In vitro antifungal susceptibility profiles of Cryptococcus neoformans and Cryptococcus gattii clinical isolates of in Guangxi, Southern China

Abstract

Abstract: Objective To analyze the clinical characteristics, pathogenic strain identification and in vitro antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii in Guangxi. Methods One hundred and three Cryptococcus strains were isolated from 86 patients with confirmed cryptococcosis (14 were HIV-positive and 72 were HIV-negative) and their clinical characteristics were analyzed. 92 strains were identified as Cryptococcus neoformans grubii variant and 11 strains were identified as Cryptococcus gattii using MALDI-TOF MS and ITS region sequencing molecular identification methods. The strains were tested for in vitro antifungal susceptibility to the commonly used antifungal drugs including fluconazole, amphotericin B, 5-fluorocytosine, itraconazole, voriconazole, posaconazole, and esaconazole referring to the CLSI M27-A4 method. Results There were 59 males and 27 females aged 21-84 years in 86 patients, 55 of them had no underlying disease. Referring to the interpretation criteria in CLSI and EUCAST, all strains were sensitive to most antifungal drugs. The minimum inhibitory concentrations of antifungal drugs against Cryptococcus neoformans var. grubii ranged from 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, and 0.0020-0.125 μg/mL for estaconazole. The minimum inhibitory concentrations of antifungal drugs against Cryptococcus gattii ranged from 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, 0.0156-0.125μg/mL for voriconazole, 0.0156-0.25 μg/mL for posaconazole,and 0.0078-0.125 μg/mL for esaconazole. Conclusion MALDI-TOF MS is a rapid and reliable method for the identification of Cryptococcus. Cryptococci isolated in Guangxi are sensitive to clinical antifungal drugs, and the antifungal sensitivity test helps to detect drug-resistant strains at an early stage, which is important for the effective treatment of cryptococcosis.

Key words: Cryptococcus gattii, variant identification, in vitro drug sensitivity, C. neoformans var. grubii, guangxi

CLC Number:

R519.4

ZHENG Dongyan, CAO Cunwei, LI Xiuying, ZHENG Yanqing, PAN Kaisu, LIAO Wangqing. In vitro antifungal susceptibility profiles of Cryptococcus neoformans and Cryptococcus gattii clinical isolates of in Guangxi, Southern China[J]. Chinese Journal of Mycology, 2022, 17(3): 188-194.

