to 5-FC. For clinical practice, we therefore recommend using 5-FC even if high MICs were found during susceptibility testing (147). When possible, serum flucytosine levels should be measured after 3-5 days as well as hematologic parameters because of bone-marrow toxicity.

Fluconazole

Fluconazole (FCZ) has a fungistatic effect on C. neoformans. In general, C. neoformans isolates are susceptible to fluconazole in vitro and most isolates have MIC <16ug/L. Cut-off for “resistance” definition is variable in different studies (8μg/L, 32μg/L). Primary “resistance” is reported, but not in our hands. Acquisition of resistance during fluconzole monotherapy may be observed. VNI genotype shows lower MICs than other strains, but no mutations have been observed when fluconazole MIC <16ug/L (42).  There is a correlation between high fluconazole MICs or increase of fluconazole MICs during therapy with relapse of cryptococcosis in patients treated by initialfluconazole monotherapy (8, 29). Mutation in the ERG11 gene alters affinity of fluconazole to its target enzyme. Efflux mechanisms have also been demonstrated. A mechanism of heteroresistance to fluconazole has been recently described: under in vivo stress and fluconazole treatment the yeast can acquire resistance to fluconazole through gene duplications in the target or efflux pumps (155).  Interestingly, when the isolated yeast grows under in vitroconditions, it returns to normal susceptibility. Azole cross resistance is rare, but does exist between fluconazole and Itraconazole (126). C. gattii isolates are susceptible to fluconazole in vitro. VGII case clusters exhibit higher MICs for fluconazole (82).  Resistance mechanisms such as ERG11 mutations or efflux are probably not similar between C. neoformans and C. gattii. An implication for drug dosing and clinical outcome is not determined. Fluconazole reaches high concentrations in serum and tissues, including the CSF. Its long half-life enables once daily dosing. This drug is used with success for both consolidation and maintenance therapy for cryptococcal meningitis in HIV-infected patients.

Itraconazole

Itraconazole has good activity against C. neoformans. It has been compared with fluconazole as consolidation therapy and has shown less effectiveness in preventing relapse (140).   Itraconazole is proposed as an alternative in consolidation therapy when there are formal contraindications. It requires careful serum drug level monitoring.

Posaconazole

Posaconazole has a good activity against C. neoformans. However there is a lack of clinical experience in cryptococcal meningitis. It has been used as salvage therapy in some studies with small sample size (128).  Drug-drug interactions and serum level should be closely monitored.

Voriconazole

Voriconazole shows the best in vitro activity of all azole agents against C. neoformans. However there is a lack of clinical experience during cryptococcal meningitis. It has been used as salvage therapy in some studies with small sample sizes (125).  Voriconazole might be superior to fluconazole and need to be evaluated in different phase of therapy for C. gattii and C. neoformans (18).  Drug-drug interactions and serum level should be closely monitored.

Isavuconazole

Not yet registered, isavuconazole has an interesting in vitro activity against C. neoformans and C. gattii (44).

Echinocandins

Echinocandins are ineffective in cryptococcal infections. C. neoformans β(1-3)-glucan synthase is inhibited by echinocandins, but this yeast is able to grow in the presence of high concentrations of these molecules (95).

CRYPTOCOCCOSIS TREATMENT

Infectious Diseases Society of America (IDSA) in 2010 and WHO (World Health Organization) in 2011 have published practice guidelines for the management of cryptococcal diseases  (125, 170).   Table 7 is adapted from these recommendations with some variations taking into account recent relevant studies. Overall cryptococcosis management is determined by the host immune status, the severity of illness, infection site and unfortunately the availability or not of appropriate antifungals (88, 90).

Cryptococcosis in AIDS patients

Meningo-encephalitis

Treatment involves three parts: induction, consolidation and maintenance therapy.

Induction Phase

Combined AmB and flucytosine (5-FC) are currently the « gold standard » therapy for cryptococcal meningitis in AIDS and non-AIDS patients. In vivo experimental models showed a synergy of AmB and 5-FC which was the best effective association in a murine model (147).

Regarding difficulties to prove benefit in survival because of a lack of homogeneity in studies, the use of rate of clearance of infection or early fungicidal activity is now applied to test antifungal drug combinations in phase II studies. Bicanic et al. found that early fungicidal activity (in the first weeks of induction therapy) was closely correlated with survival (9).

Van der Horst in 1997, suggests that AmB + 5-FC combination was more effective than AmB alone, in a prospective study involving AIDS patients with cryptococcal meningitis (56).  At week 2, the sterilization of CSF was higher in the AmB + 5-FC group versus AmB alone. There were no differences in mortality, probably due to high CSF opening pressure in the two groups with high mortality rates. Another study from Brouwer et al. in 2004 demonstrated that early fungicidal activity was strongly higher in AmB + 5-FC combination compared with AmB alone, AmB + FCZ (400 mg/d) and a tritherapy of AmB + 5-FC + FCZ (16).

Concerning the optimal AmB dose, a prospective study from South Africa compared a regime with AmB 0,7mg/kg/d + 5-FC versus AmB 1mg/kg/d + 5-FC (10). The high dose regimen was more rapidly fungicidal. There were limited data on toxicity due to the small size of the samples.

Liposomal AmB (L-AmB) is associated with fewer adverse effects especially nephrotoxicity, infusion-related reaction when compared with deoxycholate AmB (AmB-d). In 2010, Hamill et al. demonstrated in a study conducted between 1995 and 1998 no differences in efficacy between 3 monotherapy regimens: AmB-d, L-AmB 3mg/kg/d, L-AmB 6mg/kg/d (51).  The CSF rate of sterilization was similar between the three groups with fewer adverse events in the L-AmB groups. Lack of 5-FC use and introduction of early HAART therapy are the main limiting factors of this study, and could explain the absence of difference in mortality. The high cost of L-AmB limits its expansion in a lot of regions of the world. L-AmB 3mg/kg/d is preferred because of lower levels of toxicities than the 6mg/kg/d regimen.

