Cryptococcosis is an opportunistic and potentially life-threatening infection, not only occurring in immunocompromised patients, including those with HIV/AIDS and autoimmune diseases, and transplant recipients, but also posing threat to apparently immunocompetent subjects (Bratton et al., 2012; Kwon-Chung et al., 2017; Beardsley et al., 2019; Ellis et al., 2019). The current genus Cryptococcus contains 10 species, of which seven are pathogenic to humans and animals: the two members of the Cryptococcus neoformans species complex that are C. neoformans sensu stricto (serotype A; AFLP1/VNI, AFLP1A/VNB/VNII, AFLP1B/VNII), Cryptococcus deneoformans (serotype D; AFLP2/VNIV); and the six species in the Cryptococcus gattii species complex: Cryptococcus gattii sensu stricto (serotype B; AFLP4/VGI), Cryptococcus bacillisporus (serotype B&C; AFLP5/VGIII), Cryptococcus deuterogattii (serotype B; AFLP6/VGII), Cryptococcus tetragattii (serotype C; AFLP7/VGIV) and Cryptococcus decagattii (AFLP10/VGIV/VGIIIc) (Hagen et al., 2015, 2017; Kwon-Chung et al., 2017; Ashton et al., 2019). Furthermore, a new lineage of Cryptococcus gattii species complex (serotype B, VGV) was discovered by Farrer et al. in 2019, while cryptococcosis by other species has rarely been documented till date (Farrer et al., 2019). Multilocus sequence typing (MLST) based on seven housekeeping genes allows for the classification of most clinical Cryptococcus strains into varied sequence types (STs), among which ST5 is mainly reported from mainland China (Fan et al., 2016).

The genus Cryptococcus usually invades the central nervous system (CNS) and results in cryptococcal meningitis and then a high mortality rate (Lahiri et al., 2019). In addition, meningitis by C. neoformans species complex occurred more frequently than that by C. gattii species complex (Yuchong et al., 2012; Hagen et al., 2015; Smith et al., 2015; Ferreira-Paim et al., 2017; Thanh et al., 2018; Rakotoarivelo et al., 2020), and this might be explained by that the former is globally distributed, while the latter seems to be geographically restricted (Kidd et al., 2007; Chen et al., 2014; Smith et al., 2015; Firacative et al., 2016; May et al., 2016; Souto et al., 2016). Furthermore, both amphotericin B and fluconazole remain the mainstay treatment in cryptococcal meningitis (Yao et al., 2014; Beardsley et al., 2019). The resistance patterns to them are documented worldwide, and geographic variability is noted (Fan et al., 2016; Nyazika et al., 2016).

Understanding the epidemiological characteristics of local Cryptococcus strains and clinical features of cryptococcal meningitis is essential for the development of efficient diagnosis and treatment strategy. Studies on that are rare in China (Fan et al., 2016; Guo et al., 2016; Liu et al., 2017; Cao et al., 2019), and they focused on pediatric patients (Guo et al., 2016), clinical features (Liu et al., 2017; Cao et al., 2019), or molecular and antifungal resistance characteristics (Fan et al., 2016), respectively. Continuous and comprehensive monitoring of the epidemical changes is crucial for the treatment and prevention of cryptococcosis. The present study involved 110 cryptococcal meningitis cases from 2013 to 2018 in Xiangya Hospital of Central South University (XHCSU), Hunan, China. The molecular characteristics and antifungal agent susceptibility data of Cryptococcus strains, and the clinical, demographic features and therapeutic outcomes were documented to help improve timely diagnosis and reduce the mortality rate.

Cryptococcus, usually acquired by inhalation, causes pneumonia and cryptococcemia, and exhibits a propensity to disseminate to the brain and presents as meningitis (Fan et al., 2016; O’Halloran et al., 2017; Cao et al., 2019; Tsai et al., 2019). Cryptococcal meningitis is the most severe and common form of cryptococcosis (Yuchong et al., 2012; Rajasingham et al., 2017). Of 204 cases of cryptococcosis in a US hospital from 1996 to 2009, 62% (126/204) were cryptococcal meningitis (Bratton et al., 2013). In the current retrospective study, we analyzed the clinical features of patients with culture-confirmed meningitis. Meanwhile, we also provided information about the microbial characteristics of C. neoformans/gattii species complex isolated.

