In vitro Antifungal Susceptibility Profiles of Cryptococcus neoformans var. grubii and Cryptococcus gattii Clinical Isolates in Guangxi, Southern China

This study analyzed the in vitro drug sensitivity of Cryptococcus spp. from Guangxi, Southern China. One hundred three strains of Cryptococcus were recovered from 86 patients; 14 were HIV positive and 72 were HIV negative. Ninety-two strains were identified as Cryptococcus neoformans var. grubii, while 11 strains were identified as Cryptococcus gattii (5 C. gattii sensu stricto and 6 Cryptococcus deuterogattii). The recovered strains were tested against commonly used antifungal drugs (fluconazole, amphotericin B, 5-fluorocytosine, itraconazole, and voriconazole) and to novel antifungal drugs (posaconazole and isavuconazole) using CLSI M27-A4 method. The results showed that all isolates were susceptible to most antifungal drugs, of which the minimum inhibitory concentration (MIC) ranges were as follows: 0.05–4 μg/ml for fluconazole, 0.25–1 μg/ml for amphotericin B; 0.0625–2 μg/ml for 5-fluorocytosine, 0.0625–0.25 μg/ml for itraconazole, 0.0078–0.25 μg/ml for voriconazole, 0.0313–0.5 μg/ml for posaconazole, 0.0020–0.125 μg/ml for isavuconazole for C. neoformans var. grubii isolates, and 1–16 μg/ml for fluconazole, 0.125–1 μg/ml for 5-fluorocytosine, 0.25–1 μg/ml for amphotericin B, 0.0625–0.25 μg/ml for itraconazole, 0.0156–0.125 μg/ml for voriconazole, 0.0156–0.25 μg/ml for posaconazole, and 0.0078–0.125 μg/ml for isavuconazole for C. gattii isolates. Furthermore, some C. neoformans var. grubii isolates were found to be susceptible-dose dependent to 5-fluorocytosine and itraconazole. In addition, a reduction in the potency of fluconazole against C. gattii is possible. We observed no statistical differences in susceptibility of C. neoformans var. grubii and C. gattii in the tested strains. Continuous observation of antifungal susceptibility of Cryptococcus isolates is recommended to monitor the emergence of resistant strains.


INTRODUCTION
Cryptococcosis is a common opportunistic invasive fungal infection caused mainly by Cryptococcus neoformans (C. neoformans, serotypes A, AD, and D) and Cryptococcus gattii (C. gattii serotypes B and C) (Guinea et al., 2010). The latter mainly affects otherwise healthy individuals, whereas C. neoformans is more common in immunocompromised patients (Park et al., 2009;Guinea et al., 2010;Maziarz and Perfect, 2016). Although the incidence of cryptococcosis has declined with the introduction of highly active antiretroviral therapy (HAART), immunocompromised individuals remain at risk, and mortality rate remain unacceptably high despite the continued research on cryptococcosis (Park et al., 2009;Selb et al., 2019). C. neoformans and C. gattii are genetically related to each other; however, they vary in the ecological niche geographic distribution, natural habitat, host infectivity, and pathogenicity (Gutch et al., 2015). For instance, C. neoformans has a global distribution, while C. gattii strains are more common in North America and Australia (Ellis and Pfeiffer, 1990). Moreover, C. gattii seems to be less sensitive to therapy and requires more aggressive management than C. neoformans (Speed and Dunt, 1995). China has the worlds' largest population with large humid tropical and subtropic regions rich with vegetation, a climate favorable for the growth and spread of fungi. In addition, the number of immunocompromised (HIV and non-HIV) populations in China has been increasing in the past several decades, leading to an increase in the incidence and prevalence of aggressive fungal infections such as cryptococcosis, which is a heavy burden to public health (Fang et al., 2015). Furthermore, cryptococcosis can be easily misdiagnosed due to the vague and diversity of the clinical manifestations, leading to the delay of proper treatment. Therefore, early diagnosis and timely treatment play an important role in the prognosis of the disease. Several studies have been performed to investigate the microbiological, epidemiological, and clinical characteristics of C. neoformans and C. gattii strains in China (Chen et al., 2008(Chen et al., , 2018Feng et al., 2008;Wu et al., 2015Wu et al., , 2021Fang et al., 2020;Jin et al., 2020;Xu et al., 2021). Unfortunately, relatively little is known about the pathogenic Cryptococcus species and their sensitivity to antifungal chemotherapy in Guangxi, Southern China. Guangxi province is located in the subtropical zone with a warm, humid climate where cryptococcosis is significantly common but might be severely underreported. Recently, new antifungal agents have been introduced, suggesting that the antifungal susceptibility profiles need to be researched and updated. Therefore, the current study aims to analyze the clinical characteristics of Cryptococcus infection and in vitro drug susceptibility of Cryptococcus spp. in Guangxi, Southern China.