There is currently no convenient regimen constituting a good alternative to the « gold standard » association of AmB and 5-FC. 5-FC remains largely unavailable in large parts of Asia and Africa while AmB requires careful clinical surveillance and laboratory monitoring (90). Their costs are also a barrier to access. Alternative combinations with AmB are needed in case of absence of 5-FC availability. Loyse et al. showed similar efficacy of theses alternatives in a study involving 80 HIV-seropositive patients presenting with cryptococcal meningitis (92). Patients were randomized to 4 treatment arms of 2 weeks duration: group 1, AmB (0.7–1 mg/kg) and 5-FC (25 mg/kg 4 times daily); group 2, AmB (0.7–1 mg/kg) and fluconazole (800 mg daily); group 3, AmB (0.7–1 mg/kg) and fluconazole (600 mg twice daily); and group 4, AmB (0.7–1 mg/kg) and voriconazole (300 mg twice daily). There were no statistically significant differences in early fungal activity and in survival (although not powered for) between these 4 groups. These results supports the use of regimens containing AmB + Fluconazole as alternative regime at a minimal dose of 800mg/d(121),  but they are contrasting with the work of Brouwer et al. which showed  significantly less fungicidal activity of this association (with 400 mg/d of fluconazole) when compared to 5-FC + AmB. Loyse et al. study was limited by its small size and the lower fungal burden in CSF of patients.

A regimen with AmB and fluconazole could be an alternative option. An open label phase II trial in Thailand enrolling 143 patients with cryptococcal meningitis showed satisfactory outcomes.  The success primary end point was a composite of survival, neurologic stability, and negative cerebrospinal fluid culture results after 14 days of therapy. Patients were assigned in three groups: AmB 0.7 mg/kg alone, AmB 0,7mg/kg + FCZ (400mg/d), AmB 0,7mg/kg + FCZ 800mg/d administered daily for 14 days, followed by fluconazole alone at the randomized dosage (400 or 800 mg per day) for 56 days. The 14-day end point of success was 41%, 27% and 54% respectively. High-dose fluconazole + AmB group showed good outcomes, although combination low-dose fluconazole and AmB seems to be antagonistic. On contrary in vivo mice model have shown no antagonism effect of this combination.

A recent study, enrolling 375 HIV positive patients from Vietnam with cryptococcal meningitis, showed for the first time benefit in global survival with the combination of AmB + 5-FC (35).   Patients were assigned to three groups for induction therapy: AmB 1mg/kg/d for 4 weeks, AmB + 5-FC for 2 weeks, AmB + FCZ (800mg/d) for 2 weeks. Then a maintenance therapy with fluconazole 800mg/d was continued after the induction phase. Mortality was lower at day 14 and 70 in the AmB + 5-FC group versus AmB alone (15 vs. 25 deaths by day 14; hazard ratio, 0.57; 95% confidence interval [CI], 0.30 to 1.08; unadjusted P = 0.08; and 30 vs. 44 deaths by day 70; hazard ratio, 0.61; 95% CI, 0.39 to 0.97; unadjusted P = 0.04). Fungal clearance of CSF was greater in the AmB + 5-FC group compared to the others. There was no survival benefit of AmB + FCZ regimen compared to AmB monotherapy but fewer deaths at 10 weeks occurred in the AmB + Fluconazole group. Theses results suggest that AmB monotherapy should no more be considered as a good alternative of the 5-FC + AmB regimen, as proposed in the 2010 IDSA guidelines.

High-dose fluconazole monotherapy is the worst alternative regimen. Between 2010 and 2011 in Malawi, 60 patients were recruited and treated with fluconazole 800 mg/d for 2 weeks, followed by 400 mg/d for 6 weeks, then secondary prophylaxis with 200 mg/d indefinitely. Mortality was very high, 43% of patients died by 4 weeks (139). 58.0% and 77% died or failed treatment by 10 or 52 weeks respectively. Despite some studies showing a possible efficacy, mortality rates are unacceptable to recommend this option and emergence of resistance under monotherapy is frequently reported. Another study reports faster fungicidal effect with high dose fluconazole (1200mg/d) compared with fluconazole (800mg/d) but higher mortality at week 10 (54%) although not statistically different (84).

A promising association could be represented by the FCZ + 5-FC combination as induction therapy. In an open study, Larsen et al. first found that this combination was clinically superior to results that had been previously reported for fluconazole alone. Fluconazole (400 mg daily) was combined with 5-FC (150 mg/kg daily). After 10 weeks of therapy, 75% of 32 patients CSF cultures were negative. The median time to negativity of the CSF culture was 23 days(75).  In 1998, a randomized Ugandan study showed superiority of a combination therapy with fluconazole (200mg/d) for 2 month with 5-FC (150mg/kg/d) for the first two weeks compared to fluconazole monotherapy. 30 patients were randomized. Survival rates at 6 months were 32% in the 5-FC group whereas 12% of patients with monotherapy of fluconazole survived (96).  More recently Nussbaum et al. demonstrated in a randomized single study in Malawi that combination of fluconazole (1200mg/d) + 5-FC (100mg/kg/d) was more effective than fluconazole (1200mg/d) alone (116). The mean rate of decrease of log CFU or early fungicidal activity was significantly greater for patients receiving combination (-0.28 log CFU per day) versus monotherapy (-0.11 log CFU per day; p<0.001). There was also a better survival at weeks 2 (10% vs 37%) and 10 (43% vs 58%) in the combination group but mortality still remained high.

Short course therapy of AmB (7 days) can also be an effective alternative in some countries. Jackson et al. designed a study enrolling 81 patients in a two-step study (58). 4 regimens were compared: fluconazole(1200mg/d), FCZ (1200mg/d) + 5-FC (100mg/kg/d), FCZ (1200mg/d) + AmB 1mg/kg  (7days), FCZ (1200mg/d) + 5-FC (100mg/kg/d) + AmB (7 days). Endpoint was early fungicidal activity which was faster in the « short course “AmB group” and in the « triple » combination group. Although, there were no statistically differences in survival at 2 weeks and 10 weeks, this study provides some arguments for the short course regimen containing AmB. In Malawi, Zambia and Cameroon a promising randomised phase III trial is comparing five groups :  FCZ (1200mg/d) + 5FC (100mg/kg/d), AmB (7 days) + FCZ (1200mg/d) or 5-FC(100mg/kg/d), AmB (14 days) + FCZ (800mg/j) or 5-FC(100mg/kg/d) for the initial treatment of AIDS-associated cryptococcal meningitis (ACTA-Advancing Cryptococcal meningitis Treatment for Africa, www.controlled-trials.com/ISRCTN45035509). This study should provide convincing arguments for policy makers involved in limited resource countries.

