Detection of mPCR amplified products by agarose gel electrophoresis.

<p>An agarose gel of mPCR amplified products using template DNA from reference strain of <i>C</i>. <i>parapsilosis</i> (lane CP), <i>C</i>. <i>orthopsilosis</i> (lane CO), <i>C</i>. <i>metapsilosis</i> (lane CM) and <i>L</i>. <i>elongisporus</i> (lane LE). Lane M is 100 bp DNA marker and the position of migration of 100 bp, 300 bp and 600 bp fragments are marked.</p

Comparison between various phenotypic and genotypic methods used for identification of <i>C</i>. <i>parapsilosis</i> complex isolates.

<p>Comparison between various phenotypic and genotypic methods used for identification of <i>C</i>. <i>parapsilosis</i> complex isolates.</p

Simple, Low-Cost Detection of <i>Candida parapsilosis</i> Complex Isolates and Molecular Fingerprinting of <i>Candida orthopsilosis</i> Strains in Kuwait by ITS Region Sequencing and Amplified Fragment Length Polymorphism Analysis

<div><p><i>Candida parapsilosis</i> has now emerged as the second or third most important cause of healthcare-associated <i>Candida</i> infections. Molecular studies have shown that phenotypically identified <i>C</i>. <i>parapsilosis</i> isolates represent a complex of three species, namely, <i>C</i>. <i>parapsilosis</i>, <i>C</i>. <i>orthopsilosis</i> and <i>C</i>. <i>metapsilosis</i>. <i>Lodderomyces elongisporus</i> is another species phenotypically closely related to the <i>C</i>. <i>parapsilosis</i>-complex. The aim of this study was to develop a simple, low cost multiplex (m) PCR assay for species-specific identification of <i>C</i>. <i>parapsilosis</i> complex isolates and to study genetic relatedness of <i>C</i>. <i>orthopsilosis</i> isolates in Kuwait. Species-specific amplicons from <i>C</i>. <i>parapsilosis</i> (171 bp), <i>C</i>. <i>orthopsilosis</i> (109 bp), <i>C</i>. <i>metapsilosis</i> (217 bp) and <i>L</i>. <i>elongisporus</i> (258 bp) were obtained in mPCR. Clinical isolates identified as <i>C</i>. <i>parapsilosis</i> (n = 380) by Vitek2 in Kuwait and an international collection of 27 <i>C</i>. <i>parapsilosis</i> complex and <i>L</i>. <i>elongisporus</i> isolates previously characterized by rDNA sequencing were analyzed to evaluate mPCR. Species-specific PCR and DNA sequencing of internal transcribed spacer (ITS) region of rDNA were performed to validate the results of mPCR. Fingerprinting of 19 clinical <i>C</i>. <i>orthopsilosis</i> isolates (including 4 isolates from a previous study) was performed by amplified fragment length polymorphism (AFLP) analysis. Phenotypically identified <i>C</i>. <i>parapsilosis</i> isolates (n = 380) were identified as <i>C</i>. <i>parapsilosis</i> sensu stricto (n = 361), <i>C</i>. <i>orthopsilosis</i> (n = 15), <i>C</i>. <i>metapsilosis</i> (n = 1) and <i>L</i>. <i>elongisporus</i> (n = 3) by mPCR. The mPCR also accurately detected all epidemiologically unrelated <i>C</i>. <i>parapsilosis</i> complex and <i>L</i>. <i>elongisporus</i> isolates. The 19 <i>C</i>. <i>orthopsilosis</i> isolates obtained from 16 patients were divided into 3 haplotypes based on ITS region sequence data. Seven distinct genotypes were identified among the 19 <i>C</i>. <i>orthopsilosis</i> isolates by AFLP including a dominant genotype (AFLP1) comprising 11 isolates recovered from 10 patients. A rapid, low-cost mPCR assay for detection and differentiation of <i>C</i>. <i>parapsilosis</i>, <i>C</i>. <i>orthopsilosis</i>, <i>C</i>. <i>metapsilosis</i> and <i>L</i>. <i>elongisporus</i> has been developed.</p></div

Amplified fragment length polymorphism (AFLP)-based fingerprinting of <i>C</i>. <i>orthopsilosis</i> isolates.

<p>An UPGMA-derived dendrogram based on AFLP fingerprints for 19 clinical <i>C</i>. <i>orthopsilosis</i> isolates obtained from 16 patients in Kuwait. The reference <i>C</i>. <i>orthopsilosis</i> strain (ATCC 96139) was also included in AFLP analysis and a clinical <i>C</i>. <i>metapsilosis</i> isolate (Kw164-7/12) was used as an outer group. Similarity is presented in percentages using the scale bar in the upper left corner. The columns after the AFLP patterns represent the isolate number, species name, hospital name, source of the isolates and AFLP and ITS genotypic grouping.</p

Specific features and DNA sequences of the mPCR primers used in this study.