Ethics Statement
This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. The clinical data in this study were obtained with written consent from the patients or their families, and data collected concerning them was anonymized.

Isolates and Clinical Data
Between May 2014 and May 2018, clinical isolates of Cryptococcus spp. from patients admitted to the First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, the Fourth People's Hospital of Nanning, and People's Hospital of Guangxi Zhuang Autonomous Region were collected and recovered for this study. We assessed the patients' medical records to collect clinical information. Strains with insufficient clinical data of patients were excluded.

Activation and Identification
A total of 120 Cryptococcus strains were taken out of the refrigerator at −80 • C, resuscitated at 20 • C for 24 h, transferred to Sabouraud dextrose agar (SDA) medium, and incubated at 27 • C for 48-72 h, then transferred onto L-canavanineglycine-bromothymol blue (CGB) medium for 3-7 days to differentiate C. neoformans from C. gattii species as previously mentioned (Klein et al., 2009). Three consecutive purifications were made using the streak plate technique to ensure the growth and purity of the strains. Multiple colonies were transferred to SDA medium for cultivation at 27 • C and enrichment for later use.

Extraction of Cryptococcus Neoformans Protein
A sterile loop full of the purified isolated strains (about 5 mg) was added to an Eppendorf tube containing 300 µl of distilled H 2 O and was mixed thoroughly. Nine hundred microliters of absolute ethanol was added to the tube, mixed, and centrifuged at 12,000 r/min for 2 min. After discarding the supernatant, the tube was centrifuged again at 12,000 r/min for 2 min and later placed at room temperature to dry. Following the addition of 50 µl of 70% formic acid and 50 µl of acetonitrile, the tube was centrifuged for 2 min at 12,000 r/min. The supernatant was then transferred to a new tube for use.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Identification
The samples were overlaid with 1 µl of matrix solution consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% trifluoroacetic acid and again allowed to air dry prior to analysis. For each isolate, a spectrum with a mass-to-charge range of 2,200-22,000 Da was generated as an average of 240 laser shots in an automatic acquisition mode. When poor spectra (fewer than 10 well-defined peaks above 1,000 arbitrary units) were obtained, analysis was repeated with an extra wash step during the protein extraction procedure, which improved the quality of the spectra. The MALDI Biotyper 3.0 database was used to compare the data and record the mass spectrometry identification results. According to the instruction manual, a log score ≥1.70 indicates correct identification; a log score <1.70 indicates that the identification is incorrect or unable to be identified (McTaggart et al., 2011).

