Rapid duplex flap probe-based isothermal assay to identify the Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is a life-threatening invasive fungal infection with significantly increasing mortality worldwide, which is mainly caused by Cryptococcus neoformans and Cryptococcus gattii. These two species complexes have different epidemiological and clinical characteristics, indicating the importance of accurate differential diagnosis. However, the clinically used culture method and cryptococcal capsular antigen detection couldn't achieve the above goals. Herein, we established a novel duplex flap probe-based isothermal assay to identify the Cryptococcus neoformans and Cryptococcus gattii within 1 hour. This assay combined the highly sensitive nucleic acid isothermal amplification and highly specific fluorescence probe method, which could effectively distinguish the sequence differences of the two species complexes using two different fluorescence flap probes in a single reaction system. This novel method showed excellent detection performance with sensitivity (10 copies/µL each) and specificity (100%) compared to traditional culture and sequencing methods. Furthermore, we applied this method to spiked clinical samples, 30 cerebrospinal fluids and 30 bronchoalveolar lavage fluids, which kept good detection performance. This novel rapid duplex flap probe-based isothermal assay is a promising and robust tool for applications in differential diagnosis of the Cryptococcus neoformans and Cryptococcus gattii in clinical settings, especially when clinical suspicion for cryptococcal disease is high and epidemiological studies.

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Cryptococcus gattii strains with a high phagocytosis phenotype by macrophages display high pathogenicity at the early stage of infection in vivo.

Cryptococcus gattii (Cg) is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages, causing life-threatening cryptococcosis in both immunocompetent and immunocompromised individuals. Cryptococcus-macrophage interactions are crucial for cryptococcosis prognosis. However, the relationship between Cg pathogenicity and phagocytosis by macrophages has not yet been investigated in depth. In this study, a series of in vitro and in vivo experiments were conducted to investigate the interaction between macrophages and Cg. Flow cytometry was used to detect the phagocytic phenotypes of the Cg strains within macrophages. Scanning electron microscopy, transmission electron microscopy, and immunofluorescence were used to observe phagocytosis and proliferation, respectively. Survival and lung fungal burden tests were also performed. Our results show that Cg cells display different phagocytosis phenotypes, which are independent of the molecular type. Within macrophages, the high phagocytosis phenotype (HP) strains obtain higher intracellular proliferation than the low phagocytosis phenotype (LP) strains. At the early stage of infection in vivo, HP-inducing permissive granulomas within the lungs seldom limit the dissemination of cryptococci. In addition, HP strains could inhibit the formation of M1-type macrophages, proliferate intracellularly and disseminate extracellularly, and cause hypoxia induced by mucus and acidic polysaccharide accumulation in pulmonary alveoli much earlier than LP strains in vivo. Our work reveals that Cg displays diverse interactions with macrophages, which may enhance our understanding of the pathogenicity of this life-threatening pathogen.

Virulence profiling of Cryptococcus gattii isolates in China: insights from a multi-center study.

IMPORTANCE: Our study indicates that the molecular typing of Cryptococcus gattii is unrelated to virulence. The integration of animal experiments and clinical prognosis demonstrated that pathogenicity did not exhibit a direct correlation with in vitro virulence phenotypes or molecular genotypes, emphasizing the intricate nature of virulence. In conclusion, our research holds the potential to provide valuable insights into understanding the microbiological attributes of C. gattii in China.

A severe case of disseminated cryptococcosis in a young French bulldog living in South-East Queensland caused by Cryptococcus gattii VGII.