Consolidation Phase

Consolidation phase should be considered only if there is a global improvement and ideally sterile CSF fungal cultures at day 14. Theses recommendations can be adapted to every single case. Combination therapy with 5-FC + AmB should be continued (1-6 weeks) in case of: reduced consciousness, clinical deterioration, refractory raised intracranial pressure, fungal culture positive at day 14. 80% of patients have negative cultures at day 14. It has been shown that positive culture at day 14 was a risk factor for relapse (133).   Fluconazole (400mg/d) for 8-10 weeks is recommended for consolidation therapy. Patients not receiving induction with AmB + 5-FC should be treated withfluconazole 800mg/d for consolidation. In addition, some investigators like our group suggest to give 800 mg/d fluconazole until the start of antiretrovirals (i.e. for a minimum of 2 weeks).

Maintenance Phase

Fluconazole (200mg/d) is for maintenance phase (6-12 months). Maintenance therapy should be continued if immune status is not improved. For AIDS patients, stopping maintenance therapy may be considered after one year of antifungal and HAART therapy with CD4 count cell > 100/mm3, undetectable viral load sustained for 3 months and a CRAG <1/512 (87).  A careful follow-up need to be provided regarding some tardive relapses after discontinuation of maintenance therapy according previous criteria  (106).   A reinstitution of maintenance therapy is required if CD4 decrease to <100/mm3.   Itraconazole (400mg/d) or AmB (1mg/kg/week) are less effective. A first study has demonstrated superiority of fluconazole (200mg/d) over AmB (1mg/kg/week) with more relapse in the AmB group (18% vs 2%)  (130).  A second study has shown that fluconazole was superior to ITZ with also more relapse in the ITZ group (23% vs 4%) (140).

Preemptive therapy/Primary prophylaxis

As seen before, an asymptomatic positive cryptococcal antigen in body fluids should lead to a control and when appropriate to exhaustive work-up. In AIDS patients with low CD4 count cell, preemptive therapy might be an option. There is currently no evidence to support primary prophylaxis (see below).

Extra meningeal cryptococcosis in AIDS patients

Systematic evaluation is needed, including blood, urine at least in male patients, and CSF cultures, measurement of serum and CSF cryptococcal antigen.

A mild or moderate pulmonary involvement or a skin involvement can be treated with fluconazole 400mg/d for 12 months. Severe forms, cryptococcocemia, dissemination even if isolated should be treated as a meningoencephalitis. Itraconazole (200 mg twice per day), voriconazole (200 mg twice per day), and posaconazole (400 mg twice per day) are acceptable alternatives with a particular attention to monitoring drug serum and drugs interactions. Surgical resection for voluminous pulmonary abscess should be considered if there is no response to antifungal treatment.

Criteria for disruption of therapy are the same that those mentioned above (« HIV and fungal control »).

When to start HAART – IRIS

HAART, inducing both a decrease in HIV viral load and an increase in CD4 cell counts, has led to a dramatic decrease in incidence of opportunistic infections (OIs), the progression to AIDS, and the overall mortality for HIV-infected patients. For these reasons, clinicians are often eager to start HAART in AIDS patients being treated for OIs. Some evidence of immune reconstitution syndromes in these patients, however, raises concerns about the proper place of antiretrovirals in the setting of acute OIs.

Two forms of IRIS are described: Unmasking IRIS is the discovery of an unknown infection (like cryptococcosis) shortly after HAART introduction during the first weeks following the diagnosis of HIV infection. Paradoxical or classical IRIS occurs during therapy of cryptococcal disease in patients who initially improve on antifungal therapy and is characterized by clinical deterioration and/or new clinical manifestations after 1 or 2 months of HAART therapy (maximum 9 months). Frequency would approach 6% to 45% in AIDS patients (83). It can mimic a relapse or another OIs, but fungal cultures of CSF or pulmonary samples are negative. Mortality ranges 0-36% and is closely related to the risk of neglected intracranial hypertension. Some recent studies seem to show no differences in early mortality between patients who did and did not develop IRIS but it remains unclear to distinct whether deaths are due to cryptococcosis by itself or IRIS (11). Major risk factors for paradoxical IRIS include: low CD4 count at baseline (<100/mm3), high fungal burden (positive culture at J14 and/or fungemia and/or high cryptococcal antigen >1/512), low CSF white cell count and proteins at baseline (85).

Minor manifestations may resolve in a few weeks without any treatment. Corticosteroid are recommended in case of clinical deterioration at a dose of 0.5–1.0 mg/kg during a short period of 2 to 6 weeks (125). Thalidomide is sometimes used as salvage therapy in refractory or steroid-dependant IRIS (19). The question of when to start HAART therapy is now solved. Starting early HAART increases the risk of IRIS while delayed HAART increases the risk of complications or others OIs. A recent Cochrane review compared in pooled analysis two eligible randomized controlled trials (N=89 patients)  (115). There was no statistically significant difference in mortality in the group with early initiation of HAART (<4 weeks after starting antifungal treatment) compared to the group with delayed initiation (> 4 weeks). One major limit is the heterogeneity of antifungal treatment in these studies. A trial was stopped early based on excess mortality in a group of patients starting HAART one or two weeks after diagnosis of cryptococcosis (11). This study enrolled 177 HIV patients randomly assigned in “earlier HAART” (1 or 2 weeks after diagnosis) or “delayed HAART” (until 5 weeks after diagnosis). Mortality at week 26 was significantly higher in the « earlier HAART » group (45% [40 of 88 patients] vs. 30% [27 of 89 patients]; hazard ratio for death, 1.73; 95% confidence interval [CI]). The mortality was also higher among subgroup of patients with lack of CSF inflammation (cell count<1mm3). This study highlights the need of differing HAART after 4 weeks in a very convincing way. We recommend therefore that it should be later in patients with unfavorable outcome and lack of CSF inflammation.

