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Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.

PurposeThe diagnosis of pulmonary cryptococcosis (PC) remains challenging, particularly in patients presenting with lobar or patchy consolidation on chest radiographs. Biopsies are sometimes performed for histopathologic examination and microbiological culture to differentiate infections, including PC, from lung cancers. However, to date, the clinical value of small biopsy samples and their reasonable processing methods for detecting Cryptococcus are rarely evaluated. Furthermore, the cryptococcal antigen (CrAg) test has been widely used in cryptococcosis diagnosis due to its high specificity. This 6-year retrospective study aimed to assess the efficacy of four tests commonly used for detecting Cryptococcus in the diagnosis of pulmonary cryptococcosis, and reveal that the combination of 2 or 3 methods would raise diagnosis sensitivity.MethodsThe results of CrAg test, histopathologic examination and routine cryptococcal culture of sputum/bronchoalveolar lavage fluid (BALF) were collected from hospitalized patients between June 2019 to May 2024. Additionally, the results of 4 above-mentioned methods were analyzed to compare their effectiveness in PC diagnosis.ResultsAmong 1508 patients whose biopsy specimens were sent for pathogen detection, 63 PC cases were diagnosed, and 24 C. neoformans strains were cultivated using the Myco/F Lytic culture, which was more than those by sputum/BALF culture (9 strains). CrAg was positive in 82.5% (52/63) PC patients and remained the most sensitive method. The combination of CrAg and biopsy culture will increase the overall diagnostic yield to 95.2% (60/63).ConclusionsIn summary, for those having biopsy tissue collected, the combination of CrAg and biopsy culture using Myco/F could effectively identify most PC cases.

To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) in pulmonary cryptococcosis (PC) using bronchoalveolar lavage fluid (BALF) and lung biopsy tissue specimens. In this retrospective study, 321 patients diagnosed with lower respiratory tract diseases who underwent mNGS using BALF and LBT samples, between January 2021 and December 2023 were included. Individuals were classified into PC and non-PC groups according to the diagnostic criteria for PC, and conventional fungal cultures were performed. A serum/BALF cryptococcal antigen (CrAg) test was performed in some patients with PC. The diagnostic efficiencies of three methods for PC (mNGS, conventional culture, and CrAg) were compared. Additionally, two mNGS methods were used in this study: original mNGS (OmNGS, testing time from January 2021 to December 2022) and modified mNGS (MmNGS, testing time from January to December 2023). The diagnostic efficiency of the two mNGS methods on PC was simultaneously compared. Among the 321 patients, 23 (7.2%) had PC and 298 (92.8%) did not. Compared with the composite reference standard for PC diagnosis, the sensitivity, specificity, and accuracy of mNGS for PC were 78.3% (95% confidence interval [CI], 55.8%-91.7%), 98.7% (95% CI, 96.4%-99.6%), and 97.2% (95% CI, 94.7%-98.7%), respectively. The sensitivity of mNGS was similar to that of CrAg (80.0%, 12/15) ( > 0.05). The diagnostic sensitivity of both mNGS and CrAg was higher than that of conventional culture (35.0%, 7/20) ( = 0.006, = 0.016), and the combined detection of mNGS and CrAg further improved the diagnostic sensitivity of PC (93.3%, 14/15). The area under the receiver operating characteristic curve of mNGS was superior to that of conventional culture (0.885 vs. 0.675). In addition, the diagnostic sensitivity of PC was higher than that of OmNGS ( = 0.046). The sensitivity of mNGS is better than that of conventional culture. The combination of mNGS and CrAg improves the testing sensitivity of . MmNGS could further improve the detection of . Conventional PC detection methods are indispensable and mNGS can be used as a rapid and accurate auxiliary diagnostic method for PC.

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease. Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

Cryptococcosis represents a fungal disease acquired from the environment with animals serving as host sentinels for human exposure. The aim of this study was to investigate the genetic characteristics of Cryptococcus isolates from veterinary sources (cats, dogs and birds) to understand their epidemiology and the genetic variability of the casual isolates. Mating‐type PCR in connection with MLST analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex was used to genotype 17 C. neoformans isolates. In the absence of an MLST typing scheme Cryptococcus adeliensis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus and C. uniguttulatus strains were typed using M13 PCR fingerprinting. All C. neoformans isolates were MATα mating type, but hybrids possessed αADa and aADα mating and serotypes. Two C. neoformans molecular types VNI, VNIV and VNIII and VNII/VNIV hybrids were identified. Amongst the 66 non‐C. neoformans strains investigated 55 M13 PCR fingerprinting types were identified. The wide variety of MLST types of C. neoformans and the occurrence of αADa and aADα hybrids in our study supports the notion of genetic recombination in the area studied. The heterogeneity of the non‐C. neoformans isolates remains open to further investigations and should be taken into consideration when identifying emergent pathogens.