BACKGROUND: Cryptococcus is one of the most common systemic mycosis worldwide, infecting young adults of the large to giant breed dogs. Infection is commonly acquired from the environment via the sinonasal cavity as the main portal of entry. It either remains there, or spreads to the central nervous system (CNS) and the eye (optic nerve and retina) by penetration of the cribriform plate, or haematogenously to other viscera. Lung involvement is uncommon in cats and dogs in contrast to human and equine patients. Whilst there is a wide genetic diversity amongst Cryptococcus neoformans and Cryptococcus gattii isolates along the West Coast and Northern parts of Australia, the molecular diversity of C. gatti is considered very low on the East Coast of Australia, with a huge preponderance of VGI cases. We report on a young small breed brachycephalic dog that presented with extreme gastrointestinal and respiratory signs, but no CNS involvement. It is the first reported case of C. gattii VGII genotype in a companion animal from Queensland. CASE REPORT: A 9-month old female entire French Bulldog presented initially for diarrhoea. Clinical progression was accompanied by the development of respiratory signs, so the patient was referred to a 24 h care facility. Following hospitalisation, the patient became hypoxemic requiring mechanical ventilation. A bronchoalveolar lavage performed antemortem confirmed abundant Cryptococcal spp. Further culturing and genotyping identified the species as Cryptococcus gattii VGII. Post-mortem findings indicated gross gastrointestinal and mesenteric involvement, with possible dissemination to the local mesenteric lymph node and lungs. CONCLUSION: This case describes a rare example of a Cryptococcus spp suspected of disseminating from the gastrointestinal tract to the lungs, without involvement of the CNS. The observation of this finding in a small brachycephalic breed is unusual, and the finding of genotype VGII on the East Coast of Queensland is extremely unusual as there is no prior travel history of the dog or owners. The presence of a miliary lung pattern with primary gastrointestinal disease in a small breed dog warrants adding cryptococcosis to the differential diagnosis.

Reduced Susceptibility to Azoles in Cryptococcus gattii Correlates with the Substitution R258L in a Substrate Recognition Site of the Lanosterol 14-α-Demethylase.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 µg/mL) and voriconazole (1 µg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

The small heat shock protein Hsp12.1 has a major role in the stress response and virulence of Cryptococcus gattii.

Cryptococcus gattii is one of the etiological agents of cryptococcosis. To achieve a successful infection, C. gattii cells must overcome the inhospitable host environment and deal with the highly specialized immune system and poor nutrients availability. Inside the host, C. gattii uses a diversified set of tools to maintain homeostasis and establish infection, such as the expression of remarkable and diverse heat shock proteins (Hsps). Grouped by molecular weight, little is known about the Hsp12 subset in pathogenic fungi. In this study, the function of the C. gattii HSP12.1 and HSP12.2 genes was characterized. Both genes were upregulated during murine infection and heat shock. The hsp12.1 Δ null mutant cells were sensitive to plasma membrane and oxidative stressors. Moreover, HSP12 deletion induced C. gattii reactive oxygen species (ROS) accumulation associated with a differential expression pattern of oxidative stress-responsive genes compared to the wild type strain. Apart from these findings, the deletion of the paralog gene HSP12.2 did not lead to any detectable phenotype. Additionally, the double-deletion mutant strain hsp12.1 Δ /hsp12.2 Δ presented a similar phenotype to the single-deletion mutant hsp12.1 Δ, suggesting a minor participation of Hsp12.2 in these processes. Furthermore, HSP12.1 disruption remarkably affected C. gattii virulence and phagocytosis by macrophages in an invertebrate model of infection, demonstrating its importance for C. gattii pathogenicity.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

The Cryptococcus gattii species complex: Unique pathogenic yeasts with understudied virulence mechanisms.

Members of Cryptococcus gattii/neoformans species complex are the etiological agents of the potentially fatal human fungal infection cryptococcosis. C. gattii and its sister species cause disease in both immunocompetent and immunocompromised hosts, while the closely related species C. neoformans and C. deneoformans predominantly infect immunocompromised hosts. To date, most studies have focused on similarities in pathogenesis between these two groups, but over recent years, important differences have become apparent. In this review paper, we highlight some of the major phenotypic differences between the C. gattii and neoformans species complexes and justify the need to study the virulence and pathogenicity of the C. gattii species complex as a distinct cryptococcal group.

Comparison of features and outcomes between HIV-negative patients with Cryptococcus gattii meningitis and Cryptococcus neoformans meningitis in South China.

OBJECTIVES: The objective of this study is to compare the epidemiologic, clinical, laboratory, and imaging features, and outcomes in patients with Cryptococcus gattii meningitis (CGM) and Cryptococcus neoformans meningitis (CNM). METHODS: We performed a retrospective study of HIV-negative patients with CGM and CNM (2015-2021) distinguished by metagenomic next-generation sequencing in cerebrospinal fluid in South China. RESULTS: A total of 81 patients (17 CGM, 64 CNM) were enrolled (72.8% male, median age 49 years, range 21-77 years), and CGM patients were younger (median, 43 vs 53 years, p = .005). Of 17 CGM, VGI and VGII accounted for 70.6% and 29.4%, respectively. CGM patients had less underlying diseases (7/17 [41.2%] vs 48/64 [75%], p = .018) and focal neurologic deficit (3/17 [17.6%] vs 35/64 [54.7%], p = .022), had higher intracranial pressure (15/17 [88.2%] vs 25/64 [39.1%], p = .002), more meningeal enhancement (14/17 [82.4%] vs 32/64 [50%], p = .034), less parenchymal involvement (median, 1 vs 3, p = .018), more lung cryptococcomas (6/12 [50%] vs 6/47 [12.8%], p = .014), faster CSF fungal clearance (p = .004), less complications (median, 1 vs 3, p < .001), and more favourable outcomes (16/17 [94.1%] vs 41/64 [64.1%], p = .035). CONCLUSIONS: This study demonstrated that species identification helps to guide therapy and predict outcomes.