References

[1] GUINEA J, HAGEN F, PELAEZ T, et al. Antifungal susceptibility, serotyping, and genotyping of clinical Cryptococcus neoformans isolates collected during 18 years in a single institution in Madrid, Spain[J]. Med Mycol,2010,48(7):942-948.
[2] MAZIARZ E K, PERFECT J R. Cryptococcosis[J]. Infect Dis Clin North Am,2016,30(1):179-206.
[3] VPARK B J, WANNEMUEHLER K A, MARSTON B J, et al. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS[J]. Aids,2009,23(4):525-530.
[4] SELB R, FUCHS V, GRAF B, et al. Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017[J]. Int J Med Microbiol,2019,309(6):151336.
[5] GUTCH R S, NAWANGE S R, SINGH S M, et al. Antifungal susceptibility of clinical and environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Jabalpur, a city of Madhya Pradesh in Central India[J]. Braz J Microbiol,2015,46(4):1125-1133.
[6] ELLIS D H, PFEIFFER T J. Natural habitat of Cryptococcus neoformans var. gattii[J]. J Clin Microbiol,1990,28(7):1642-1644.
[7] SPEED B, DUNT D. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans[J]. Clin Infect Dis,1995,21(1):28-36.
[8] FANG W, FA Z, LIAO W. Epidemiology of Cryptococcus and cryptococcosis in China[J]. Fungal Genet Biol,2015,78:7-15.DOI:10.1016/j.fgb.2014.10.017.
[9] CHEN Y H, YU F, BIAN Z Y, et al. Multilocus sequence typing reveals both shared and unique genotypes of Cryptococcus neoformans in Jiangxi Province, China[J]. Sci Rep,2018,8(1):1495.
[10] FENG X, YAO Z, REN D, et al. Genotype and mating type analysis of Cryptococcus neoformans and Cryptococcus gattii isolates from China that mainly originated from non-HIV-infected patients[J]. FEMS Yeast Res, 2008,8(6):930-938.
[11] WU S Y, KANG M, LIU Y, et al. Molecular epidemiology and antifungal susceptibilities of Cryptococcus species isolates from HIV and non-HIV patients in Southwest China[J]. Eur J Clin Microbiol Infect Dis,2021,40(2):287-295.
[12] JIN L, CAO J R, XUE X Y, et al. Clinical and microbiological characteristics of Cryptococcus gattii isolated from 7 hospitals in China[J]. BMC Microbiol,2020,20(1):73.
[13] KLEIN K R, HALL L, DEML S M, et al. Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing[J]. J Clin Microbiol,2009,47(11):3669-3672.
[14] MCTAGGART L R, LEI E, RICHARDSON S E, et al. Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry[J]. J Clin Microbiol,2011,49(8):3050-3053.
[15] FAVALESSA O C, de PAULA D A, DUTRA V, et al. Molecular typing and in vitro antifungal susceptibility of Cryptococcus spp from patients in Midwest Brazil[J]. J Infect Dev Ctries. 2014,8(8):1037-1043.
[16] PFALLER M A, MESSER S A, BOYKEN L, et al. Global trends in the antifungal susceptibility of Cryptococcus neoformans(1990 to 2004)[J]. J Clin Microbiol,2005,43(5):2163-2167.
[17] DIAS A L T, MATSUMOTO F E, MELHEM M S C, et al. Comparative analysis of Etest and broth microdilution method (AFST-EUCAST) for trends in antifungal drug susceptibility testing of Brazilian Cryptococcus neoformans isolates[J]. J Med Microbiol,2006,55(Pt 12):1693-1699.
[18] REVANKAR S G, KIRKPATRICK W R, MCATEE R K, et al. Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method[J]. J Clin Microbiol, 1998,36(1):153-156.
[19] RODRIGUEZ-TUDELA J L, MARTINEZ-SUAREZ J V, DRONDA F, et al. Correlation of in-vitro susceptibility test results with clinical response:a study of azole therapy in AIDS patients[J]. J Antimicrob Chemother,1995,35(6):793-804.
[20] ESPINEL-INGROFF A, CHOWDHARY A, CUENCA-ESTRELLA M, et al. Cryptococcus neoformans-Cryptococcus gattii species complex:an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine[J].Antimicrob Agents Chemother,2012,56(6):3107-3113.
[21] ESPINEL-INGROFF A, ALLER A I, CANTON E, et al. Cryptococcus neoformans-Cryptococcus gattii species complex:an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole[J]. Antimicrob Agents Chemother,2012,56(11):5898-5906.
[22] ESPINEL-INGROFF A, CHOWDHARY A, GONZALEZ G M, et al. Multicenter study of isavuconazole MIC distributions and epidemiological cutoff values for the Cryptococcus neoformans-Cryptococcus gattii species complex using the CLSI M27-A3 broth microdilution method[J]. Antimicrob Agents Chemother,2015,59(1):666-668.
[23] HUANG C, TSUI C K M, ChEN M, et al. Emerging Cryptococcus gattii species complex infections in Guangxi, southern China[J]. PLoS Negl Trop Dis,2020,14(8):e0008493.
[24] ChEN J, VARMA A, DIAZ M R, et al. Cryptococcus neoformans strains and infection in apparently immunocompetent patients, China[J]. Emerg Infect Dis, 2008,14(5):755-762.
[25] WU S Y, LEI Y, KANG M, et al. Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province, China[J]. Mycoses,2015,58(5):280-287.
[26] DROMER F, MATHOULIN S, DUPONT B, et al. Epidemiology of cryptococcosis in France:a 9-year survey (1985-1993). French Cryptococcosis Study Group[J]. Clin Infect Dis, 1996,23(1):82-90.
[27] MOOSA M Y, COOVADIA Y M. Cryptococcal meningitis in Durban, South Africa:a comparison of clinical features, laboratory findings, and outcome for human immunodeficiency virus (HIV)-positive and HIV-negative patients[J]. Clin Infect Dis,1997,24(2):131-134.
[28] SINGHAL N, KUMAR M, KANAUJIA P K, et al. MALDI-TOF mass spectrometry:an emerging technology for microbial identification and diagnosis[J]. Front Microbiol,2015,6:791.DOI:10.3389/fmicb.2015.00791.
[29] FIRACATIVE C, TRILLES L, MEYER W. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex[J]. PLoS One,2012,7(5):e37566.
[30] CHENG K, CHUI H, DOMISH L, et al. Recent development of mass spectrometry and proteomics applications in identification and typing of bacteria[J]. Proteomics Clin Appl,2016,10(4):346-357.
[31] PERKINS A, GOMEZ L A, MELLADO E, et al. Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans[J]. J Antimicrob Chemother,2005,56(6):1144-1147.
[32] LIA M, CARETTE A, TANG H, et al. Functional dissection of the chromosome 13q14 tumor-suppressor locus using transgenic mouse lines[J]. Blood,2012,119(13):2981-2990.
[33] SAR B, MONCHY D, Vann M, et al. Increasing in vitro resistance to fluconazole in Cryptococcus neoformans Cambodian isolates:April 2000 to March 2002[J]. J Antimicrob Chemother, 2004,54(2):563-565.
[34] HERKERT P F, MEIS J F, LUCCA O S G, et al. Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil[J]. J Med Microbiol,2018,67(4):560-569.
[35] ARSIC A V, PEKMEZOVIC M G, MEIS J F, et al. Molecular epidemiology and antifungal susceptibility of Serbian Cryptococcus neoformans isolates[J]. Mycoses, 2014,57(6):380-387.
[36] THOMPSON G R 3rd, WIEDERHOLD N P, FOTHERGILL A W, et al. Antifungal susceptibilities among different serotypes of Cryptococcus gattii and Cryptococcus neoformans[J]. Antimicrob Agents Chemother,2009,53(1):309-311.