Cryptococcosis in Solid Organ Transplant Patients

Meningo-encephalitis

In solid organ transplant (SOT) patients, treatment has been extrapolated from AIDS patients, as there are no randomized trials but some retrospective studies. In  solid organ transplant patients (especially renal transplant), L-AmB (3mg/kg/d) should be preferred as first-line in combination with 5-FC (100mg/kg/d given in four divided doses) for a minimum of 14 days. It has been demonstrated in a non-randomized study that the use of lipid formulations was independently associated with lower mortality at day 90 (61).

Consolidation therapy with fluconazole (400-800mg/d for 8 weeks) is followed with maintenance therapy (200-400mg/d) for a minimum of 6-12 months adjusted for renal function. Duration of maintenance therapy should depend closely on clinical, mycological evolutions. A careful follow-up after cessation is recommended. With this strategy, risk of relapse is under 1.5% (54).

A careful management is required in solid organ transplant patients for drug-drug interactions and drug dosage adjustment in patients with renal impairment. Whenever possible, therapeutic drug monitoring (immunosuppressive and antifungal therapy) should be performed.

IRIS has also been reported in  solid organ transplant-related cryptococcosis with a prevalence of 4.8% (153). It follows reduction of cessation of immunosuppresive drugs during antifungal therapy. Previous therapy with calcineurin-inhibitor agents (tacrolimus), mycophenolate-mofetil, corticosteroids and alemtuzumab have been recognized as a risks factors for IRIS in solid organ transplant patients (125). In case of severe IRIS, steroids should be introduced for at least one month.  Recently, we successfully used adalimumab in a steroid-refractory IRIS case in this context (145).

Reduction of Immunosuppression

Reduction of immunosuppression in  solid organ transplant patients is the perfect reflection of starting HAART therapy in AIDS patients. An insufficient reduction should lead to a bad control of the fungal clearance and an excessive reduction should increase the risk of IRIS. Whenever possible a gradual tapering of corticosteroids is recommended first, since it has been shown that calneurin inhibitors have an inherent antifungal effect. Moreover, a recent study has shown that discontinuation of calcineurin inhibitors is the only modification of immunosuppression that influences the occurring of IRIS (adjusted OR, 5.11 [95% CI, 1.33–19.69]; P = .02). Therefore, a continuation of calcineurin inhibitors can be preferred to discontinuation, because IRIS might be a risk for allograft rejection (160).

Cryptococcosis in non-HIV and non-transplant patients

Meningo-encephalitis

In non-HIV and non-transplant patients, therapy is a challenge since this population is very heterogeneous: it ranges from people otherwise healthy to people with variable immunodeficiency (organ dysfunction, corticosteroids, chemotherapy, diabetes mellitus, primary immunodeficiency, etc.). Immunocompetent patients are diagnosed later (81 days vs 34 days), the rate of permanent CSF shunts is higher than in immunocompromised population (118). These datas have suggested that induction therapy could be longer than 2 weeks.  Prospective studies in non-HIV and non-transplant patients are old (6, 38). Mortality was higher before “azoles for consolidation and maintenance” and “high dose AmB for induction” eras. They therefore suggested treating 4-6 weeks during the induction phase. Based on clinical trials in HIV-patients, others experts suggest a “standard” 2 weeks induction phase. In IDSA guidelines published in 2010, there was no consensus between experts on this topic. The consolidation and maintenance phase are similar as described above. Higher dose of fluconazole (800 mg per day) for consolidation is recommended if the 2-week induction regimen was used and if there is normal renal function (125).

Extra meningeal cryptococcosis

An asymptomatic and isolated pulmonary involvement could be treated for less time (3 months). In mild-to-moderate involvement, duration of therapy could be reduced to 6 months if there is no sustaining immunodeficiency. Surprisingly, a retrospective study performed between 1976 and 2001 has shown that 20% of non immunocompromised patients with pulmonary cryptococcosis might heal spontaneously (107).  However, physicians have to give careful attention to non-HIV non-transplant patients especially those with severe pulmonary disease and treat them as a CNS disease when required.

C. gattii cryptococcosis therapy

No randomized controlled trials are available for therapy of C. gattii infections. Recommendations are extrapolated from IDSA guidelines (Infectious Diseases Soci­ety of America) for C. neoformans and small case series for C. gattii(63, 72, 104, 125).  Cornerstone of induction therapy is also based upon combination of Amphotericin-B + 5-FC. Management of HIV-patients with C. gattii should be similar to those with C. neoformans infections, because of comparable features in this sub-group. Unlike C. neoformans infections in HIV-patients, C. gattii infections in non-immunocompromised patients display two main differences: the duration of induction phase and the concept of eradication phase. 

Proposed regimen for C. gattii infections are summarized in table n°8.

CNS disease

Patients with large and multiple cryptococcomas associated with C. gattii seem to require a longer therapy. C. gattii infection in non-HIV patients is also associated with more neurological complications and higher incidence of neurosurgical intervention (158).  Expert opinion propose 6 weeks of initial combination of AmB + 5-FC in line with IDSA guidelines which recommend for non-immunosuppressed patients with C. neoformans CNS disease 4 to 6 weeks of induction therapy (125).  We always recommend to perform a lumbar puncture every two weeks for fungal culture during the induction phase.

For some experts, maintenance phase and consolidation phase should be grouped in a unique phase of eradication (27). Unlike patients with prolonged immunosuppression, this concept could make sense since eradication is the main objective in immunocompetent patients. Eradication therapy should be longer (>12 months) based on two studies with early relapse (14%) and high mortality (22-43%) enrolling patients with a duration of treatment <12 months(72, 77).  Fluconazole (400mg/d) should be given for this phase while others azoles (voriconazole, posaconazole, itraconazole) should be considered in case of clinical deterioration or relapse associated with high MICs for fluconazole.

A retrospective Australian study published in 2013 supports these findings (26).  Overall 73 patients with C. gattii CNS disease, AmB was initiated in 64 patients (84%) and in combination with 5-FC in 57 patients (78%). The median duration of combination therapy was 6 weeks. On the 9 patients (12%) received induction therapy with  fluconazole, 7 patients were reinduced with AmB + 5-FC (one had IRIS). There were no relapses and complete/partial clinical response in 80% of patients. However there is currently no evidence to generalize these recommendations to epidemic strains of C. gattii and others genotypes.

