| Summary: | Histoplasmosis is a systemic fungal disease caused by the pathogen <i>Histoplasma</i> spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on <i>Histoplasma</i>-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of <i>Histoplasma</i> strains but not in other genera. Two of the genes, <i>PPK</i> and <i>CFP4</i>, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 <i>H. capsulatum</i> isolates using purified DNA. We also obtained positive detections of 2/2 confirmed <i>H. capsulatum</i> clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.
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