<p>Specific features and DNA sequences of the mPCR primers used in this study.</p

Minimum spanning tree showing wide genotypic diversity both in the clinical <i>A</i>. <i>terreus</i> isolates from India and those outside India.

<p>The figure shows the 115 different genotypes (circles), the number of strains belonging to the same genotype (sizes of the circles), and origin of isolates (circles in yellow indicate Indian isolates; green indicating European isolates including France (n = 4), Slovenia (n = 1), Germany (n = 2), Italy (n = 2), Norway (n = 2), Spain (n = 4), Netherlands (n = 5); pink indicate isolates from Australasia, including New Guinea (n = 2), New Zealand (n = 1), Taiwan (n = 1), China (n = 1), Thailand (n = 1); bright blue indicates isolate from Panama (Latin America; n = 1); dark blue indicates North American (n = 20) isolates). Gray-zone indicates microsatellite cluster representing minimal 2 isolates that differ maximum by 1 microsatellite marker out of 9. Thick and medium-thick branches indicate 1 or 2 microsatellite marker differences, respectively. Thick dashed line indicates 3 marker differences between two genotypes; 4 or more microsatellite markers differences between genotypes are indicated by medium thick and thin dashed lines, respectively.</p

Analysis of genotypic relationship between <i>A</i>. <i>terreus</i> isolates from India (n = 101), Europe (20), USA (20), Panama (n = 1), New Zealand (n = 1), Papua New Guinea (n = 2), China (n = 1), Taiwan (n = 1) and Thailand (n = 1) using STR typing.

<p>The dendrogram is based on a categorical analysis of 9 microsatellite markers in combination with UPGMA clustering. The scale bar indicates the percentage identity.</p

Phylogenetic tree based on partial sequence of <i>calmodulin</i> gene using maximum likelihood analysis depicting intraspecies variation among <i>A</i>. <i>terreus</i> isolates.

<p><i>Aspergillus terreus</i> (CBS 601.65^T), <i>Aspergillus alabamensis</i> (UAB38), <i>A</i>. <i>terreus</i> var. <i>africanus</i> (syn. <i>A</i>. <i>neoafricanus</i> CBS 130.55^T), <i>A</i>. <i>aureoterreus</i> (CBS 265.81^T), <i>A</i>. <i>hortai</i> (IBT16744, IBT16745 and IBT26384) of <i>A</i>. <i>terreus</i> section <i>Terrei</i> were taken as outliers for the analysis. Bootstrap values are shown above the branches. Environmental isolates are denoted by E, and clinical isolates are denoted by C.</p

Amplified fragment length polymorphism analysis showing genotypic diversity among 122 clinical and environmental Indian A. terreus isolates.

<p><i>Aspergillus terreus</i> (CBS 601.65^T), <i>A</i>. <i>terreus</i> var. <i>africanus</i> (syn. <i>A</i>. <i>neoafricanus</i> CBS 130.55^T), <i>A</i>. <i>terreus</i> var. <i>floccosus</i> (syn. <i>A</i>. <i>floccosus</i> CBS 116.37^T) were used for the analysis. The dendrogram was constructed by using UPGMA (unweighted pair group method with averages) in combination with the Pearson correlation coefficient and was restricted to fragments of 60–400 bp. Scale bar indicates the percentage similarity.</p

Image_1_Low-Cost Tetraplex PCR for the Global Spreading Multi-Drug Resistant Fungus, Candida auris and Its Phylogenetic Relatives.JPEG

<p>Candida auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii are closely related and highly multidrug resistant yeast pathogens. The high cost and low accuracy of current diagnostics may underestimate their prevalence, especially in medical resource-limited regions. In this study, we used 172 C. auris stains and its relatives and 192 other fungal strains to establish and validate a novel multiplex end-point PCR. A prospective and a retrospective clinical screenings using this assay were further performed in China and Iran respectively. We identified the first isolate of C. pseudohaemulonii in China and the first isolate of C. haemulonii in Iran from 821 clinical isolates in total, without any false positive. Animal models of C. auris and C. haemulonii were established for validation. The overall positive rates of the assay for mice blood and tissue were 28.6 and 92.9%, respectively. Compared with previously developed assays, our assay is more available and affordable to the developing countries, and may contribute to a better understanding of the epidemiology of C. auris and its relatives in these regions.</p