P151 Molecular epidemiology of environmental strains of Cryptococcus isolated from Varanasi, Uttar Pradesh, India

Abstract Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Cryptococcosis affects more than one million people per year worldwide. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. The ecological niche of the etiological agents of C. neoformans and C. gattii are not well established yet in various parts of India. Methodology The present study was performed on the C. neoformans isolated from the Eucalyptus terreticornis at Varanasi, India. A total of 265 samples including flowers, leaves, bark, and nearby soil of E. terreticornis. The fungal pathogens were identified by both the conventional and molecular methods. The isolates were also grown in Staib's and tobacco agar medium. In addition, all isolates were identified on the basis of different biochemical tests such as urease test and canavanine-glycine-bromothymol blue (CGB). Further, molecular characterization was carried out by using PCR and DNA sequence analyses. Results Of all the 265 samples, 11 were positive for C. neoformans. 8% (4/50) for E. tereticornis bark, 5% (3/60) for nearby soil, 6.66% (2/30) for debris, and 3.07% (2/65) for decayed wood from tree hollows. C. neoformans was not recovered from the leaves and flowers of E. tereticornis. The presence of brown colonies on Staib's and tobacco agar media confirmed that the isolated pathogen is Cryptococcus. Further the isolates of Cryptococcus were tested for urease production using Christensen urea citrate agar medium in which all isolates were found to hydrolyze urea rapidly and reddish pink color was obtained confirming it to be C. neoformans. All isolated yeast were negative for Canavanine glycinebromothymol blue (CGB) as they fail to produce a color change in the medium. Five strains were identified as C. neoformans var. grubii and one of the isolates was identified as C. neoformans var. neoformans. Molecular typing grouped the isolates into two major genotypes. Five were molecular type VNI (serotype A, var. grubii), one of the isolates was molecular type VNIV (serotype D, var. neoformans). Figure 3 shows the Gel electrophoresis image of ITS1- ITS4 amplicons. The nucleotide sequences were submitted in NCBI GenBank with accession numbers MZ824412, MZ848145, MZ823611, MZ882370, MZ882372, and MZ817986. Conclusion In spite of a major Eucalyptus growing region of India, there is no report of C. neoformans/C. gattii-Eucalyptus association from Eastern Uttar Pradesh. Therefore, the current study would certainly be helpful in the establishment of molecular epidemiology of Cryptococcus in this area.