In this study, of 110 Cryptococcus strains from meningitis subjects, C. neoformans s.s. predominated (95.5%, 105/110), while C. deuterogattii (4, 3.6%) and C. deneoformans (1, 0.9%) were rarely identified. By comparison, Chen et al. also showed that 93.0% (120/129) C. neoformans s.s. and 7.0% (9/129) C. gattii (VGI) were isolated from 1980 through 2006 from cryptococcosis patients in 16 provinces of China (Chen et al., 2008). In Taiwan during 1997–2010, C. neoformans and C. gattii species complexes accounted for 95.9% (210/219) and 4.1% (9/219), respectively, and the predominant molecular type was also VNI (94.1%, 206/219) (Tseng et al., 2013). However, the species and molecular types distribution of C. neoformans and C. gattii species complex might be geographically varied, presenting a peculiar epidemiological profile (Chen et al., 2014; Smith et al., 2015; Alves Soares et al., 2019). For example, in 62 Cryptococcus strains collected from meningitis cases from 2006 to 2010 in Brazil, C. gattii species complex accounted for 21%, tremendously higher than that in mainland China (Matos et al., 2012). In addition, there are several studies in Brazil also showing a high prevalence of C. neoformans species complex (Andrade-Silva et al., 2018; Ponzio et al., 2019). Infection due to C. gattii species complex is more prevalent in the Northeast of Brazil (Matos et al., 2012), despite has been described in the Southeast (Vilas-Boas et al., 2020). Furthermore, in the current study, all four C. gattii species complex strains belonged to VGII molecular type; however, in Colombia, VGII molecular type was identified only in 54.3% C. gattii species complex strains (Escandon et al., 2018). Also, Cryptococcus deuterogattii molecular type is the most prevalent molecular type in Brazil and it seems to be transmitted from South America to North America (Souto et al., 2016; David and Casadevall, 2019).

ITS and MLST are often applied in the evaluation of the genetic relationship among Cryptococcus strains (Fan et al., 2016). Differences in ST distribution have previously been noted across varied populations (Beale et al., 2015; Desnos-Ollivier et al., 2015; Fan et al., 2016). In the current study, Cryptococcus strains showed a low degree of genetic diversity, and only seven STs were detected in 110 Cryptococcus strains, in which 89.1% were represented by ST5. The ST5 lineage is the predominant ST reported from China. For example, in a study in China, in 303 C. neoformans strains from 10 hospitals over 5 years, only 12 STs were identified, and ST5 accounted for 89.2% (272/305) (Fan et al., 2016). In another Chinese study, MLST analysis assigned 41 C. neoformans strains into 5 STs, and ST5 accounted for 82.9% (34/41) (Wu et al., 2015). There were two HIV/AIDS patients among the non-ST5 C. neoformans cases (25%), significantly more than in the ST5 group (2.2%, 2/91). In addition, in 183 clinical and environmental isolates of Cryptococcus strains from Thailand, Southeast Asia, population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only 10 nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating their strains (Simwami et al., 2011). In an article in Laos, the strains were dominated (83%) by STs 4 and 6, while in Vietnam, the strains were dominated by the ST4/ST6 (35%) and ST5 (48%) lineages (Thanh et al., 2018). Taken together, the molecular type VNI C. neoformans species complex strains with low diversity of STs predominate in China and around Asia, and the predominant STs of Cryptococcus strains might differ geographically. The molecular diversity across Southeast Asia has been explained by ecological rather than human host factors (Thanh et al., 2018).

