Genotyping of cryptococcus neoformans species complex Thai isolates

Abstract

To investigate the genetic polymorphism of human, animal, and environmental isolates of Cryptococcus neoformans in Thailand during 2003 – 2005, Multilocus Sequencing Typing (MLST) and Multilocus Microsatellite Typing (MLMT) techniques were used. 481 isolates of C. neoformans species complex from clinical and environmental sources were screened for their variety using URA5-RFLP, mating type. All the isolates were identified as C. neoformans var. grubii serotype A, molecular type VNI (98%) and molecular type VNII (2%) with mating type α. Intra-species level were studied by M13-fingerprinting technique. Nine subtypes, subtype A to I, were observed. Subtype A, B, C, D, E, and F were grouped in molecular type VNI while subtype G, H and I were molecular type VNII. The most frequency subtype was subtype A (88.25%). Totally 31 isolates from all individual subtype was recruited for MLST and MLMT study. For MLST, the analytical fragments (Sequence Type, ST) of each individual isolate were obtained by PCR reaction using primers from eight of housekeeping and virulence genes. ST is composed of the combination of allele types (AT) or allele profiles from any distinct genes. The genetic variation of 12 STs was demonstrated. Comparing our STs with MLST database website, new 13 ATs were revealed. For MLMT method, the allele of each DNA locus (microsatellite allele; MA) were analyzed in the combination of locus; resulting Microsatellite type (MT). In this study, five loci showed polymorphisms, totally 13 MTs found in the study. The concordance of M13-fingerprinting, MLST and MLMT method was found with the discriminatory power of 0.75 and 0.86 and 0.82 respectively. Thus, the genetic information among Thai isolates was classified in the same genetic cluster shared and grouped. In addition, the genetic relationship with global isolates was also demonstrated. In conclusion, C. neoformans var. grubii was the majority variety in Thailand according with the previous report. Genetic polymorphism revealed 12 STs and 13 MTs. MLST is one of the promising typing methods for epidemiological study and the result can be comparable and also applicable in the e-network.