Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection

Our study included 135 C. gattii cases in which patients’ clinical history had been well documented (Table S1 in the supplemental material). They were from 5 continents, including 40 cases from Africa, 50 from North and South America, 12 from Asia, 7 from Europe, and 26 from Australia (Fig. 1A). Of these, 132 had central nervous system (CNS) infection, 2 isolated pneumonia, and 1 had a spinal epidural abscess. Of the original 135 isolates, 2 isolates from the patients SCA-2 (26) and LDS-107 (this study) had been discarded after being identified to the species level, and only 133 were available for molecular typing. We analyzed molecular types of 124 isolates in this study using the URA5 restriction fragment length polymorphism (RFLP) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and those of the remaining 9 isolates had been reported previously (24). Eighty-six (63.7%) of the 135 patients were originally considered to be lacking underlying risks (Fig. 1B to F; Table S1), and 49 (36.3%) patients were diagnosed as having recognized risks for cryptococcal infection, including HIV/AIDS, corticosteroid receipt, diabetes mellitus, cancer, or other immunosuppression. The 135 isolates represented all four major C. gattii lineages, which include 62 VGI (45.9%), 29 VGII (21.5%), 19 VGIII (14.1%), and 23 VGIV (17.0%), and 2 molecular type unknown (1.5%) (Fig. 2A). The molecular type determined by URA5 RFLP analysis and MALDI-TOF MS score agreed for all isolates tested except for one (99.2% agreement), PW-039 (Table S2; Fig. S1). The distribution of each molecular type in different geographic regions agreed with previous findings in that the VGI is not only the most frequent but is especially common in Asia, Europe, and Australia, while VGII and VGIII are most prevalent in North and South America (6, 33). The isolates of VGIV we collected were all from Africa, where VGIV is known to be the most common molecular type of C. gattii among clinical isolates, especially from people with HIV/AIDS (6, 15, 33, 34).

The 86 isolates from patients considered to be immunocompetent included 43 VGI (69.4% of total cases due to VGI), 22 VGII (75.9% of total cases due to VGII), and 19 VGIII (100% of total cases due to VGIII) (Fig. 3); this included no VGIV and 2 isolates of molecular type unknown. Although the distribution of molecular type across patients with and without immunosuppression is similar to the previous report by Chen et al. (6), our isolates from the patients considered immunocompetent were lower in VGI and VGII (69.4% and 75.9% versus 92.8% and 88.9%) while higher in VGIII (100% versus 75.8%) than the previous reports (6, 35) (Fig. 3). The total number of VGI isolates included in the previous report (6) was 77 from immunocompetent and 6 from immunocompromised patients, and VGII was reported from 96 immunocompetent and 12 immunosuppressed (6) compared to our isolates of 43 and 19, respectively, for VGI and 22 and 7, respectively, for VGII. Fisher’s exact test indicated the difference between the two data sets is statistically significant (P = 0.0012). The reason for this significant difference is likely due to differences in the patients’ geographic and clinical backgrounds. Although VGI is not known to be the most common molecular type in Africa (10, 33), nearly 20% of VGI isolates we collected for this study were from patients with HIV infection in South Africa (Table S1). The differences between the previous report and our data on the frequency of patients recognized as immunocompetent versus immunocompromised infected by VGII were also statistically significant (P = 0.0168). The difference can be explained by sampling difference in this study. Our collection contained more isolates of VGII from patients without recognized risks than those from immunosuppressed patients since our interest was in determining the prevalence of GM-SCF autoantibody as a hidden immune defect. A previous study (36) has shown that the patients infected by VGII in the United States are less likely to have recognized immunosuppression than those infected by VGI or VGIII. There was no significant difference between previous reports and this study in the proportion of VGIII (P = 0.7259) and VGIV (P = 0.1314) molecular types from immunocompetent versus immunosuppressed patients.

