A New <i>Aspergillus fumigatus</i> Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP)

<div><p><i>Aspergillus fumigatus</i> is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For <i>A</i>. <i>fumigatus</i> genotyping, the short tandem repeat method (STR<i>Af</i>) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. <i>A</i>. <i>fumigatus</i> isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated <i>A</i>. <i>fumigatus</i> strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for <i>A</i>. <i>fumigatus</i> has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.</p></div

Tandem repeats and flanking sequence for CSP types identified among 175 <i>A</i>. <i>fumigatus</i> isolates.

<p>Tandem repeats and flanking sequence for CSP types identified among 175 <i>A</i>. <i>fumigatus</i> isolates.</p

MP2 alleles identified among 175 <i>A</i>. <i>fumigatus</i> isolates.

<p>MP2 alleles identified among 175 <i>A</i>. <i>fumigatus</i> isolates.</p

Summary data for TRESP typing method using unrelated strains.

<p>Summary data for TRESP typing method using unrelated strains.</p

Minimum spanning tree (MST) showing the genotypic relationship between the azole-resistant and azole-susceptible <i>A</i>. <i>fumigatus</i> isolates.

<p>Each circle corresponds to a unique genotype, and the size of the circle proportionally represents the number of isolates with that genotype (1 to 21). Connecting lines correspond to the number of differences between the genotypes. Short bold line, 1 difference; black line, 2 differences; long grey line, 3 differences; dotted line, 4 or more differences. Grey circles, azole resistant TR₃₄/L98H, (n = 27); black circles, azole resistant non TR₃₄/L98H, (n = 22); white circles, azole susceptible strains, (n = 126). Four clusters were found (A-D).</p

Number of sequences on each set of identical N450 and M-F NCR sequences associated to the MVs/Madrid.ESP/46.10/ sub-lineage.

<p>Number of sequences on each set of identical N450 and M-F NCR sequences associated to the MVs/Madrid.ESP/46.10/ sub-lineage.</p

M-F NCR in the MeV D4 genotype strains obtained from GenBank.

<p>27 M-F NCRs of MeV genotype D4 strains were identified in GenBank; 23 had non-standard length M-F NCR sequences, 22 were of European origin, and 1 was from India. * indicates that the M-F NCR was extracted from the complete genome sequence. ** indicates MeV cases imported from India.</p

Molecular characterization of the samples analyzed in the study.

<p>Molecular characterization of the samples analyzed in the study.</p

M-F NCR types.

<p>Eight types of nucleotide sequences were identified in this study. Type 8 corresponds to the sequences of the D4-Bucharest lineage, which did not show the atypical M-F NCR structure. Numbers under the alignment indicate nucleotide positions in the full-length genome of the MeV Edmonston strain (GenBank Accession No. AY486083).</p

Phylogenetic analysis of the N-450 and M-F NCR.

<p>Panel A: N-450 dendrogram. The Kimura 2P nucleotide substitution model was used to build the tree. The MeV genotype D8 and D4 references (black diamonds) and D4 named strains accepted in MeaNS (black squares) were included in the analysis. The tree was rooted with respect to the genotype D8 WHO reference sequence. The number (n) of sequences of each set of identical sequences or named strain is shown in brackets. Panel B: M-F NCR dendrogram. The TN93 nucleotide substitution model with gamma distribution was used to build the tree. Genotype D4 M-F NCR sequences from GenBank were included in the analysis. A genotype D8 M-F NCR sequence from GenBank was used to root the tree. The number (n) of identical sequences of each set of identical sequences or variant is shown.</p