IDSA guidelines also recommend increased radiologic and follow-up evaluations for patients with large cryptococcomas and/or hydrocephalus.

Pulmonary disease

Immunocompromised patients with pulmonary disease could be treated as for CNS disease. For immunocompetent patients, large and multiple cryptococcomas can be treated with a 4 to 6 weeks combination of AmB + 5-FC (125).A 2 weeks induction therapy with AmB + 5-FC seems to be as effective in a recent study (99).  In case of isolated small cryptococcoma of the lung, monotherapy of fluconazole could be an alternative regime. Fluconazole should be administered for eradication therapy more than 12 months since a recent study did not show any relapse in this case (99). Very recently, a retrospective study enrolling patients from Pacific Northwest infected with outbreak strains has shown a good correlation between an inappropriate initial therapy and increased mortality (156).   The authors observed that patients not receiving IDSA guideline-recommended therapy for pulmonary infection seem to have increased three-month mortality compared to the others (42% vs. 10%, p = 0.07). This study emphases the role of initial evaluation in emerging C. gattii infections in USA and others regions. It focuses on the need to disseminate recommendations among clinicians in non-endemic areas.

C. gattii immune reconstitution inflammatory syndrome (IRIS)

Risk factors for IRIS have been studied in a recent study (26).   Female gender, brain involvement at baseline and unexpectedly high CD4 count are reported as determinants of IRIS during C.gattii infection. IRIS is not restricted to immunocompromised hosts and do not correlated with fungal burden. IRIS has to be evoked in case of clinical deterioration 4 weeks to 12 months after starting antifungal therapy. IRIS is often confused with relapse but fungal cultures of sample are always negative.

ADJUNCTIVE THERAPY

Intracranial Hypertension-Hydrocephalus

Intracranial hypertension, (defined as intracranial pressure of ≥ 25 cmH2O), occurs in at least 50% of HIV-patients with cryptococcal meningitis (49). Its control is one of the major determinants of outcome. Although these patients often show neurological symptoms such as headache, papilledema hearing loss, visual changes and seizure, they can also be asymptomatic. In addition, CT scan or MRI of the majority of these patients shows no evidence of hydrocephalus (49). Thus, the absence of symptoms or a normal head scan does not exclude the presence of intracranial hypertension. Post mortem histopathology studies have shown that intracranial hypertension was related to obstruction to CSF reabsorption by large numbers of cryptococcal cells at the level of arachnoid granulations (91). There is also a direct correlation between high fungal burden in CSF and increase of intracranial pressure (7). In all cases of suspicion of cryptococcosis when a lumbar puncture is performed, it is essential to always measure opening pressure. IV tubing sets are a good alternative when manometers are not available (100). When opening pressure(OP) is ≥ 25 cmH2O, CSF drainage is needed with repeated CSF withdrawal. The objective is to decrease OP below 20 cmH20 otherwise 50% of baseline if it is extremely high (125). In practice it is unclear if a maximum of 30ml of CSF withdrawal could be recommended, some teams suggest rechecking pressure every 10ml of CSF. CSF removal should be performed every 2 days (and even daily in worse cases) in case of intracranial hypertension but no trials have assessed optimal frequency of therapeutic lumbar puncture (119). More recent attention has focused on therapeutic lumbar puncture effect on acute mortality regardless of initial intracranial pressure (137).  Short term survival was improved among patients who underwent more than one therapeutic lumbar puncture.In other words baseline raised intracranial pressure or related symptoms are not the only indications for therapeutic lumbar punctures that might be beneficial even in absence of elevated OP (119). In case of persistent pressure elevation despite iterative CSF removals, consideration should be given to rapid temporary percutaneous lumbar drains or ventriculostomy (94).  Permanent ventriculo-peritoneal shunts must be considered if the patient is receiving approopriate antifungal therapy, when clinically needed even in absence of complete sterilization.

Compared to a previous study (49) which found an increased risk of mortality in patients with elevated opening pressure, a recent study enrolling 163 HIV-patients showed that with an appropriate strategy of control of intracranial pressure, there were no statistically significant differences between “normal” and “raised” opening pressure groups (7). These findings are supported by another study showing improved mortality at day 11 among patients who underwent at least one therapeutic lumbar puncture (137).

There are currently no strong evidences supporting medical adjunctive therapy of intracranial pressure such as acetazolamide, mannitol and corticosteroids  (111). The CryptoDex study aiming to explore if adding dexamethasone to standard therapy reduce mortality in HIV-patients with cryptococcal meningitis has prematurely been interrupted (34). Hydrocephalus is also a complication of cryptococcal meningitis. Major symptoms are decreased mental acuity and gait ataxia. The diagnosis is easily made by CT-scan or MRI showing enlarged cerebral ventricles not attributable to atrophy. Usually HIV-patients do not have hydrocephalus, which is more common during C. gattii infections(22, 89). Hydrocephalus can compromise brain vessels and lead to brain ischemia and deterioration of neurological status. Placement of a VPS improves the outcome only if placed early, before deterioration of consciousness occurs. VPS placement in patients with Glasgow coma scale <8-9 and/or poor level of consciousness for greater than 48 hours does not appear to improve outcome (80).

C. gattii specificities

A study for C. gattii infections has recently shown the need of shunt or drain for 6 in 13 patients with elevated OP managed with iterative removal of CSF  (8). Hydrocephalus identified by cerebral imaging requires mostly drains or shunts in around two thirds of cases (8).  Early removal of large cryptococcomas should be considered in case of inadequate response to antifungal therapy or compression of vital structures. Neurosurgery or thoracotomy for excision should be contemplated in the first weeks in this case. Patients with excised cryptococcomas might be required shorter duration of therapy.
A retrospective study suggests a potential effect of corticosteroids on visual outcome in patients with clinical deterioration, but no randomized trial supports this findings (151). Other studies report a significant improvement in patients receiving dexamethasone for severe CNS disease associated with intracranial hypertension (127). However in context of IRIS-related symptoms with negative fungal cultures, corticosteroid should be recommended in case of clinical deterioration at a dose of 0.5–1.0 mg/kg during a short period of 2 to 6 weeks (125).

