Molecular type distribution and fluconazole susceptibility of clinical Cryptococcus gattii isolates from South African laboratory-based surveillance, 2005-2013

Abstract

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC50 and MIC90 values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC50 value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93-55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa's most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.

Fig 1. Neighbour joining phylogenetic tree based on multilocus sequence typing data showing clustering of the molecular types for 99 different sequence types (STs) from 146 South African clinical Cryptococcus gattii isolates.

Cryptococcus neoformans strain WM 05.474, molecular type VNI serves as the outgroup. A standard strain for each molecular type was also included. Bootstrap analysis was performed with 100 bootstrap replicates. Bootstrap values are shown next to the branches.

Fig 2. Rooted maximum parsimony tree generated from the WGS SNP analysis of 98 South African clinical VGIV Cryptococcus gattii isolates (coloured circles and bolded tip labels) and 47 global samples.

IND107 was used as the reference. BWA was used to align reads to the reference and SAMtools was used to call SNPs. There were 123,891 SNPs in total with a coverage breadth of 61%. Bootstrap analysis was peformed with 100 bootstrap replicates. The bootstrap values are shown next to the branches. The consistency index was 0.35 and the number of phylogenetic informative positions were 123,891. BO–Botswana, SA–South Africa, CO–Colombia, AU–Australia, IN–India, ZA–Zambia, U—Unknown.

Fig 3. All clinical cases of Cryptococcus gattii infection (n = 508) collected through laboratory-based surveillance during 2005–2013 from three South African provinces highlighting the cases that clustered closely by genomic data.

A: Unrooted maximum parsimony tree generated from the WGS SNP analysis of ten South African clinical VGIV C. gattii isolates from patients living in the Gauteng and Limpopo provinces. Strain number 918 was used as the reference. There were 1600 SNPs in total with a coverage breadth of 96%. The consistency index was 0.99 and the number of phylogenetic sites were 1600. Maroon–Gauteng, purple–Limpopo. B: Unrooted maximum parsimony tree generated from the WGS SNP analysis of eight South African clinical VGIV C. gattii isolates (six from this study and two from a previously-published South African study [48]). Isolates from this study are indicated in bold. Strain number 976 was used as the reference. There were 1649 SNPs in total with a coverage breadth of 96%. The consistency index was 1.00 and the number of phylogenetic sites were 1649. Yellow–Mpumalanga Province. C: Map of South Africa showing the clustered cases in yellow and all C. gattii cases (n = 508) in grey from Gauteng, Limpopo and Mpumalanga provinces (https://cdn.arcgis.com/sharing/rest/content/items/7dc6cea0b1764a1f9af2e679f642f0f5/resources/styles/root.json). This map was created using ArcGIS software by ESRI (www.esri.com). ArcGIS and ArcMap are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com.