Cryptococcosis is a significant invasive fungal infection with noteworthy morbidity and mortality, primarily caused by Cryptococcus neoformans (C. neoformans)and Cryptococcus gattii.1,2 Although cryptococcosis may occur in healthy hosts,3 the majority of infections occur in patients with significant underlying predisposing factors, such as organ transplantation, hematologic malignancies, and advanced human immunodeficiency virus (HIV) disease.2,4–8 In Europe and the in United States about 80% of the cryptococcosis cases were associated with HIV/AIDS.2,9 Nonetheless, in China, the majority of patients with cryptococcosis are not infected with HIV.10,11 This infection has been increasing steadily over the past years in China.

In recent years, there is a growing concern among clinicians regarding the potential for the emergence of antifungal resistance among C. neoformans because of high rates of fungal persistence and frequent disease relapse.12–14 Several reports on the emergence of resistance to amphotericin B, fluconazole, flucytosine, or itraconazole in C. neoformans during treatment has been published.15,16 Consequently, widespread increase in fluconazole resistance among C. neoformans became a public health concern calling for more-widespread surveillance. However, studies on the trends in antifungal drug susceptibility of Cryptococcus are limited in China. In 2016, Fan et al. reported a proportional increase of cryptococcal isolates with non-wild-type MICs to fluconazole (non-WT, MIC>8μg/ml) in China from 2009 to 2014,15 but clinical characteristics of these cryptococcal isolates were not collected. Now, the trend in antifungal drug susceptibility of clinical Cryptococcus strains in China is still uncertain, and further research on the correlation of the antifungal drug susceptibility trend and clinical outcomes is necessary.

In this study, we analyzed the trends in antifungal drug susceptibility of 217 consecutive cryptococcal isolates from a medical center in China between 2013 and 2017, of which 98 isolates were conserved for molecular evaluation. The present study also assessed the clinical manifestations of the 98 patients during the study period.

In the past years, the incidence of cryptococcosis in China is on the rise, and the fungal persistence and frequent disease relapse of cryptococcosis is the main clinical problem. Several reports of the emergence of resistance to the antifungal agents in HIV-infected patients have been published.12,16,20 However, in China, cryptococcosis mostly occurs in patients without HIV infection, and there are limited data on the molecular epidemiology of cryptococcosis in China. In our study, the susceptibility to the five antifungal agents in use (amphotericin B, fluconazole, flucytosine, itraconazole, and voriconazole) of 217 cryptococcal isolates from patients admitted to an university hospital between 2013 and 2017 were evaluated. The trend in the MIC range of fluconazole showed gradual increase from 2013 to 2017, while 16 C. neoformans strains were dose-dependent susceptible to fluconazole (MIC 8μg/ml) and five C. neoformans strains with non-wild-type MICs to fluconazole (MIC 16μg/ml) were detected in 2016–2017. This phenomenon was worth to be conveyed to clinicians, especially in cryptococcosis cases with fungal persistence and relapse. The Infectious Diseases Society of America (IDSA) recommends amphotericin B and 5-flucytosine as the preferred agents for the induction therapy, whereas the azoles (especially fluconazole) are used in the consolidation and maintenance phases of therapy, or as primary prophylaxis.21 However, in resource limited settings, azoles are often used as initial therapy.22 In the past years in our hospital, a relatively low fluconazole dose (400mg per day and even 200mg per day) was commonly administrated for therapy or prophylaxis of fungal infection, which might lead to the gradual upward MIC drifts of fluconazole. In several reports, high-dose of fluconazole (800mg per day or even 1200mg per day) was confirmed more efficacious to treat cryptococcosis.22,23 Higher doses of fluconazole and longer durations of treatment are suggested.21,23 The gradual upward MIC drifts of fluconazole might also explain the reason that some patients with cryptococcosis were cured or improved with higher dose of fluconazole. Amphotericin B maintained 1.0% susceptibility in the period of 2013–2016, but one isolate was shown to be resistant to amphotericin B (MIC 8μg/ml) in 2017.

As the results of species and varieties identification by MALDI-TOF MS and ITS sequencing, the conserved 98 clinical cryptococcal strains included 97 C. neoformansstrains (96 var. grubii strains and one var. neoformans strain) and one C. gattii strain. Analysis of MLST showed that 91.8% of the isolates belonged to ST5 genotype VNI, which was the main ST of clinical C. neoformans in China, consistent with the related reports.15C gattii, an emerging agent of cryptococcosis, was initially reported only in tropical and subtropical zones and rarely reported in China.24 The patient infected with C. gattii had been worked in Cambodia and suffered before going back to China. So, this case of C. gattii cryptococcosis might have been imported.

In previous reports, about 80% of the cryptococcosis cases were associated with HIV/AIDS in Europe and the United States.9,25 In contrast, in our study most of patients (71.4%) with cryptococcosis were not HIV-infected; moreover, 32.7% of the cryptococcosis patients had no underlying diseases, as shown in other reports from China.6,26 All of the 28 HIV-infected cryptococcosis patients with were male, and younger than the patients without HIV. There was no apparent sex difference in the cryptococcosis patients without HIV. The incidence rate of disseminated cryptococcosis, as well as the mortality rate, among HIV-infected patients were significantly higher than that in patients without HIV infection.

In contrast to the findings of Lee et al., no relationship between MICs of antifungal agents and outcome of patients with cryptococcosis could be demonstrated in this study. The discrepancy might be due to different choices of treatment regimens.14 In this study, four of the five patients infected with C. neoformans with higher MICs for fluconazole were still cured or improved following the regimen recommended by IDSA with amphotericin B and 5-flucytosine as the preferred agents for the induction therapy and fluconazole or voriconazole used in the consolidation and maintenance phases. Moreover, the patient infected with C. neoformans resistant to amphotericin B (MIC 8μg/ml) responded to voriconazole. However, the shifting trend in MIC ranges of antifungal agents, especially fluconazole, warrants attention as it was also reported in other countries and might be related to the dose selection of fluconazole for effective therapy.27 Hence, the dynamic real time surveillance of susceptibility for antifungal agents is necessary in clinic practice.

This study has some limitations. It was a single center retrospective study conducted in a major teaching hospital in China. Hence, the results in this study are exploratory. In future, further investigations with larger samples including more hospitals in China will be necessary to generalize the results for the Chinese population.

The authors report no conflicts of interest in this work.