mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province, China Si-Ying Wu, Yao Lei, Mei Kang, Yu-Ling Xiao and Zhi-Xing Chen Division of Clinical Microbiology, Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China
Summary
Previous reports on the molecular characteristics of clinical isolates of Cryptococcus species in China have focused on isolates from southeast China. To obtain a more detailed molecular epidemiology, a total of 92 cryptococcal isolates were collected from Sichuan province. A total of 24 isolates from 12 other provinces were collected for comparative study. Genotypes and mating types of 116 Cryptococcus isolates were determined. Among the 116 isolates, 43 isolates (19 isolates from Sichuan and 24 isolates outside of Sichuan) were analysed by multi-locus sequence typing (MLST). All 116 clinical isolates were mating type a. Most isolates (114/116) were molecular type VNI and the remaining two isolates were VGI and VGII respectively. MLST results revealed five sequence types (STs) of C. neoformans including two novel STs, with most isolates identified as ST5. The two C. gattii isolates identified in our study were ST44 and ST159. Based on our report and previous studies, there are 15 C. neoformans STs in China which can be divided into three subgroups. The C. gattii isolate from Sichuan could be a scattered subtype of VGII (ST44). Our findings demonstrated that C. neoformans isolates in Sichuan are genetically homogeneous, and ST5 is the epidemic clone of C. neoformans in China.
Key words: Cryptococcus neoformans, Cryptococcus gattii, Sichuan, Multi-locus sequence typing, sequence type, molecular epidemiology.
Introduction Cryptococcosis is an invasive fungal disease affecting approximately 1 million individuals worldwide each year.1 Infections due to Cryptococcus spp., which were previously considered rare fungal pathogens, have gradually increased over the past 20 years in China.2 Wang et al. [3] reported that, in China, the most frequently identified fungal pathogens belong to the genus Candida, followed by Cryptococcus spp. (7.7%). Cryptococcosis is primarily caused by two Cryptococcus Correspondence: Mei Kang, Division of Clinical Microbiology, Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. Tel.: +0086 28 85422618. Fax: +0086 28 85422618. E-mail: [email protected] Submitted for publication 4 December 2014 Revised 9 February 2015 Accepted for publication 11 February 2015
© 2015 Blackwell Verlag GmbH
species, C. neoformans and C. gattii.4 C. neoformans includes molecular types AFLP1/VNI, AFLP1A/VNB/ VNII, AFLP1B/VNII (C. neoformans var. grubii, serotype A); AFLP2/VNIV (C. neoformans var. neoformans, serotype D); and AFLP3/VNIII (hybrid, serotype AD) while C. gattii includes molecular types AFLP4/VGI, AFLP6/ VGII and AFLP10/VGIV (serotype B), AFLP5/VGIII and AFLP7/VGIV (serotype C).5,6 C. neoformans is an important opportunistic pathogen that commonly causes infections of the central nervous system, especially in patients with HIV while C. gattii generally results in pulmonary disease.7 C. gattii genotypes AFLP4/VGI and AFLP6/VGII usually occur in patients without detectable predisposing factors, whereas genotypes AFLP5/VGIII, AFLP7/VGIV and AFLP10/VGIV are commonly found among immuno-compromised individuals, similar to C. neoformans.8 Several molecular methods have been used for the detection of specific genetic sequences of C. neoformans/C. gattii species complex. The most commonly