Performance of Metagenomic Next-Generation Sequencing for the Diagnosis of Cryptococcal Meningitis in HIV-Negative Patients.

Objectives: Metagenomic next-generation sequencing (mNGS) has been applied more and more widely for the diagnosis of infectious diseases, but its performance in the diagnosis of cryptococcal meningitis (CM) remains unclear. Methods: Cerebrospinal fluid (CSF) samples from 197 HIV-negative patients with suspected central nervous system infections were tested simultaneously by mNGS and routine methods [India ink staining, fungal culture, or cryptococcal antigen (CrAg) tests]. The performance of mNGS was evaluated. Results: Of the 197 enrolled cases, 46 (23.4%) cases were finally diagnosed with CM, including 43 (93.5%) Cryptococcus neoformans infections and 3 (6.5%) Cryptococcus gattii infections. The sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of mNGS were 93.5% [95% confidence interval (CI) at 86.4%~100.0%], 96.0% (95% CI at 92.9%~99.1%), 87.8%, 98.0%, and 95.4%, respectively. Comparing to the conventional diagnostic methods, the sensitivity and concordance rate of mNGS were slightly lower than those of CrAg tests (97.4%) but higher than those of India ink (63.0%) and culture (76.7%). Besides, mNGS had a sensitivity of 100.0% against culture. It should be noted that mNGS could identify Cryptococcus at species level; C. gattii of the 3 cases was only distinguished by mNGS. Conclusions: CSF mNGS can be considered as a supplementary test to diagnose CM and directly distinguish C. gattii from C. neoformans in clinical specimens.

Case Report: Invasive Cryptococcosis in French Guiana: Immune and Genetic Investigation in Six Non-HIV Patients.

Objectives: We describe the clinical, mycological, immunological, and genetic characteristics of six HIV-negative patients presenting with invasive cryptococcosis. Methods: Patients with cryptococcosis without any of the classical risk factors, such as HIV infection, followed at Cayenne Hospital, were prospectively included. An immunologic and genetic assessment was performed. Results: Five male patients and one female patient, 5 adults and one child, were investigated. All presented a neuromeningeal localization. Cryptococcus neoformans var. gattii and C. neoformans var. grubii were isolated in two and three patients, respectively, whereas one patient could not be investigated. Overall, we did not observe any global leukocyte defect. Two patients were found with high levels of circulating autoantibodies against Granulocyte macrophage-colony stimulating factor (GM-CSF), and none had detectable levels of autoantibodies against Interferon gamma (IFN-γ) Sequencing of STAT1 exons and flanking regions performed for four patients was wild type. Conclusion: To better understand cryptococcosis in patients with cryptococcosis but otherwise healthy, further explorations are needed with repeated immune checkups and strain virulence studies.

Gene, virulence and related regulatory mechanisms in Cryptococcus gattii.

Cryptococcus gattii is a kind of basidiomycetous yeast, which grows in human and animal hosts. C. gattii has four distinct genomes, VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7. The virulence of C. gattii is closely associated with genotype and related stress-signaling pathways, but the pathogenic mechanism of C. gattii has not been fully identified. With the development of genomics and transcriptomics, the relationship among genes, regulatory mechanisms, virulence, and treatment is gradually being recognized. In this review, to better understand how C. gattii causes disease and to characterize hypervirulent C. gattii strains, we summarize the current understanding of C. gattii genotypes, phenotypes, virulence, and the regulatory mechanisms.

A Possible Link between the Environment and Cryptococcus gattii Nasal Colonisation in Koalas (Phascolarctos cinereus) in the Liverpool Plains, New South Wales.