A sex bias is observed in cryptococcal studies. The prevalence of cryptococcosis is consistently common in males in both HIV-positive and -negative patients (Guess et al., 2018). In our study, male patients accounted for 60.9% (67/110). Similarly, male predominance was also shown in Wuhan, China (75.6%, 68/90 cryptococcal meningitis cases) (Cao et al., 2019), in Beijing, China (73%, 38/52 pediatric patients with disseminated cryptococcosis) (Gao et al., 2017), in Colombia (79.6% in the general population and 84.4% in HIV/AIDS patients) (Escandon et al., 2018), and in Brazil (69% in 5,755 recorded deaths related to cryptococcosis) (Alves Soares et al., 2019). Furthermore, in our study, 16.4% (18/110) were elderly patients (≧65 years, 13 males and 5 females). In a study in Taiwan, elderly patients were more vulnerable to cryptococcal meningitis than those aged <65 years, and fewer males were affected in the elderly group (57.9%, 22/38) than in non-elderly group (78.7%, 48/61) (Tsai et al., 2019). The sex bias has been explained by the increased incidence of the HIV epidemic in males (Liu et al., 2017; Guess et al., 2018). Nevertheless, in our study, only 4.55% (5/110) were HIV-positive. Furthermore, Cryptococcus species are commonly distributed in the environment and associated with bird excreta (mostly pigeon droppings), soil and wood debris (Alves Soares et al., 2019), and males are more likely to work outside. This could also partially explain the sex disparity (Guess et al., 2018). In the present study, only 9 (8.2%) had been in contact with pigeon droppings, and similarly, only 10 (10/52, 19.2%) cases had a history of exposure to pigeon droppings in pediatric patients in China (Gao et al., 2017). Therefore, the reasonable explanation of male predominance remained elusive.

Cryptococcosis presents in both immunocompromised and immunocompetent subjects (Lahiri et al., 2019). Some cryptococcal meningitis patients might have predisposing factors, which change over time and are geographically varied (O’Halloran et al., 2017; Ellis et al., 2019). The common risk factors for cryptococcosis generally involve HIV/AIDS, organ transplant, corticosteroid use, and malignancy (Hong et al., 2017; Kashef Hamadani et al., 2018; Ellis et al., 2019). In our study, 60% (66/110) of the strains were isolated from patients with apparent risk factors, and the underlying status included hepatitis and cirrhosis (13, 11.8%), autoimmune disorders (12, 10.9%) and diabetes mellitus (5, 4.5%). Comparatively, in Taiwan during 1997–2010, HIV infection was the most common underlying condition (54/219, 24.6%), and among HIV-negative patients, liver diseases (HBV carrier or cirrhosis) were common (30.2%) and 15.4% did not have any underlying condition (Tseng et al., 2013). The current study was conducted in XHCSU, China. It is not a reference service for HIV. HIV-affected patients will be transferred to the specific infectious disease hospital. In another study in China, 71% (91/129) of cryptococcosis cases during 1985–2006 had no apparent risk factor and only 8.5% (11/129) were HIV/AIDS patients (Chen et al., 2008). In addition, in a study in the Southwest of China, among 85 patients with CM were identified, 32 (37.6%) were HIV-uninfected patients (Liu et al., 2017). The less HIV-infected patients might be explained by the bias in the population studied. As previously reviewed, cryptococcosis in non–HIV-infected patients, compared to those HIV-infected, the substantial differences in terms of natural history, clinical course, diagnosis, and outcome should be noted in China, especially the transplant recipients with cryptococcosis (Pappas, 2013).

Different from C. neoformans species complex, C. gattii species complex-caused cryptococcosis occurs mainly in non-elderly and immunocompetent hosts (Chen et al., 2008; Alves Soares et al., 2019). We compared the clinical and laboratory data of meningitis cases caused by C. neoformans and C. deuterogattii, and found that four C. deuterogattii-infected patients had no underlying diseases, significantly fewer than among C. neoformans cases (100 vs. 36.4%). Similarly, in Colombia between 1997 and 2011, 91.1% (41/45) C. gattii species complex-caused cases had no predisposing factors and only 6.7% (3/45) were HIV-positive (Lizarazo et al., 2014). It is documented in a previous study that dual tubercular/cryptococcal meningitis was the most frequent (54.0%) and most easily misdiagnosed (95.2%, 40/42) co-infection (Fang et al., 2017). However, in our study, only three tuberculosis patients (2.73%, 3/110) were detected, which could be explained by geographical differences (Fang et al., 2017).