Plasma was available for detection of GM-CSF autoantibodies only from 32 of 135 C. gattii patients, which included 3 patients with known immunosuppressive conditions, such as lymphoma and receipt of corticosteroid, and 29 previously healthy with no recognized immunosuppression (Table 1; Fig. 2B). Four of the 29 previously healthy patients considered immunocompetent had other medical issues, including menometrorrhagia, increased regulatory T cells (Treg), and cerebrospinal eosinophilia, and 25 had no known comorbid conditions at the time of diagnosis for cryptococcosis. Although considered immunocompetent, 20 (69%) of those 29 patients were positive for neutralizing antibodies against the GM-CSF (Table 1; Fig. 4A and B), indicating certain immune system abnormality. Except for the patient with menometrorrhagia, all patients with other medical issues before the onset of cryptococcosis, such as increased Treg and CSF eosinophilia or history of corticosteroid therapy, were negative for GM-CSF autoantibodies. This indicates that 19 (76%) of the 25 C. gattii patients without any known other medical issue had GM-CSF autoantibody as a hidden risk factor (Table 1; Fig. 4A). Our observation also raises the question of whether increased Treg or CSF eosinophilia played any role in the patient’s susceptibility to C. gattii or they were the by-product of C. gattii infection. Corticosteroid therapy is a known risk for cryptococcosis (2, 37), although it is poorly understood whether short-term therapy for skin lesions, which does not result in clear immunosuppression, could predispose one to cryptococcosis. Figure 2C and Table 1 show that 9 of the 20 GM-CSF antibody-positive patients were infected by VGI (45%); the remaining 11 (55%) patients were infected by VGII (4, 20%), VGIII (5, 25%), and 2 (10%) by isolates of unknown molecular type. Frequency of the three molecular types, VGI to VGIII, among the cases with antibody-positive plasma reflected the geographic origin of the patients; nearly 67% of the antibody-positive VGI patients were from Australia/Asia, where VGI is the most prevalent type, and 33% were from North America (Table 1). Antibody-positive patients infected by VGII and VGIII were all from North America where VGII and VGIII are most common, except for one VGII from Australia (6, 33). The lack of VGIV among the GM-CSF antibody-positive patients is expected since the patients’ plasma for the antibody test all came from the geographic regions where cryptococcosis due to VGIV is rarely reported (6, 33). Our data clearly suggest that the presence of GM-CSF-neutralizing antibody is a hidden risk for C. gattii infection in otherwise healthy individuals and suggest that the antibody test should be included in the immunologic profiling when previously healthy patients present with C. gattii infections. It is important to detect the presence of anti-GM-CSF autoantibody since it could lead to pulmonary alveolar proteinosis (PAP), a chronic lung disease characterized by abnormalities of surfactant metabolism. PAP impairs the function of alveolar macrophages and can cause respiratory insufficiency (29). It is also noteworthy that one patient considered immunocompetent and lacking GM-CSF antibodies had a splice-site mutation in STAT3 when the whole exon of his genome was sequenced (data not shown). This suggests that exon sequencing may reveal other hidden risk factors in those seemingly immunocompetent patients infected by C. gattii.

Of the 135 patients, 49 (36.3%) had known immunosuppression. The major immunosuppression identified among patients infected by the molecular types VGI, VGII, VGIII, and VGIV were similar to those associated with C. neoformans infection, including HIV infection, cancer, corticosteroid therapy, and other immunosuppressive conditions (Table S1). There was a clear relationship between geographic origin of patients and the prevalence of C. gattii molecular type, but no clear relationship was found between C. gattii molecular type and the immunosuppressive conditions except that all VGIV isolates we studied were from HIV-associated cryptococcosis. The predominance of VGIV among the HIV-associated C. gattii cases was expected since nearly 90% of the HIV-associated C. gattii isolates we have collected were from South Africa and Botswana, where VGIV is the predominant molecular type found in AIDS patients (10, 15, 33). A strong association of VGIV with AIDS patients is supported by the fact that we found no VGIV isolates in our stock cultures from the pre-HIV era (38). The VGIV isolates are serotype C and are found mostly in tropical and subtropical regions, such as India, Sub-Saharan Africa, Mexico, and Columbia, but infrequently in temperate zones (10). Finding no VGIV infection among our 86 patients originally diagnosed as immunocompetent (Fig. 3) supports the fact that VGIV most commonly affects HIV+ patients more than any other type of immunosuppressed patients.