Cryptococcosis caused by yeasts of the Cryptococcus gattii species complex is an increasingly important mycological disease in humans and other mammals. In Australia, cases of C. gattii-related cryptococcosis are more prevalent in the koala (Phascolarctos cinereus) compared to humans and other animals, likely due to the close association that both C. gattii and koalas have with Eucalyptus species. This provides a cogent opportunity to investigate the epidemiology of spontaneous C. gattii infections in a free-living mammalian host, thereby offering insights into similar infections in humans. This study aimed to establish a link between nasal colonisation by C. gattii in free-ranging koalas and the tree hollows of Eucalyptus species, the key environmental source of the pathogen. We (i) detected and genotyped C. gattii from nine out of 169 free-ranging koalas and representative tree hollows within their home range in the Liverpool Plains, New South Wales, and (ii) examined potential environmental predictors of nasal colonisation in koalas and the presence of C. gattii in tree hollows. Phylogenetic analyses based on multi-locus sequence typing (MLST) revealed that the koalas were most likely colonised by the most abundant C. gattii genotypes found in the Eucalyptus species, or closely related genotypes. Importantly, the likelihood of the presence of C. gattii in tree hollows was correlated with increasing hollow size.

Cryptococcus spp. and Cryptococcosis: focusing on the infection in Brazil.

Cryptococcosis is a global fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii yeast complex. This infection is acquired by inhalation of propagules such as basidiospores or dry yeast, initially causing lung infections with the possibility of progressing to the meninges. This infection mainly affects immunocompromised HIV and transplant patients; however, immunocompetent patients can also be affected. This review proposes to evaluate cryptococcosis focusing on studies of this mycosis in Brazilian territory; moreover, recent advances in the understanding of its virulence mechanism, animal models in research are also assessed. For this, literature review as realized in PubMed, Scielo, and Brazilian legislation. In Brazil, cryptococcosis has been identified as one of the most lethal fungal infections among HIV patients and C. neoformans VNI and C. gattii VGII are the most prevalent genotypes. Moreover, different clinical settings published in Brazil were described. As in other countries, cryptococcosis is difficult to treat due to a limited therapeutic arsenal, which is highly toxic and costly. The presence of a polysaccharide capsule, thermo-tolerance, production of melanin, biofilm formation, mechanisms for iron use, and morphological alterations is an important virulence mechanism of these yeasts. The introduction of cryptococcosis as a compulsory notification disease could improve data regarding incidence and help in the management of these infections.

Domestic Birds as Source of Cryptococcus deuterogattii (AFLP6/VGII): Potential Risk for Cryptococcosis.

Cryptococcosis is an infection caused by encapsulated basidiomycetous yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. It is acquired through inhalation of infectious propagules, often resulting in meningitis and meningoencephalitis. The ecological niche of these agents is a wide variety of trees species, as well as pigeon, parrot and passerine excreta. The objective of this study was to isolate Cryptococcus yeasts from excreta of commercially traded parrots and passerines. The 237 samples were collected between October 2018 and April 2019 and processed using conventional methodologies. Nineteen colonies with a dark brown phenotype, caused by phenol oxidase activity, were isolated, suggesting the presence of pathogenic Cryptococcus yeasts. All isolates tested positive for urease activity. URA5-RFLP fingerprinting identified 14 isolates (68.4%) as C. neoformans (genotype AFLP1/VNI) and 5 (26.3%) as C. deuterogattii (genotype AFLP6/VGII). Multi-locus sequence typing was applied to investigate the relatedness of the C. deuterogattii isolates with those collected globally, showing that those originating from bird-excreta were genetically indistinguishable from some clinical isolates collected during the past two decades.

Multilocus sequence typing of strains from the Cryptococcus gattii species complex from different continents.

BACKGROUND: Cryptococcus neoformans and Cryptococcus gattii species complexes are pathogens causing cryptococcal meningitis, a fungal infection that leads to death unless treated. Worldwide, it is estimated to kill over 180,000 individuals annually. OBJECTIVES: We aim to investigate the molecular diversity of C. gattii isolates from strains isolated from 1995 to the present day from different continents. METHOD: In this study, we analysed the molecular diversity by MLST and antifungal susceptibility by using the broth microdilution method according to the CLSI M27-A4 protocol of a total of 26 strains from Cryptococcus gattii species complex from both clinical and environmental sources. RESULTS: Genotyping showed that most of the strains (17/26; 65.4%) belonged to serotype B and were distributed between three genotypes: VGI (13/17; 76.5%), VGII (3/17; 17.6%) and VGVI (1/17; 5.9%). The serotype C strains (9/26; 34.6%) were distributed between the VGIII (1/9; 11.1%) and VGIV (8/9; 88.9%) genotypes. The 26 strains belonged to 17 different MLST subtypes, and we highlight four new MLST genotypes (ST553, 554, 555 and 556). The two environmental strains were identified as serotype B and genotype VGI, but were of ST 51 and 154. All isolates have wild-type MIC of fluconazole and flucytosine. Regarding amphotericin B, five VGI strains showed MICs to AMB equal to 1 µg/ml, and according to the ECV for these genotypes, they were considered non-wild-type strains. CONCLUSIONS: The current study reveals the genetic diversity and new sequence types among strains from the C. gattii complex species.