Cryptococcal meningitis is highly lethal without early diagnosis and proper treatment. Its clinical presentation is often not specific (Liu et al., 2017; Alves Soares et al., 2019). In the current study, headache was the most common presentation (85, 77.3%), followed by fever (52, 47.3%), and nausea/vomiting (32, 29.09%), in accordance with another study involving 90 cryptococcal meningitis patients (Cao et al., 2019). In 45 cryptococcosis cases caused by C. gattii species complex, their clinical features also included headache (80.5%) and nausea/vomiting (56.1%) (Lizarazo et al., 2014). Additionally, in our study, in 103 patients, the mortality rate was 35.0% (36/103), lower than that in Colombia (47.5%) (Escandon et al., 2018). In Taiwan, patients infected with C. gattii species complex, compared to those with C. neoformans species complex, were more likely to have a higher 10-week mortality rate (44.4 vs. 22.2%) (Tseng et al., 2013). As is the case with our study, in four C. deuterogattii meningitis subjects, two died.

The emergence of Cryptococcus strains with resistance or elevated MIC above ECVs is of concern as well. Antifungal susceptibility revealed species-specific differential susceptibility, but the acquired resistance was still an infrequent phenomenon (Hagen et al., 2016). Fluconazole and amphotericin B are the most commonly used antifungal agents for the treatment of cryptococcal meningitis. As shown in Table 4 and Figure 4, 8.6% (9/105), 6.6% (7/105), and 8.6% (9/105) of C. neoformans s.s. strains had higher MIC values than the recommended ECVs of fluconazole, 5-fluorocytosine, and amphotericin B, respectively. All four C. deuterogattii strains were uniformly designated as WT to the above antifungal agents. In a study from Denmark, all 108 clinical C. neoformans and C. gattii species complex strains were amphotericin B susceptible (Hagen et al., 2016). In a Chinese study from 10 hospitals over 5 years, among 303 C. neoformans species complex strains, 7.6% (23/303) were non-WT to fluconazole, however, seven C. gattii species complex strains had WT MICs to all drugs tested except for one C. gattii species complex strain with a fluconazole MIC of 16 μg/ml (Fan et al., 2016). By comparison, the susceptibilities of the 52 Cryptococcus spp. strains in Zhejiang, mainland China, only one C. neoformans s.s. (1/51, 2.0%) was non-WT to amphotericin B (1.0 mg/L) and one (2.0%) non-WT to fluconazole (16 μg/ml) (Fu et al., 2019). Taken together, the elevated MIC values for antifungal agents are rarely observed and partially related to the use of unstandardized regimens, which should attract more attention.

It is recommended that amphotericin B and 5-flucytosine are the preferred agents for the initial or induction therapy, whereas the azoles (especially fluconazole) are generally used in the consolidation and maintenance phases of therapy (Beale et al., 2015; Fan et al., 2016). In 204 adults with cryptococcosis from 1996 to 2009 in the United States of America, 5-flucytosine exposure was demonstrated to be associated with a lower mortality rate (Bratton et al., 2013). In a previous study, the proportion of patients receiving amphotericin B-containing regimen was 70.6% (48/68), and had a lower 30-day mortality rate than those treated with other regimens, but the difference was not statistically significant (16.7%, 8/48 vs. 25.0%, 5/20) (Fu et al., 2019). In our study, the regimen containing amphotericin B, fluconazole and 5-flucytosine demonstrated a lower mortality rate. Furthermore, as revealed in Table 6, the elevated MIC value for fluconazole was statically related to a higher mortality rate (22.8 vs. 18.9%, P = 0.034). Nevertheless, the conclusion should be interpreted with caution due to the small size of the present study.

In summary, the present study demonstrated that more C. neoformans species complex isolates (mainly C. neoformans s.s.) were observed than C. gattii species complex (mainly C. deuterogattii). Low prevalence of HIV patients with cryptococcal meningitis and relatively high non-WT rates to Amphotericin B and fluconazole in Cryptococcus strains in China were also noted. The study will be helpful for understanding the genetic diversity of Cryptococcus strains and for decision making in the context of the diagnosis, treatment and prevention strategies in cryptococcosis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

The institutional review boards at the XHCSU approved the study protocol. The written informed consent from participants was waived and the data were analyzed anonymously.

YL and MZ isolated the Cryptococcus spp. and performed the tests. JY collected the clinical data. ZL collected the laboratory data. YL and BL made substantial contributions to conception and design, and drafted the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.