We aimed to determine the environmental sources of C. neoformans and C. gattii species complexes in Uganda. One hundred fifty (n = 150) environmental specimens of chicken droppings, marabou stork bird droppings and eucalyptus tree barks in Uganda were examined phenotypically. The specimens were grown on Sabouraud dextrose agar and the colonies examined by India ink microscopy, urea hydrolysis and formation of blue colonies on chromogenic L-Canavanine glycine blue bromothymol blue agar of C. gattii species complex. The prevalence of C. neoformans species complex was 4% (6/150). Of the positive environmental samples from which C. neoformans species complex were isolated; the predominant source was marabou stork droppings; 8% (2/25) followed by eucalyptus tree barks; 3.96% (4/101). However, there was no Cryptococcus gattii species complex; 0% (0/150). In Uganda, the environmental sources of C. neoformans species complex are marabou stork droppings and eucalyptus tree barks, however, there seems to be no Cryptococcus gattii species complex.

The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status.

There are approximately 1 million cryptococcal infections per year among HIV+ individuals, resulting in nearly 625,000 deaths. Cryptococcus neoformans and Cryptococcus gattii are the two most common species that cause human cryptococcosis. These two species of Cryptococcus have differences in pathogenicity, diagnosis, and treatment. Cryptococcal infections are usually difficult to identify because of their slow growth in vitro. In addition, the long detection cycle of Cryptococcus in clinical specimens makes the diagnosis of Cryptococcal infections difficult. Here, we used positively charged silver nanoparticles (AgNPs + ) as a substrate to distinguish between C. neoformans and C. gattii in clinical specimens directly via surface-enhanced Raman scattering (SERS) and spectral analysis. The AgNPs + self-assembled on the surface of the fungal cell wall via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substrate negatively charged silver nanoparticles (AgNPs). The SERS spectra could also be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. This novel SERS detection method can clearly distinguish between the two Cryptococcus species using principal component analysis. The accuracy of the training data and test data was 100% after a tenfold crossover validation.

The purpose of this study was to investigate the molecular characteristics of 83 clinical Cryptococcus neoformans / C. gattii species complex isolated in Beijing, China, between 2007 and 2013. Restriction fragment length polymorphism of the gene URA5 ( URA5 -RFLP), multilocus sequence typing (MLST), and automated repetitive polymerase chain reaction (rep-PCR; DiversiLab system) were performed to genotype these cryptococcal isolates. There was an excellent correlation amongst the three methods; however, PU157 was assigned as VNII according to URA5 -RFLP, while it was classified as VNI by the DiversiLab system analysis. PU157 was finally identified as VNB by seven-locus MLST analysis. Moreover, though AD hybrids could not be processed by MLST, ideal results could be obtained by the DiversiLab system. The genotype VNI accounted for 95.2 % (79/83) of isolates. Besides one strain of VNB, VNIII, and VGI each, a strain of VGII was detected in our study, which was isolated from a patient from the temperate region in North China. In addition, the most common MLST sequence type (ST) was ST5, accounting for 91.6 % (76/83), followed by ST31, ST63, ST182, ST295, ST296, and ST332. ST295, ST296, and ST332 were new STs. Except for isolate PU157 (VNB), identical results were obtained quickly and accurately through the DiversiLab system compared to MLST and URA5 -RFLP. The discovery of VNB and VGII in the temperate climate regions of China suggested that the population structure of C. neoformans and C. gattii should be explored more extensively. Our results also showed that the DiversiLab system can be used in the genotyping of C. neoformans and C. gattii .

Cryptococcosis has been reported to be mostly associated with non-HIV-related patients in China. However, little is known about the molecular characteristics of clinical isolates from the Cryptococcus neoformans species complex in this country. In this study, 115 clinical isolates were included. Molecular type VNI was the most representative (n=103), followed by VGI (n=8), VNIII (n=2), VNIV (n=1), and VGII (n=1). With the exception of a serotype D mating type a isolate, all possessed the MATα locus. Multilocus sequence typing (MLST) revealed that most Cryptococcus gattii isolates from China shared identical MLST profiles with the most common MLST genotype reported in the VGI group, and the only one VGII isolate resembled the Vancouver Island outbreak minor genotype. The C. gattii strains involved in this study were successfully grouped according to their molecular type and mating types by PCR-restriction fragment length polymorphism (RFLP) analysis of the GEF1 gene. Our results suggest that (1) in China, cryptococcosis is mostly caused by C. neoformans var. grubii (molecular type VNI), and mating type α; (2) The most common causative agents of C. gattii infection in China are closely related to a widely distributed MLST genotype; and (3) The PCR-RFLP analysis of the GEF1 gene has the potential to identify the molecular and mating types of C. gattii simultaneously.