High Prevalence of HIV-Related Cryptococcosis and Increased Resistance to Fluconazole of the Cryptococcus neoformans Complex in Jiangxi Province, South Central China.

Background: Cryptococcosis is caused by a fungi of the Cryptococcus neoformans/Cryptococcus gattii complex and is a severe concern for public health worldwide. C. neoformans species are globally distributed, and C. gattii species are mostly found in America, Australia, and Sub-Saharan Africa. Cryptococcus usually infects an immunocompromised population; however, the majority of cryptococcosis in China has been reported in patients without any recognizable immunosuppression, i.e., HIV infection. To date, very few studies investigated this disease in South Central China. Methods: The present study recruited 230 clinically suspected cryptococcosis cases in the last 5 years at two hospitals in Jiangxi Province, South Central China. All isolated strains were subjected to multilocus sequence typing (MLST) and phylogenetic analysis. Serotype and mating type were assessed by PCR, in vitro antifungal susceptibility was assessed by the CLSI-M27-A3 protocol. Results: A total of 230 patients were identified as infected by C. neoformans, including 12 cases with Talaromyces marneffei coinfection. All seven MLST markers were successfully amplified and used to identify the ST genotype in 199 strains. C. gattii strains were not detected. In contrast to previous studies, 59.3% of the patients had an immunocompromised status, and 61.9% of these patients were infected with HIV. All isolates manifested serotype A and mating type α. The ST5 genotype was common (89.5%) in the Jiangxi region, and three novel genotypes (ST656, ST657, and ST658 in six isolates) were detected in the present study. A total of 86 of the isolates (43.2%) were not sensitive to fluconazole at a MIC50 ≥ 8 µg/ml, most of the isolates were resistant to amphotericin B, and nearly all isolates were resistant to itraconazole and posaconazole. Resistances to 5-Flucytosine and voriconazole were very rare. Conclusions: The results of the present study indicated that C. neoformans is the predominant species for cryptococcosis in Jiangxi Province, and a large proportion of the strains were not sensitive to fluconazole, which may be related to treatment failure and relapse. A high percentage of HIV-related C. neoformans infections was reported in Jiangxi, supporting a previous hypothesis that cryptococcosis is more frequent among the HIV-infected population in China. Continuous monitoring of species distribution and antifungal sensitivity is important for the investigation of this severe disease in the Jiangxi region.

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

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Cryptococcosis by Cryptococcus neoformans/Cryptococcus gattii Species Complexes in non-HIV-Infected Patients in Southeastern Brazil.

INTRODUCTION: The clinical manifestations of cryptococcosis are usually associated with the infecting agents Cryptococcus neoformans (CN) and C. gattii (CG) species complexes and the host. In this study, non-HIV-infected patients, at a university hospital in southeastern Brazil, had epidemiological and clinical data associated with cryptococcal disease and isolated Cryptococcus species: CN - 24 patients and CG - 12 patients. METHODS: The comparison was comprised of demographic data, predisposing factors, clinical and laboratory manifestations, and outcomes of cryptococcosis patients treated between 2000 and 2016. Immunocompetent and immunosuppressed patients were also compared, irrespective of the infecting species. Cryptococcus spp. were genotyped by PCR-RFLP analysis of the URA5 gene. RESULTS: Infections by the CN species complex (100% VNI genotype) were associated with drug immunosuppression and fungemia, and patients infected with the CG species complex (83% VG II and 17% VGI genotypes) had more evident environmental exposure and higher humoral response. CN and CG affected patients with or without comorbidities. CONCLUSIONS: Diabetes mellitus, other chronic non-infectious diseases, and alcoholism were likely predisposing factors for infection by both CN and CG species. Immunocompetent patients, independent of the infecting Cryptococcus species complexes, showed a higher occurrence of meningitis and a trend toward less fungal dissemination and longer survival than immunosuppressed hosts.