MSP dendrograms grouping A) mass spectra of <i>Cryptococcus neoformans</i> and B) <i>C. gattii</i> strains according to their major molecular type.

<p>MSP dendrograms grouping A) mass spectra of <i>Cryptococcus neoformans</i> and B) <i>C. gattii</i> strains according to their major molecular type.</p

Scores obtained for the identification of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> isolates, using the original un-supplemented MALDI Biotyper BDAL MSP library (Bruker).

*<p>2.300–3.000: highly probable species identification; 2.000–2.299: secure genus identification, probable species identification; 1.700–1.999: probable genus identification.</p

Gel view of the MSP of the obtained mass spectra of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> strains according to their major molecular type.

<p>Gel view of the MSP of the obtained mass spectra of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> strains according to their major molecular type.</p

Association between MLST clusters and HRCA curves.

<p>(<b>A</b>) Unrooted neighbor-joining tree inferred from the combined sequences of<i>CAP59</i>, <i>GPD1</i>, <i>LAC1</i>, <i>SOD1</i>, <i>URA5</i>, <i>PLB1</i> 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, <i>URA5</i> RFLP is grouping them incorrectly to VGIV due to a point mutation in the RFLP restriction site; (<b>B</b>) Amplification curves for representative strains of each major haploid molecular type, <i>C. neoformans</i> molecular types VNI, VNII and VNIV and <i>C. gattii</i> molecular types VGI, VGII, VGIII and VGIV obtained with the respective HRCA probes. (<b>C</b>) <i>C. neoformans/ C. gattii</i> VNI+VGII hybrid strain (WM 05.532), positive amplification with the HRCA probes VNI-PLB and VGII-PLB. (<b>D</b>) <i>C. neoformans</i> 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.</p

Detection limit of the HRCA after semi-nested PCR.

<p>(<b>A</b>) Real-time-PCR amplification curves for the semi-nested PCR amplification of the <i>PLB1</i> 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>B</b>) <i>PLB1</i> locus semi-nested PCR products from serial DNA dilutions of WM 179 strain separated on a 1.4% agarose gel.</p

Identification of the Major Molecular Types of <i>Cryptococcus neoformans</i> and <i>C. gattii</i> by Hyperbranched Rolling Circle Amplification

<div><p>The agents of cryptococcosis <i>C. neoformans</i> and <i>C. gattii</i> are important agents of meningoencephalitis in immunocompromised and immunocompetent hosts, respectively. They are grouped into eight major molecular types, VNI-VNIV for <i>C. neoformans</i> and VGI-VGIV for <i>C. gattii</i>. 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 <i>PLB1</i> 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 <i>PLB1</i> 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.</p></div

Strains tested in this study and the results obtained by HRCA.

<p><b>Note: +</b>: Positive signal with the specific probe; -: No signal with the specific probe;</p><p>*VGIII only when determined by the ISHAM MLST scheme, <i>URA5</i> RFLP is grouping them incorrectly to VGIV due to a point mutation in the RFLP restriction site; ^S: standard strain for the major molecular type (Meyer <i>et al.</i> 2009).</p

Padlock probes and padlock probe primers.

<p>Note: The 5′- and 3′-ends of the probes that are complementary to the target sequences are underlined. The regions where the two padlock probe-specific primers (RAC1 and RCA2) bind for real-time amplification are in lower case letters. The 5′-end of probe, p-indicates phosphorylation. Ambiguous positions were introduced according to the target sequences (in bold): M = A or C, K = G or T, R = A or G, S = C or G.</p

Sampling locations in the city of Santa Isabel do Rio Negro (circles) with the 3 positive houses (dark circles), DW650, SI-444 and SI-443 with the respective sequence types (ST) identified amongst the samples, and location of Santa Isabel do Rio Negro in the Amazonas state, Northern Brazil.

<p>The maps were adapted from Bing Maps to reflect the specific study site situation.</p

Gene network placing the Thai<i>C.</i><i>neoformans</i> isolates in global context.

<p>Gene network constructed from all <i>C. neoformans</i> ST types identified by MLST analysis in the current study in-cooperating the STs previously obtained from Thai cryptococcal isolates <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002297#pntd.0002297-Simwami1" target="_blank">[33]</a> and standard strains based on the combined seven ISHAM consensus MLST loci using the program Network 4.5.1.6, showing the close relationships between STs present in Thailand and globally.</p