Immunomodulatory Therapy

Although HAART and aggressive antifungal therapy improves the outcome of AIDS patients with cryptococcal meningitis, this disease still has significant morbidity and mortality. New immunomodulatory therapies could be promising. Cytokines responses patterns in human are mixed during C. neoformans infections, but IFN-γ appears to be essential and its elevation in CSF is correlated with a positive outcome.

In 2004, a phase-2 randomized controlled study enrolling 75 AIDS patients suggested that use of rIFN-γ1b could induce a more rapid-sterilization of CSF compared with a placebo (120). Treatment was well tolerated. The 2010 IDSA guideline proposed a level B-III recommendation for the use of rIFN-γ for persistent infection defined as positive culture at 4 weeks of appropriate antifungal therapy (125). The recommended regimen is a dosage of 100 ug/m2 for adults who weigh >50 kg (for those who weigh <50 kg, consider 50 ug/m2) 3 times per week for 10 weeks. A larger recent study supported the results of the 2004 phase 2 study. Between 2007 and 2010, 90 HIV-patients were randomized in South-Africa (62).  The primary outcome measure was mean rate of decrease in CSF cryptococcal CFU (Early Fungicidal Activity). Difference in EFA was -0.15 [confidence interval (95% CI) -0.02 to -0.27, P=0.02] between standard treatment and IFNγ two doses, and -0.15 (95% CI -0.05 to -0.26, P=0.006) between standard treatment and IFNγ six doses. To conclude, the rate of fungal clearance was faster with IFNγ and as well effective with a 2 doses regime of IFNγ. 

As showing in animal models and in vitro assays, monoclonal antibodies (mAb) against glucuronoxylmannane, glucosylceramides, melanin or B-glucan seem to provide a protective effect. A phase 1 mAb 18B7 (murine-derived antibody against capsular glucurunoxylomannan) dose escalation study in HIV infected patients has shown a modest and transient reduction of serum cryptococcal antigen titres in patients receiving doses of 1 and 2 mg/kg (76).

C. gattii specificities

Although recombinant IFN-γ has been used as salvage therapy in some cases of HIV-patients with C. neoformans infections, there is no study supporting its efficiency in C. gattii infections. Some experts recommend its use for unresponsive infections with prolonged courses of antifungal therapy (157).

ENDPOINTS FOR MONITORING THERAPY

Prognosis factors

Without treatment, meningo-encephalitis is always fatal in AIDS patients. Despite HAART therapy, early global mortality approaches 20% in developed countries and much over 50% in low-resource countries lacking of HAART and antifungal reference therapy (15).  A prospective study performed in France between 1997 and 2001 has shown that serotype A (vs serotype D), CRAG antigen >1/512 in CSF and lack of 5-FC during induction phase are major risks factors associated with lack of CSF sterilization at day 14 in HIV-infected patients (40).  Concerning determinants of early and late mortality during cryptococcal meningitis, this study enrolling 230 patients in France reported a death rate of 6,5% and 18% at day 14 and week 10 respectively. Majors risks factors at baseline were abnormal neurological status and abnormal brain imaging. Another study developed in low resource countries (Thailand, Uganda, Malawi, and South Africa) has followed 501 HIV-patients for a minimum of ten weeks between 2002 and 2010 (59). Mortality was 17% at 2 weeks and 34% at 10 weeks. High CSF fungal burden, altered mental status, age>50 years and reduction of rate of clearance of infection predict acute mortality at week 2 in HIV-associated cryptococcal meningitis. The median time of death was 13 days suggesting that earlier appropriate therapy is critical. A more recent study has demonstrated that CSF CRAG LFA titers (>1:1280) at baseline correlate with fungal burden and are associated with mortality at 2 and 10 weeks (65). A retrospective single center study (1996-2010) compared outcomes between HIV patients, organ-transplant patients and non-HIV non-transplant patients (15).  At baseline cryptococcemia, high intracranial pressure and altered mental status increase mortality rates in all groups. Non-HIV non-transplant patients display higher rates of mortality compared to the others (27% at day 90). This is consistent with others studies that have shown an increase risk of mortality in non-HIV non-transplant patients probably because of delayed diagnosis and treatment (13, 79).

Treatment response monitoring

A close clinical evaluation is needed to observe response to treatment and to anticipate IRIS or relapse. A lumbar puncture with a microbiological assessment (fungal culture) at week 2 and week 10 is recommended as well as measuring opening pressure. There is no evidence to follow serum cryptococcal antigen titres that can be positive several months. A  positive India ink examination despite a clinical improvement and negative fungal cultures canalso be linked with dead yeasts.

VACCINES

There are no vaccines for Cryptococcus.

PREVENTION

Fluconazole: primary prophylaxis or preemptive treatment?

Several factors influence the decision of whether to implement primary prophylaxis against opportunistic infections: 1) the prevalence of disease, 2) the associated morbidity and mortality, 3) the efficacy of the prophylactic regimen and its impact on the overall outcome, and 4) the cost of the prophylactic regimen (monetary cost, side effects, and emergence of resistant organisms).

The indication for cryptococcal prophylaxis is not as apparent as for Pneumocystis prophylaxis. The prevalence of cryptococcal infection among AIDS patients was historically between 5 to 10% in the US and Europe, less than that of Pneumocystis.

In a randomized trial conducted in USA during AIDS epidemic, primary prophylaxis with fluconazole (200mg/d) in patients with CD4 <200/mm3, significantly reduced cryptococcal infections (breakthrough rate of 0.9%, 2/217) compared to placebo (breakthrough rate 7%, 15/ 211) (129). A survival benefit could not be demonstrated, however, as most infections resolved once therapy was instituted. Primary prophylaxis with a lower dosage of fluconazole (200 mg thrice weekly) given only to patients with CD4 ≤ 100/mm3 was as efficacious, and much less costly (152). An area of concern is the emergence of fluconazole-resistance with prolonged use of fluconazole (5). Although fluconazole-resistance was not documented in the breakthrough cryptococcal isolate in the study by Singh et al., the follow-up time of 12.1months was short (152).

