Identification of the major molecular types of Cryptococcus neoformans and C. gattii by Hyperbranched rolling circle amplification

Abstract

The agents of cryptococcosis C. neoformans and C. gattii are important agents of meningoencephalitis in immunocompromised and immunocompetent hosts, respectively. They are grouped into eight major molecular types, VNI-VNIV for C. neoformans and VGI-VGIV for C. gattii. These major molecular types differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. To enable a rapid identification of the major molecular types and potential hybrids within the two species specific probes based on the PLB1 gene in combination with hyperbranched rolling circle amplification (HRCA) were developed. HRCA was applied to 76 cryptococcal strains, 10 strains each representing the 7 haploid major molecular types, 4 VNIII hybrid strains and 2 inter-species hybrid strains. All strains were correctly identified to the major molecular type and or hybrid type using HRCA alone. To increase the sensitivity a semi-nested PCR step was developed, which will enable the identification of the molecular types/hybrids directly from clinical samples, harboring a low copy number of DNA (40 copies). Thus, HRCA based on the PLB1 locus alone and in combination with a semi-nested PCR showed to be a specific and sensitive methodology, with a great potential to be used on clinical specimens for the direct diagnosis of the agents of cryptococcosis, including hybrid strains, enabling a rapid and patient tailored treatment choice of this disease.

Figure 1. Association between MLST clusters and HRCA curves.

(A) Unrooted neighbor-joining tree inferred from the combined sequences ofCAP59, GPD1, LAC1, SOD1, URA5, PLB1 genes and IGS of 76 strains tested in this study. Numbers on branches are bootstrap support values obtained from 1,000 pseudoreplicates. *VGIII only when determined by the ISHAM MLST scheme, URA5 RFLP is grouping them incorrectly to VGIV due to a point mutation in the RFLP restriction site; (B) Amplification curves for representative strains of each major haploid molecular type, C. neoformans molecular types VNI, VNII and VNIV and C. gattii molecular types VGI, VGII, VGIII and VGIV obtained with the respective HRCA probes. (C) C. neoformans/ C. gattii VNI+VGII hybrid strain (WM 05.532), positive amplification with the HRCA probes VNI-PLB and VGII-PLB. (D) C. neoformans VNIII hybrid strain (WM 628), positive amplification with the HRCA probes VNI-PLB and VNIV-PLB. Positive results are indicated when the fluorescence signals increased exponentially.

Figure 2. Detection limit of the HRCA after semi-nested PCR.

(A) Real-time-PCR amplification curves for the semi-nested PCR amplification of the PLB1 locus from serial DNA dilutions of the strain WM 178 with the HRCA VGII probe. Lower curves correspond to dilutions with less than 40 copies of DNA (4×10⁻¹, 4×10⁻², 4×10⁻³, 4×10⁻⁴); (B) PLB1 locus semi-nested PCR products from serial DNA dilutions of WM 179 strain separated on a 1.4% agarose gel.