Global VGIIa isolates are of comparable virulence to the major fatal Cryptococcus gattii Vancouver Island outbreak genotype

Abstract

The ongoing cryptococcosis outbreak on Vancouver Island, BC, Canada, is caused by two VGII sub-genotypes of the primary pathogen, Cryptococcus gattii: VGIIa isolates predominate, whereas VGIIb isolates are rare. Although higher virulence of the VGIIa genotype has been proposed, an unresolved key question is whether VGIIa isolates from other regions are also more virulent than VGIIb isolates. We report the relationship between genotype and virulence for a global collection of C. gattii VGIIa and VGIIb isolates (from Australia, Argentina, Brazil, Canada, Thailand and the USA). In vitro and in vivo virulence studies were conducted. At 37°C, growth [at 18 h: 0.2 optical density (OD) difference, p 0.026; at 36 h: 0.6 OD difference, p 0.036) and mean melanin production (OD = 0.25 vs. OD = 0.15, p 0.059] of VGIIa isolates was greater than that of VGIIb isolates. The inhibitory effect of high temperature on melanin production of VGIIa isolates was less than that of VGIIb isolates (OD = 0.36 vs. OD = 0.69; p 0.001). Capsule production at 37°C of VGIIa isolates was less than that of VGIIb isolates. All VGIIa isolates were fertile, whereas only 17% of VGIIb isolates were fertile (p <0.001). In vivo virulence studies using the BALB/c mice nasal inhalation model revealed that VGIIa isolates were more virulent than VGIIb isolates (p <0.001) independent of their clinical (p 0.003) or environmental origin (p <0.001). This study established a clear association between genotype and virulence of the primary fungal pathogen, C. gattii.

Cryptococcus gattii Infections

“…This clinical observation is consistent with the much decreased survival and, hence, greater virulence of C. gattii VGIIa in mice and a substantially increased ability to replicate within J774 macrophages (1,38,84,149). A more extensive study of VGIIa and VGIIb isolates from around the world confirmed these findings (150). More recently, zebrafish larvae have been proposed as a tractable model in which to study cryptococcal interactions with the innate immune system (151).…”

“…In early studies of C. gattii VGI, phospholipase production per se quantified in vitro was correlated with virulence in mice (154). Differences between the two closely related VGII Vancouver Island outbreak subtypes were noted; all VGIIa isolates were fertile, compared with only 17% of VGIIb isolates, and at 37°C, growth rates, melanin production, and capsule size were increased (150). In another study of Vancouver Island outbreak strains, capsule size; melanin, phospholipase, and proteinase production; and other enzyme activities were analyzed in 39 C. gattii strains of variable virulences and different molecular types.…”

Phenotypic Differences of Cryptococcus Molecular Types and Their Implications for Virulence in a Drosophila Model of Infection

“…Forty-nine Cryptococcus isolates obtained from clinical, environmental, and veterinary sources were used (Table 1). Molecular types were identified as described in prior studies (11). Mating type was determined by PCR using two pairs of mating-type-specific primers per cryptococcal species as described previously (13,14).…”

“…Melanin synthesis was quantified using a modified laccase activity test as previously described (11,19). Each isolate was grown in YPD broth for 24 h at 30°C.…”

“…VGI and VGII primarily affect immunocompetent patients, while VGIII and VGIV are most frequently found in the immunocompromised (4). Comparative studies between different cryptococcal molecular types have been limited and have primarily focused on the major outbreak strain associated with the Pacific Northwest (VGIIa) and have found VGIIa strains more lethal than VGIIb isolates (10,11). More recently, VGIIIa and VGIIIb subtypes recovered from HIV/AIDS patients in California were compared in an intranasal murine model (4), and VGIIIa isolates were found to be more virulent than VGIIIb isolates; however, comparisons of cryptococcal molecular types and levels of virulence on a broader scale have not previously been performed.…”