Indeed, there have been anecdotal reports linking failure of primary fluconazole prophylaxis to cryptococcal strains exhibiting high fluconazole MICs; these reports involved patients with very low CD4 counts (< 25/mm3) who had received primary fluconazole prophylaxis for a prolonged period of time (> 12 months).

Although the prevalence of fluconazole-resistant Cryptococcus among patients receiving primary fluconazole prophylaxis is low, the experience with fluconazole resistance might be a harbinger for the emergence of fluconazole resistance among isolates in the future. Furthermore, the cost of fluconazole prophylaxis is significant: itis estimated that 7,800 doses of fluconazole would have to be used to prevent a case of invasive fungal infection (152). The cost, the risk of emergence of fluconazole resistance, and the lack of impact on survival argue against primary fluconazole prophylaxis countries with low cryptococcosis prevalence. These issues have led the U. S. Public Health Service/Infectious Diseases Society of America task force to decide that routine antifungal prophylaxis for patients with HIV infection should not be recommended. It is worth noting that the incidence of cryptococcal meningitis in HIV-infected patients decreased in 1992-1994, before the era of protease inhibitors; the most likely cause was the rising use of fluconazole for chronic treatment of thrush and Candida esophagitis. In resource-poor countries where cryptococcal infections are common, the mortality rate is high due to the lack of optimal therapy (46).

In 2005, a Cochrane systematic review selected randomized controlled trials using antifungal interventions for the primary prevention of cryptococcal disease in adults with HIV (21). 5 studies were eligible enrolling 1316 patients. Although Itraconazole or Fluconazole are very effective in reducing incidence of cryptococcal meningitis, neither have an effect on overall mortality. Another study enrolling 1519 patients in Uganda has shown that one cryptococcosis meningitis could be prevented by treating with fluconazole 45 patients with CD4 count <200/mm3 (124). Currently new strategies consist in pre-screening for cryptococcal antigen in patients who received HIV diagnosis before symptoms of meningitis (61, 102). The high negative predictive value of the cryptococcal antigen test allows a targeted screening approach that minimizes fluconazole costs, adverse drug events and development of azole resistance. In 2012, an algorithm has been proposed to assess management of patients with positive CRAG antigen (60).  CRAG antigen must be performed in every HIV-patient with CD4 count <100/mm3. If positive, a lumbar puncture could be offered to all patients if logistically feasible. Otherwise, meningitis symptoms must be screened in each patient and they should have a lumbar puncture if needed. A CNS disease should be treated as mentioned above. If there is no CNS disease,  fluconazole (800mg/d) can be started then HAART 2 weeks later. Flluconazole (400mg/d) is continued for 8 weeks then  fluconazole 200mg/d until CD4 >200 cells/mm3. Further prospective studies involving these strategies will provide more evidences to generalize CRAG pre-screening in areas with high HIV and cryptococcosis incidence.

There is currently no specific prevention for C. gattii infections. Screening of HIV patients at diagnosis for cryptococcosis with CRAG should not differ in high prevalence areas since CRAG sensitivity is similar between C. gattii andC. neoformans. Veterinary and environmental surveillance could determine new reservoirs of C. gattii and predict emergence of infections in humans. However, there is no strict correlation between environmental exposure and pathogenicity (148).

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Tables

Table 1. Classification of pathogenic Cryptococcus spp.

Species	Varieties	Serotypes	Molecular types
Cryptococcus neoformans	grubii	A	VNI, VNII
	neoformans	D	VNIV
	-	A/D (diploid hybrid)	VNIII
Cryptococcus gattii	-	B/C	VGI, VGII, VGIII, VGIV

Table 2. Risk factors for cryptococcosis adapted from [24][73].

HIV Infection ++
Solid organ Transplantation* ++
Hematological malignancies* (mostly chronic lymphoid disorders)
Connective tissue disease*
	Sarcoidosis
	Systemic lupus erythematosus
	Rheumatoid arthritis
Primary immunodeficiencies
	Idiopathic CD4 lymphopenia
	GATA 2 deficiency IL12Rβ1 deficiency
	X-linked CD40L deficiency
	Autoantibodies against IFN-γ
	Autoantibodies against GM-CSF
Immunosuppressive therapy
	Corticosteroids ++
	Fludarabine
	Alemtuzumab (anti-CD52)
	Anti-TNF-alpha agents
Diabetes mellitus
Renal failure or peritoneal dialysis
Chronic pulmonary disease or lung cancer
Cirrhosis
Pregnancy

* In combination with immunosuppressive therapy

Table 3. Characteristics of the four C. gattii genotypes adapted from reference [27]

	VGI	VGII (VGIIa ++)	VGIII	VGIV
Main clinical presentation	CNS disease	Lung disease	CNS disease	CNS disease
Host	Immunocompetent	Both	Immunocompetent	Immunocompromised
Fluconazole MICs (vs C. neoformans)	comparable	higher	comparable	comparable
Frequency	34%	47% (All VGII genotypes)	11%	8%
Predominant zone of distribution	Australasia, Asia, Europe, USA	America (Clonal) Australia (Non clonal)	South America	Africa
Outbreak strain	Sporadic cases in USA	British Columbia, Pacific Northwest	Sporadic cases in USA	none

Abbreviations: MIC (Minimal Inhibitory Concentration), CNS (Central Nervous System)

Table 4. Exhaustive work-up in case of a positive sample with Cryptococcus spp.