The prevalence of VGI (54.5%, 12/22 cases) among the isolates from South African HIV-associated C. gattii infection, however, was significantly higher than expected.

Previous surveys record no VGI infection (39) or only 27% of all C. gattii infections in South Africa (10, 40). All 13 isolates from HIV-associated C. gattii cases in Botswana were VGIV, and VGI was lacking even though the country borders with South Africa. Only three VGII and no VGIII isolates were found among the 40 isolates from C. gattii infections in Africa. This is not surprising since VGII is infrequently found in Africa (6, 33, 39, 40), and VGIII is rarely found outside the American continent (33, 35, 41, 42).

Of the 179 isolates from patients with known clinical history in the global MLST database, 47.5% (85/179) were from patients diagnosed as immunocompetent, and 52.5% (94/179) of the isolates were from those with immunosuppressive underlying conditions (Table 2). The frequency of C. gattii species complex recovered from patients recognized as immunocompetent among the 179 (https://mlst.mycologylab.org) (Table 2) was significantly lower (P < 0.0001) than in the previous report (6) or the present study. The disparity between the previous data and the MLST database on the frequency of the two types of patients is basically due to the differences in the frequency of the two groups infected by VGI and VGII lineages, but not those infected by VGIII and VGIV. The frequency of immunocompetent versus immunosuppressed patients infected by VGIII and VGIV was not significantly different (P  =  0.0963 and 0.3693, respectively) between the two data sets, although the sample size was too small to be statistically significant. It is noteworthy that VGII isolates outnumber VGI isolates in the global MLST database. This is likely due to the disproportionately high number of VGII isolates being subjected to MLST analysis since it has attracted more attention than any other molecular type owing to the high-profile VGII epidemic in British Columbia and the Pacific Northwest from 1999 to 2007.

The frequency of VGIII from immunocompetent patients in the global MLST database was lower than the number we observed in the present study. We speculate that such disparity in the frequency of VGIII isolates from immunocompetent patients between the MLST database and our study is likely due to the difference in the isolation era of the VGIII isolates. Unlike the previous report and the global MLST database, a large portion of our VGIII isolates were from the pre-HIV era in which the number of the immunosuppressed population was lower.

As Table 2 shows, the most common underlying immunosuppression in patients infected by the C. gattii species complex was HIV infection, regardless of the molecular types of the etiologic agent. Although the number of patients infected by VGIII and VGIV in the MLST database with clinical information is too small (15 and 12, respectively), diabetes was commonly identified as an underlying condition in infection by all four lineages. Underlying conditions, such as systemic lupus erythematosus (SLE), were found among patients infected by VGI and VGII. Other underlying conditions, such as chronic myelogenous leukemia (CML), CD4 lymphopenia, and cirrhosis, were found among VGI patients. These data indicate that the underlying immunosuppressive conditions which predispose patients to infections by the C. gattii and C. neoformans species complex overlap and there is no unique condition that predisposes patients only to the C. gattii species complex.

The important finding in the present study, however, is that the seemingly immunocompetent people with anti-GMCSF autoantibodies in their plasma are more often infected by C. gattii species complex than C. neoformans species complex.

It is important to recognize the C. gattii species complex as an opportunistic pathogen. Since GM-CSF-neutralizing antibodies are one of the most common hidden underlying conditions in previously healthy C. gattii patients, the antibody test should be implemented in C. gattii patients with no known immunosuppression. This test is especially important for patient management because patients with GM-CSF antibody may eventually develop further complications, such as pulmonary alveolar proteinosis (30).

