Genotypic diversity within clinical and environmental Cryptococcus neoformans var grubii population in India

Prakash A., Hagen F., Kathuria S., Meis J.F., Chowdhary A.

Abstract: 

Background Cryptococcus neoformans is an encapsulated, ubiquitous environmental yeast that causes cryptococcosis, a potentially serious disease that affects immunocompromised individuals, especially patients with AIDS. The studies determining the genotypic diversity in C. neoformans population is important to gain insightful knowledge of this important pathogen. Microsatellite typing has become a popular molecular tool to study genotypic diversity in fungal pathogens due to its high discriminatory power and reproducibility.
Aim To investigate genetic relatedness and antifungal susceptibilities of a large collection of C. neoformans isolates from clinical and environmental sources in India collected during 2001-2010. Materials and Methods Four hundred and fifty three (224 environmental and 229 clinical) Indian Cryptococcus neoformans isolates were subjected to Amplified Fragment Length Polymorphism (AFLP) and genotyped by microsatellite typing based on nine markers specific for C. neoformans variety grubii (serotype A) or variety neoformans (serotype D). In vitro antifungal susceptibility was determined for standard antifungals using CLSI M27-A3 guidelines.
Results All 453 isolates were AFLP1/VNI genotype, representing C. neoformans variety grubii serotype A. Microsatellite typing revealed that the majority of isolates belonged to microsatellite cluster (MC) MC3 (n = 183; 40.4%), followed by MC1 (n = 160; 35.2%), MC2 (n = 24; 5.2%), MC13 (n= 19; 4.1%), MC22 (n = 7; 1.5%), and others (8 MCs, n = 20; 4.4%). Forty (9.2%) isolates could not be linked to a known MC from previous studies. MICs90 of AMB, FC, FLU, ITC, VRC, POS and ISA were 1, 16, 8, 0.25, 0.125, 0.25, and 0.06 mg l -1, respectively. Geometric mean MICs revealed that isolates in MC1 were significantly less (P < 0.0001) susceptible to ITC, ISA, FC and AMB (P = 0.002) than isolates in MC3.
Conclusions The present study is the largest series of C. neoformans var. grubii reported so far from a single country. Environmental isolates were genetically more diverse than clinical isolates comprising a larger number of MC types than clinical MCs. Two microsatellite types of C. neoformans var. grubii dominate in India and were uniformly distributed over clinical and environmental isolates. Fluconazole and flucytosine had high MICs (>8 mg l -1) in 2% and 7% of the isolates respectively, whereas the new azole Isavuconazole exhibited the lowest GM MIC of 0.06 mg l -1.

    • ICCC 9th