A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada)

Abstract

Cryptococcus gattii causes life-threatening infection of the pulmonary and central nervous systems in hosts with normal immunity and traditionally has been considered to be restricted geographically to tropical and subtropical climates. The recent outbreak of C. gattii in the temperate climate of Vancouver Island, BC, Canada, led to a collaborative investigation. The objectives of the current study were to ascertain the environmental source of the outbreak infections, survey the molecular types of the outbreak and environmental cryptococcal isolates, and determine the extent of genetic diversity among the isolates. PCR-fingerprinting and amplified fragment length polymorphism (AFLP) were used to examine the genotypes, and mating assays were performed to determine the mating type of the isolates. All outbreak and environmental isolates belonged to C. gattii. Concordant results were obtained by using PCR-fingerprinting and AFLP analysis. The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII/AFLP6, but two were caused by molecular type VGI/AFLP4. All environmental isolates belonged to molecular type VGII/AFLP6. Two or three subtypes were observed within VGII/AFLP6 among outbreak and environmental isolates. All mating-competent isolates were of the alpha-mating type. The emergence of this usually tropical pathogen on Vancouver Island highlights the changing distribution of this genotype and emphasizes the importance of an ongoing collaborative effort to monitor the global epidemiology of this yeast.

Fig. 1.

Geographical distribution of human and animal cases of cryptococcosis diagnosed between January 1999 and January 2002. All Vancouver Island cases occurred in patients and animals that lived on the east coast of Vancouver Island in the CDF biogeoclimatic zone. All mainland-based cases occurred in people who had traveled to the CDF zone on Vancouver Island within 1 year prior to the onset of disease. Environmental sampling primarily was concentrated on areas within the CDF, particularly around residences and recreational areas of patients and animals.

Fig. 2.

Examples of molecular profiles obtained from isolates from the Vancouver Island cryptococcosis outbreak. (A) PCR-fingerprint profiles amplified by using the M13 primer (5′-GAGGGTGGCGGTTCT-3′). Profiles were separated on a 1.4% agarose gel with 1× Tris-borate-EDTA (TBE) buffer [10.8 g/liter Tris base/5.5 g/liter boric acid/4 mM EDTA (pH 8.0)] to 14 cm, with a 1-kb size marker (GIBCO/BRL). (B) URA5-RFLP used enzymes HhaI and Sau96I. Profiles separated on a 3.0% agarose-TBE gel with a 100-bp mass ruler (Fermentas, Hanover, MD).

Fig. 3.

Phenogram of PCR-fingerprint profiles obtained from Vancouver Island outbreak and environmental isolates with the single primer M13, created with the program gelcompar II (Applied Maths), with Dice coefficient and UPGMA.

Fig. 4.

Clustering of AFLP patterns of isolates from a single Douglas fir tree (no. 152) by using UPGMA and band matching. Both AFLP genotypes, AFLP6A (11 isolates) and AFLP6B (1 isolate), occurred on this tree.

Fig. 5.

Comparison of PCR-fingerprint similarity between selected outbreak isolates and previously studied global VGI and VGII isolates. PCR-fingerprints were generated by using the M13 primer and analyzed with gelcompar II software (Applied Maths) by using Dice coefficient and UPGMA.