DNA Extraction and ITS Sequencing
As a reference "gold standard, " 20 strains of Cryptococcus were randomly selected to extract DNA for ITS sequencing. Strains were cultured on SDA medium at 27 • C for 72 h for DNA extraction according to the operation steps of the fungal genomic DNA extraction kit produced by Jiangsu Kangwei Century Biotechnology Co., Ltd (Changping, Beijing, China). According to the operating manual, the concentration of genomic DNA is determined using a nucleic acid protein analyzer and then placed in a refrigerator at −20 • C for later use. Ribosomal RNA (rRNA) internal transcribed spacer Region (rITS) sequencing is performed on the extracted DNA using primer sets (upstream primer sequence ITS1: 5 -TCCGTAGGTGAACCTGCGG-3 ) and (downstream primer sequence ITS4: 5 -TCCTCCGCTTATTGATATGC-3 ). A total volume of 50 µl (Taq-Mix, 25 µl; upper material, 2 µl; downstream primer, 2 µl; at least 100 ng of DNA extract and sterile deionized water) was used to perform polymerase chain reaction (PCR) for 35 cycles at 95 • C with 3 min initial denaturation, 30 s denaturation at 95 • C, 30 s annealing at 55 • C, 2 min extension at 72 • C, and final extension cycle for 10 min at 72 • C, and then stored at 4 • C. A total of 6 µl of PCR products was separated slowly into 1% agar gel spotting hole electrophoresis at 120 V for 30 min. The agar gel was placed under the UV gel imager to observe whether the PCR product and marker are clear and record the band length. PCR products are then sent with clear bands under electrophoresis to Guangzhou Kinco Biotechnology Co., Ltd. for two-way sequencing. Results were uploaded to the National Center for Biotechnology Information (NCBI) Genebank database 1 for comparison and identification. Stock solutions were prepared as follows: FLC and 5-FC were dissolved in sterile distilled water to a drug storage solution of 1,280 µg/ml, while AmB, ITC, VOC, POS, and ISA were dissolved in dimethyl sulfoxide (DMSO) (Sigma Chemical Co., United States) into a stock solution of 1,600 µg/ml and stored at −20 • C until needed. The final concentration ranges were 0.125-64 µg/ml for FLC, 0.00156-8 µg/ml for AmB and 5-FC, 0.0020-1 g/ml for ITC, VOC, POS, and ISA, respectively. The yeast inocula were adjusted to a concentration of 1 × 10 3 -5 × 10 3 cfu/ml in Roswell Park Memorial Institute (RPMI) 1640 medium as measured by a hemocytometer, and an aliquot of 0.1 ml was added to each well containing various concentrations of antifungal drugs. The 96-well plates were incubated at 37 • C. The assays were read 72 h after inoculation. Candida parapsilosis ATCC 22019 was used as a quality control strain for susceptibility tests.

Statistical Analysis
Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, United States) where p < 0.05 was considered statistically significant.

Isolates and Clinical Data
A total of 103 strains from 86 patients were included in this study ( Table 1). Fifty-nine of them were male, and twenty-seven were female. The age distribution ranged from 21 to 84 years. Of the 86 patients, 14 were HIV positive and 72 were HIV negative. A total of 55 had no underlying diseases, 21 had liver diseases, 7 were diabetic, 4 had systemic lupus erythematosus (SLE), 1 had a history of immunosuppressing therapy, 3 had liver cancer, and 1 had rheumatoid arthritis. All 86 patients came from 13 different cities in Guangxi province, Southern China (Figure 1). Of the 103 strains, 17 were episode strains (i.e., strains recovered from different parts of the same patient simultaneously or from the same part at different times). In total, 91 were isolated from cerebrospinal fluid, 7 were from lung tissue, 3 were from alveolar lavage, and 2 from the skin.

Strain Identification
Eleven strains successfully turned canavanine-glycinebromothymol blue (CGB) medium to blue and were consequently identified as C. gattii. The remaining 92 strains were identified as C. neoformans var. grubii according to the MALDI-TOF MS technique, with mass spectrum scores ≥1.7. The DNA results on the database comparison result were 100% consistent with the MALDI-TOF MS identification result, making the results of MALDI-TOF MS in our study credible. The identification of C. gattii strains and their genotypes were analyzed in detail in our previous study using multilocus sequence typing (MLST) technique, where five isolates were identified as C. gattii sensu stricto (C. gattii s.s.), and six were identified as C. deuterogattii serotype (Huang et al., 2020).

Drug Susceptibility Results
The seven antifungal agents tested retained activity against all isolates. The MIC, MIC 50 , and MIC 90 values are presented in Table 2.

C. gattii
Similar to C. neoformans var. grubii, none of the strains demonstrated resistance to the antifungal drugs (Table 2). However, we observed an MIC value above the ECVs to FLC against a single C. deuterogattii isolate.