Lumbar puncture with a minimum of 3ml (60 drops) of CSF for India-ink testing, fungal cultures, CRAG titres

Measure of CSF opening pressure

Brain imaging (MRI or CT) (before lumbar puncture if focal signs or altered vigilance (GCS<8))

Urine sample for direct examination and cultures (at least in male patients)

Blood sample for culture

CRAG testing in serum

Chest imaging +/- cultures of sputum or bronchoalveolar lavage if clinic-radiological signs

Work-up for risk factors, testing for HIV status

Study of species diagnostic (C. gattii or C. neoformans)

Start antifungal therapy after sampling

Table 5. Proposed modal MICs and ECVs for non-typed C. neoformans strains

	Modal MICa(μg/ml)	ECV 95% (μg/ml)	ECV 99% (μg/ml)
Fluconazole	4	16	32
Itraconazole	0,12	0,5	1
Posaconazole	0,12	0,25	0,5
Voriconazole	0,06	0,25	0,25
Amphotericin B	0,25	1	2
Flucytosine	4	16	32

^a : Modal MICs of strains using RPMI medium and CLSI broth microdilution technique.

abbreviations: MIC (minimum inhibitory concentration), ECV (epidemiological cut-off value)

Table 6. Proposed modal MIC and ECV for cryptococcal strains of C. gattii (regardless of genotype)

	Modal MICa(μg/ml)	ECV 95% (μg/ml)	ECV 99% (μg/ml)
Fluconazole	4	8	16
Itraconazole	0,25	0,5	1
Posaconazole	0,12	0,5	1
Voriconazole	0,12	0,25	0,5
Amphotericine B	0,25	1	2
Flucytosine	2	4	8

^a : Modal CMI of strains using RPMI medium and CLSI broth microdilution technique.

abbreviations : MIC (minimum inhibitory concentration), ECV (epidemiological cut-off value)

Table 7. Recommended treatment for cryptococcosis

Therapy in AIDS Patients	Induction (14 days minimum with negative cultures at day 14)	Consolidation (8-10 weeks)	Maintenance (6-12 months minimum until stable immune reconstitution)
Meningo-encephalitis Standard therapy	Amphotericin B* (0,7-1mg/kg/d) + 5-FC 100mg/kg/d per os given in four divided doses	FCZ 400mg/d	FCZ 200mg/j
Meningo-encephalitis Alternative	AmB 14 days + FCZ (800mg/d) 10 weeks AmB 7 days + FCZ (1200mg/d) 5-FC + FCZ (800-1200mg/d) 2-6 weeks FCZ (1200mg/d) 12 weeks	ITZ (400mg/j)	ITZ (400mg/j)
Mild to moderate isolated pulmonary disease or skin disease	FCZ (400mg/d) 6-12 months	/	/
Severe extra CNS forms or fungemia	Treat as CNS disease	Treat as CNS disease	Treat as CNS disease

Abbreviations: AIDS (acquired immune deficiency syndrome), AmB (Amphotericin B), ITZ (Itraconazole), 5-FC (Flucytosine), FCZ (Fluconazole), CNS (Central Nervous System)

Therapy in transplant patients	Induction (14 days minimum with negative cultures at day 14)	Consolidation (8-10 weeks)	Maintenance (6-12 months)
Meningo-encephalitis Standard therapy	Liposomal AmB* (3-4mg/kg/d) + 5-FC 100mg/kg/d per os given in four divided doses *ABLC given at a dose of 3-5mg/kg is equivalent	FCZ 400-800mg/d	FCZ 200-400mg/j
Mild to moderate pulmonary disease or skin disease	FCZ (400mg/d) 6-12 months	/	/
Severe extra CNS forms or fungemia	Treat as CNS disease	Treat as CNS disease	Treat as CNS disease

Note: Amphotericin B deoxycholate is not recommended as first-line because of nephrotoxicity in this population. Alternatives regimen can be derived from AIDS patients if needed. A careful attention should be given to FCZ and calcineurin inhibitors interactions with a closely monitoring of serum level.

Abbreviations: AmB (Amphotericin B), ABLC (amphotericin B lipid complex), 5-FC (Flucytosine), FCZ (Fluconazole), CNS (Central Nervous System)

Therapy in non-HIV and non-transplant patients	Induction (2-6 weeks)	Consolidation (8-10 weeks)	Maintenance (6-12 months)
Meningo-encephalitis Standard therapy	Amphotericin B* (0,7-1mg/kg/d) + 5-FC 100mg/kg/d per os given in four divided doses *liposomal AmB given at a dose  of 3-4mg/kg is equivalent	FCZ 400-800mg/d	FCZ 200-400mg/j
Mild to moderate pulmonary diseaseor skin disease	FCZ (400mg/d) 6-12 months	/	/
Severe extra CNS forms or fungemia	Treat as CNS disease	Treat as CNS disease	Treat as CNS disease

Note: Alternatives regimen can be derived from AIDS patients if needed. There is no consensus for a 2 weeks or a 4-6 weeks induction therapy.

Abbreviations: AmB (Amphotericin B), FCZ (Fluconazole), CNS (Central Nervous System)

Table 8. Recommended treatment for cryptococcosis caused by C. gattii

C.gattii therapy in non-HIV patients	Induction	Eradication
Meningo-encephalitis	Amphotericin B* (0,7-1mg/kg/d) + 5-FC 100mg/kg/d per os given in four divided doses *liposomal AmB given at a dose  of 3-4mg/kg is equivalent Duration: 6 weeks	FCZ 400mg/d   Duration (total): 18 months
Isolated Lung disease	Amphotericin B* (0,7-1mg/kg/d) + 5-FC 100mg/kg/d per os given in four divided doses *liposomal AmB given at a dose  of 3-4mg/kg is equivalent Duration: 2 weeks	FCZ 400mg/d   Duration (total): 12 months

What's New

Datta K, Bartlett KH, et al. Spread of Cryptococcus gattii into Pacific Northwest region of the United States. Emerg Infect Dis. 2009 Aug;15:1185-91.

Deresinski, S. Immune Reconstitution Inflammatory Syndrome (IRIS) in HIV-Infected Patients with Cryptococcal Meningitis. Clin Infect Dis 2009;49: iii-iv.

Sun HY, Wagener MM, et al. Cryptococcosis in Solid-Organ, Hematopoietic Stem Cell, and Tissue Transplant Recipients: Evidence-Based Evolving Trends. Clin Infect Dis. 2009 Jun 1;48:1566-76.

Kontoyiannis DP, Lewis RE, et al.  Calcineurin Inhibitor Agents Interact Synergistically with Antifungal Agents In Vitro against Cryptococcus neoformansisolates: Correlation with Outcome in Solid Organ Transplant Recipients with Cryptococcosis.Antimicrob Agents Chemother 2008;52:735-738.

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