Microevolution of Serial Clinical Isolates ofCryptococcus neoformansvar.grubiiandC. gattii

Abstract

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.

Harnessing Whole Genome Sequencing in Medical Mycology

“…In addition, genome regions showing loss of heterozygosity during passage include genes implicated in drug resistance. Another study of serial isolates of C. neoformans and gattii compared isolates during initial presentation of disease and after 120 days or more during a relapse [ 52 ]. These cases were also highly clonal, demonstrating that a second independently infecting strain was not the origin of the relapse.…”

A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands

“…In terms of base changes, the three VGVI isolates (WM1802, WM1804, and CBS11687) differed by only 419 SNPs, with the lowest number found between WM1804 and CBS11687 (n ϭ 126) and the highest number found between WM1802 and CBS11687 (n ϭ 315). These genetic differences may have occurred as a result of microevolution during or following passaging or cryopreserving, although large CNVs are common in C. gattii (24,25). All of the newly isolated VGI (n ϭ 7) and VGVA (n ϭ 3) samples from Zambia had a small, Ͻ10-kb duplication within supercontig 6 of the R265 genome (kb position 400 to position 410).…”

“…Genome Analysis Toolkit (GATK) v2.7-4-g6f46d11 (69) was used to call both variant and reference nucleotides from the 101 alignments (as previously described [24]). Briefly, the Picard tools AddOr-ReplaceReadGroups, MarkDuplicates, CreateSequenceDictionary, and ReorderSam were used to preprocess the alignments (http://broadinstitute.github.io/picard/).…”

Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level

“…grubii have used typing methods for a few genetic loci or focused on particular geographic regions or countries ( de Oliveira et al 2004 ; Litvintseva et al 2006 ; Hiremath et al 2008 ; Khayhan et al 2013 ). Recent approaches have applied whole-genome sequencing (WGS) to trace the microevolution of Cryptococcus , identifying variation that occurs during the course of infection ( Ormerod et al 2013 ; Chen et al 2017 ; Rhodes et al 2017 ) or in the environment ( Vanhove et al 2017 ). Here, we use WGS of 188 isolates to provide a comprehensive view of the population variation between the three major lineages.…”