doi:10.1111/myc.12312
S. Y. Wu et al.
used approaches were PCR fingerprinting, PCR-RFLP, AFLP, DNA sequence analysis, mating and/or serotype-specific PCRs and multi-locus sequence typing (MLST). Among these molecular techniques, MLST have been proposed as reference method.8 Extensive studies on molecular typing of Cryptococcus isolates have been reported worldwide. VNI is the predominant genotype and has been frequently reported in multiple regions globally.9–14 In China, previous studies showed that VNI caused the majority of infections in immuno-competent patients, with or without underlying conditions.15,16 Previous studies, however, were limited to southeast China. Furthermore, according to the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) programme in 2010,3 C. neoformans was recovered in
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province, China Si-Ying Wu, Yao Lei, Mei Kang, Yu-Ling Xiao and Zhi-Xing Chen Division of Clinical Microbiology, Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China
Summary
Previous reports on the molecular characteristics of clinical isolates of Cryptococcus species in China have focused on isolates from southeast China. To obtain a more detailed molecular epidemiology, a total of 92 cryptococcal isolates were collected from Sichuan province. A total of 24 isolates from 12 other provinces were collected for comparative study. Genotypes and mating types of 116 Cryptococcus isolates were determined. Among the 116 isolates, 43 isolates (19 isolates from Sichuan and 24 isolates outside of Sichuan) were analysed by multi-locus sequence typing (MLST). All 116 clinical isolates were mating type a. Most isolates (114/116) were molecular type VNI and the remaining two isolates were VGI and VGII respectively. MLST results revealed five sequence types (STs) of C. neoformans including two novel STs, with most isolates identified as ST5. The two C. gattii isolates identified in our study were ST44 and ST159. Based on our report and previous studies, there are 15 C. neoformans STs in China which can be divided into three subgroups. The C. gattii isolate from Sichuan could be a scattered subtype of VGII (ST44). Our findings demonstrated that C. neoformans isolates in Sichuan are genetically homogeneous, and ST5 is the epidemic clone of C. neoformans in China.
Key words: Cryptococcus neoformans, Cryptococcus gattii, Sichuan, Multi-locus sequence typing, sequence type, molecular epidemiology.
Introduction Cryptococcosis is an invasive fungal disease affecting approximately 1 million individuals worldwide each year.1 Infections due to Cryptococcus spp., which were previously considered rare fungal pathogens, have gradually increased over the past 20 years in China.2 Wang et al. [3] reported that, in China, the most frequently identified fungal pathogens belong to the genus Candida, followed by Cryptococcus spp. (7.7%). Cryptococcosis is primarily caused by two Cryptococcus Correspondence: Mei Kang, Division of Clinical Microbiology, Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. Tel.: +0086 28 85422618. Fax: +0086 28 85422618. E-mail: [email protected] Submitted for publication 4 December 2014 Revised 9 February 2015 Accepted for publication 11 February 2015
© 2015 Blackwell Verlag GmbH
species, C. neoformans and C. gattii.4 C. neoformans includes molecular types AFLP1/VNI, AFLP1A/VNB/ VNII, AFLP1B/VNII (C. neoformans var. grubii, serotype A); AFLP2/VNIV (C. neoformans var. neoformans, serotype D); and AFLP3/VNIII (hybrid, serotype AD) while C. gattii includes molecular types AFLP4/VGI, AFLP6/ VGII and AFLP10/VGIV (serotype B), AFLP5/VGIII and AFLP7/VGIV (serotype C).5,6 C. neoformans is an important opportunistic pathogen that commonly causes infections of the central nervous system, especially in patients with HIV while C. gattii generally results in pulmonary disease.7 C. gattii genotypes AFLP4/VGI and AFLP6/VGII usually occur in patients without detectable predisposing factors, whereas genotypes AFLP5/VGIII, AFLP7/VGIV and AFLP10/VGIV are commonly found among immuno-compromised individuals, similar to C. neoformans.8 Several molecular methods have been used for the detection of specific genetic sequences of C. neoformans/C. gattii species complex. The most commonly
doi:10.1111/myc.12312
S. Y. Wu et al.
used approaches were PCR fingerprinting, PCR-RFLP, AFLP, DNA sequence analysis, mating and/or serotype-specific PCRs and multi-locus sequence typing (MLST). Among these molecular techniques, MLST have been proposed as reference method.8 Extensive studies on molecular typing of Cryptococcus isolates have been reported worldwide. VNI is the predominant genotype and has been frequently reported in multiple regions globally.9–14 In China, previous studies showed that VNI caused the majority of infections in immuno-competent patients, with or without underlying conditions.15,16 Previous studies, however, were limited to southeast China. Furthermore, according to the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) programme in 2010,3 C. neoformans was recovered in
