Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes

Contains fulltext : 109375.pdf (publisher's version ) (Open Access)BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine

Geographically structured populations of Cryptococcus neoformans Variety grubii in Asia correlate with HIV status and show a clonal population structure.

Contains fulltext : 125490.pdf (publisher's version ) (Open Access)Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia

Distribution of microsatellite complexes (MCs) between different countries.

<p>The predominant MCs in each country are indicated in bold.</p

The MIC range, MIC₅₀, MIC₉₀, and geometric mean for all 493 <i>C. neoformans</i> isolates for seven antifungals according to clinical and environmental origin of isolates.

<p>The MIC range, MIC₅₀, MIC₉₀, and geometric mean for all 493 <i>C. neoformans</i> isolates for seven antifungals according to clinical and environmental origin of isolates.</p

Genotypic variation of <i>C. neoformans</i> isolates from different Asian countries by microsatellite typing.

<p>(A) Minimum spanning tree based on a multistate categorical analysis representing 429 <i>C. neoformans</i> var. <i>grubii</i> isolates from different countries. Each circle represents a unique genotype. The size of the circle corresponds to the number of isolates within that genotype. Numbers and connecting lines correspond to the number of different markers between genotypes. Genotypes with identical colors are part of a microsatellite complex (MC). Circles without color are unique genotypes that are not part of a MC.; (B) Same as A, but now showing the genotypes from different geographic locations. Different colors correspond to different countries.; (C) Same as A and B, but now showing the genotypes from clinical and environmental sources.; (D) Same as A, B and C, but now showing the genotypes of Thai and Japanese population from clinical and environmental sources.</p

Distribution of sequence types (STs) of <i>C</i>. <i>neoformans</i> var. <i>grubii</i> isolates according to HIV status of the patients.

<p>The predominant STs in each HIV status category are indicated in bold.</p

Primers used for MLST analysis of Asian <i>C</i>. <i>neoformans</i> var. <i>grubii</i> isolates.

<p>Primers used for MLST analysis of Asian <i>C</i>. <i>neoformans</i> var. <i>grubii</i> isolates.</p

Minimum spanning trees using the goeBURST algorithm showing MLST relationships among Asian C. neoformans var. <i>grubii</i> isolates.

<p>(A) Tree represents 476 <i>C</i>. <i>neoformans</i> var. <i>grubii</i> isolates from different countries. Each circle represents a unique genotype/sequence type (STs). The size of the circle corresponds to the number of isolates within that genotype. Different colors correspond to different countries; (B) Same as A, but now showing the genotypes from clinical and environmental sources; (C) Same as A and B, but with the addition of the genotypes of 179 <i>C</i>. <i>neoformans</i> var. <i>grubii</i> isolates from different continents (data from <a href="http://mlst.mycologylab.org" target="_blank">http://mlst.mycologylab.org</a> and previous reports by Cogliati <i>et al</i>., 2013 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072222#pone.0072222-Cogliati1" target="_blank">[44]</a> and Mihara <i>et al</i>., 2012 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072222#pone.0072222-Mihara1" target="_blank">[16]</a>).</p