The C. gattii patients considered immunocompetent and negative for the GM-CSF antibodies could carry other hidden risk factors, which may be revealed by whole-exon sequencing of the patient’s genome.

The known immunosuppressive underlying conditions that predispose patients to C. gattii infection are no different from those to C. neoformans infection except that the presence of anti-GM-CSF autoantibodies in plasma may pose more risk for infection by C. gattii species complex than the C. neoformans species complex.

Clinical data from 135 global cases of C. gattii infection were retrieved from studies approved by local institutional review boards. Isolates (n = 124) recovered from cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BAL fluid)/lung tissue, or extrapulmonary sources were available for determination/confirmation of molecular types in our laboratory. Isolates were streaked on YPD agar (glucose 2%, yeast extract 1%, and peptone 2%) to test for their purity. Purified isolates were stored in 25% glycerol at −80°C until use. The species identity of each strain was confirmed by culturing on CGB media (43) in addition to testing for urease production (44), melanin production (45), and growth at 37°C.

Clinical data correlating with infections characterized to MLST type was obtained from the cryptococcal database (https://mlst.mycologylab.org). Clinical data were available for 179 of a total of 1,454 patients whose isolates had been characterized by MLST (Table 2).

Molecular types (VGI to VGIV) of the isolates were determined via URA5 gene restriction fragment length polymorphism (RFLP) analysis (34) and MALDI-TOF MS (46). DNA from each culture was isolated (47), and the URA5 gene was amplified using two PCR primers, URA5 (5′-ATGTCCTCCCAAGCCCTCGACTCCG-3′) and SJ01 (5′-TTAAGACCTCTGAACACCGTACTC-3′). The PCR products were double digested with Sau96I and HhaI and were separated by 3% agarose gel electrophoresis (12) to compare with the URA5 RFLP patterns of WM179 (VGI), WM178 (VGII), WM161 (VGIII), and WM779 (VGIV). For MALDI-TOF MS analysis, MALDI target plates of the isolates were prepared as previously described (46). MALDI-TOF MS was performed on the MicroFlex LT mass spectrometer and analyzed by Biotyper 3.1 software (Bruker Daltonics, Inc.). Spectra were acquired over a mass/charge (m/z) ratio of 2,000 to 20,000. Spots were measured using 250 laser shots at 60 Hz in groups of 50 shots per sampling area. Both the Bruker and previously developed NIH custom databases included 22 C. gattii strains representing VGI to VGIV. The best of two scores was recorded, and a cutoff of ≥1.80 was considered acceptable.

Frozen plasma was available from 32 people with C. gattii infection: 29 people had no known immunosuppression, and 3 had lymphoma (n = 1) and/or had received corticosteroids (n = 2). Among the 32, 12 cases have already been reported (Table 1), and 20 cases were from the present study. The presence of anti-GM-CSF autoantibodies in plasma samples was tested using previously described particle-based methodology (24, 30). Briefly, fluorescent magnetic beads (Bio-Rad) were covalently coupled to 2.5 μg of human GM-CSF (R&D Systems). Beads were transferred into magnet 96-well plate and incubated for 30 min with diluted plasma (1:100) on microplate shaker, washed, and incubated with streptavidin-phycoerythrin (Bio-Rad)-labeled goat anti-human IgG (eBioscience) for an additional 30 min. The plate was then run on Bio-Plex (Bio-Rad) instrument. Normal plasma and GM-CSF autoantibody-positive plasma were used as negative and positive controls, respectively. The antibodies’ neutralizing functionality was evaluated by flow cytometry to detect the phosphorylation of STAT5 in normal peripheral blood monocytes (PBMCs) after stimulation with GM-CSF in presence of the patient’s plasma as described (24, 30).

Fisher’s exact test (Prism 8; GraphPad Software, San Diego, CA) was used to determine if there is a significant difference between the previous data and our data for the frequency of each molecular type from patients with immunosuppression versus those with no known immunosuppression. A P value of <0.05 was considered significant.