DISCUSSION
This study analyzed the epidemiology and in vitro antifungal susceptibility profiles of Cryptococcus spp. in a large-scale population from Guangxi, Southern China. Consistent with previous reports from China, our study showed that the prevalence of cryptococcosis caused by C. neoformans is higher than those caused by C. gattii (Chen et al., 2008;Fang et al., 2015;Wu et al., 2015). In addition, the disease was more observed among otherwise healthy individuals.  Furthermore, our study demonstrated that C. neoformans was responsible for the infection in immunocompetent, which is contrary to the HIV-associated cryptococcosis cases reported in the United States, Africa, and Europe (Dromer et al., 1996;Moosa and Coovadia, 1997;Hajjeh et al., 1999). Similar to reports from other parts of China, C. neoformans var. grubii was the dominant pathogenic strain in Guangxi (Chen et al., 2008). In recent years, MALDI-TOF MS has emerged as a rapid, accurate, and cost-effective method for microbial identification and diagnosis (Singhal et al., 2015). Additionally, MALDI-TOF MS can replace some traditional identification methods, which improves clinical diagnosis and treatment. Furthermore, MALDI-TOF MS can help predict drugresistant fungal isolates by identifying inflicting fungal species (McTaggart et al., 2011). Moreover, MALDI-TOF MS correctly identified 100% of Cryptococcus spp. in previous studies (Firacative et al., 2012;van Belkum et al., 2015;Cheng et al., 2016). Similarly, our study demonstrated that MALDI-TOF MS method is reliable, as we obtained a 100% consistency with results obtained using DNA sequencing.
The antifungal agents tested retained activity against all Cryptococcus isolates. Both C. neoformans var. grubii and C. gattii showed susceptibility to amphotericin B (MIC, 0.25-1 µg/ml). These results are consistent with previous reports (Thompson et al., 2009;Favalessa et al., 2014;Nascimento et al., 2017); however, greater MIC values are reported in the literature and were associated with treatment failure (Lozano-Chiu et al., 1998;Perkins et al., 2005).
High MIC values (≥64 µg/ml) have been reported for fluconazole against C. gattii (Sar et al., 2004;Tay et al., 2006;Soares et al., 2008;Lia et al., 2012), in contrast to this study, where we determined MIC values of ≤ 16 µg/ml. It is worth mentioning that MIC ≥16 µg/ml is believed to be the resistance cutoff for fluconazole; this value was observed from a single C. gattii (C. deuterogattii) isolate in this study. This observation brings the resistance concerns to attention and suggests that FLC might lose its potency, especially if used as a single treatment.
The new azoles (voriconazole, posaconazole, and isavuconazole) showed high antifungal activity against all C. neoformans and C. gattii strains, with low MIC values of ≤0.5 µg/ml, which is consistent with previous reports (Pfaller et al., 2003(Pfaller et al., , 2009Sabatelli et al., 2006;Illnait-Zaragozi et al., 2008;Torres-Rodríguez et al., 2008). Itraconazole, on the other hand, showed high activity against all isolates with low MIC values of ≤0.25, which is similar or lower than those of previous reports (Alves et al., 2001;Souza et al., 2010;Chowdhary et al., 2011;Herkert et al., 2018); however, 13 C. neoformans strains (14.13%) showed to be susceptible-dose dependent to itraconazole. Similarly, C. neoformans strains from Serbia showed to be susceptible-dose dependent to itraconazole (Arsic Arsenijevic et al., 2014). Nevertheless, itraconazole is rarely used as a single drug, especially in the treatment of cryptococcal meningitis, due to low concentration in the cerebrospinal fluid.
We observed no statistically significant difference between C. neoformans var. grubii and C. gattii, nor between C. gattii s.s. and C. deuterogattii isolates against all seven antifungal drugs (p > 0.05). Overall, according to CLSI M27-A4, most strains in this study were of wild type, except for one that might acquire resistance to FLC. A follow-up study is required to confirm if it was of non-wild type.
In summary, cryptococcosis is a complicated disease that can be easily misdiagnosed due to the lack of specificity of the clinical manifestations. In China, healthy individuals are more prone to the infection. C. neoformans var. grubii is the most predominant strain in Guangxi. MALDI-TOF MS is a rapid and reliable method to identify Cryptococcus spp. All isolates showed no resistance to commonly used antifungal drugs and were highly susceptible to the new triazoles. Antifungal susceptibility tests are desirable to early detect any resistant strains in order to ensure proper and successful therapy of cryptococcosis.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
X-YL, NA-O, C-YL, and C-YH contributed to the data collection. X-YL, X-CC, B-KL, K-SP, Y-QZ, and D-YZ contributed to the laboratory work. NA-O and X-YL wrote the manuscript. W-QL and C-WC supervised and evaluated the process of the study. All authors contributed to manuscript revision and read and approved the submitted version.