New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma 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...

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Bibliographic Details
Published in:Journal of fungi (Basel) 2021-07, Vol.7 (7), p.544
Main Authors: Gallo, Juan E., Torres, Isaura, Gómez, Oscar M., Rishishwar, Lavanya, Vannberg, Fredrik, Jordan, I. King, McEwen, Juan G., Clay, Oliver K.
Format: Article
Language:English
Subjects:
Acquired immune deficiency syndrome
AIDS
Antigens
cross-reactions
Design
emerging diseases
fungal diagnostics
Fungal infections
Fungi
Genes
Genomes
Histoplasmosis
HIV
Human immunodeficiency virus
Immunology
Laboratories
Morbidity
Mortality
Paraffin
Pathogens
PCR assays
Polymerase chain reaction
Public health
whole genome sequences
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Summary:Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma 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 Histoplasma-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 Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, 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 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum 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.
ISSN:2309-608X
2309-608X
DOI:10.3390/jof7070544