Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model

<div><p>Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x10⁶ <i>Histoplasma capsulatum</i> yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of <i>H</i>. <i>capsulatum</i> (30 strains) and other medically relevant pathogens (36 strains of related fungi and <i>Mycobacterium tuberculosis</i>) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of <i>H</i>. <i>capsulatum</i> yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from <i>H</i>. <i>capsulatum</i>-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting <i>H</i>. <i>capsulatum</i> DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.</p></div

Standardization of qPCR protocols: Sensitivity of the real time PCR using 30 different <i>Histoplasma</i> DNAs representing 6 of the 8 clades described.

<p>Example of 100-kDa qPCR protocol. NAm1: n = 4, Nam2: n = 10, LamA: n = 7, LamB: n = 5, Netherlands: n = 1, Africa: n = 3.</p

Sequences of the primers and probes for each molecular target.

<p>Sequences of the primers and probes for each molecular target.</p

Histopathological analysis and detection of <i>Histoplasma</i> DNA in FFPE lung tissues using the three molecular targets.

<p>A) Microphotographs from lung sections stained with Grocott’s methenamine silver from mice infected with 2.5x10⁶ <i>H</i>. <i>capsulatum</i> yeast cells at the first and second weeks post-infection, with the respective semiquantification measured by crosses of <i>H</i>. <i>capsulatum</i> yeast cells. Magnification 100X. B) Results of qPCR are represented with Ct values and number of copies for each protocol at the first and second weeks post-infection.</p

Fungal burden and detection of <i>Histoplasma</i> DNA in fresh lung tissues using the three molecular targets.

<p>A) Quantitative analysis of colony forming units (CFUs) evaluated in lungs of mice challenged i.n. with 2.5x10⁶ <i>H</i>. <i>capsulatum</i> yeast cells at different weeks post-infection. B) Results of qPCR represented with Ct values and number of copies for each protocol at the first and second weeks post-infection.</p

Average of crossing point (threshold cycle) for each <i>H</i>. <i>capsulatum</i> clade for the three qPCR protocols.

<p>Average of crossing point (threshold cycle) for each <i>H</i>. <i>capsulatum</i> clade for the three qPCR protocols.</p

Collection of DNA from cultures of <i>H</i>. <i>capsulatum</i> representing different clades.

<p>Collection of DNA from cultures of <i>H</i>. <i>capsulatum</i> representing different clades.</p

Whole Genome Sequence Typing to Investigate the <em>Apophysomyces</em> Outbreak following a Tornado in Joplin, Missouri, 2011

<div><p>Case reports of <em>Apophysomyces</em> spp. in immunocompetent hosts have been a result of traumatic deep implantation of <em>Apophysomyces</em> spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with <em>Apophysomyces trapeziformis</em> infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak <em>Apophysomyces</em> isolates and five additional temporally and spatially diverse <em>Apophysomyces</em> control isolates (three <em>A. trapeziformis</em> and two <em>A. variabilis</em> isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical <em>A. trapeziformis</em> isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus <em>Apophysomyces</em>.</p> </div

<i>De novo</i> assembled reference statistics for Strain Apo-097.

<p><i>De novo</i> assembled reference statistics for Strain Apo-097.</p

WGST phylogeny of outbreak and background Apophysomyces isolates.

<p>A single maximum parsimony tree was reconstructed using ∼28K SNPs from 22 whole genome sequences, resulting in a CI of 0.62. The tree was rooted with Apo-7449 (A. variabilis). The root was derived from an expanded phylogenetic analyses that included a distant outgroup, which enabled the identification of the most basal member of the samples in the dataset described in this figure (data not shown). Genomes are labeled with a Strain ID # (APO-XXXX) and a Cluster ID #-Letter (CX-A). Clusters of identical and nearly identical genome SNP profiles are also labeled. Branch lengths represent genetic distance based on the number of SNP differences; bar represents 1000 SNPs. Tree constructed using MEGA5.</p