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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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Autores principales: Gallo, Juan E., Torres, Isaura, Gómez, Oscar M., Rishishwar, Lavanya, Vannberg, Fredrik, Jordan, I. King, McEwen, Juan G., Clay, Oliver K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305531/
https://www.ncbi.nlm.nih.gov/pubmed/34356923
http://dx.doi.org/10.3390/jof7070544
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author Gallo, Juan E.
Torres, Isaura
Gómez, Oscar M.
Rishishwar, Lavanya
Vannberg, Fredrik
Jordan, I. King
McEwen, Juan G.
Clay, Oliver K.
author_facet Gallo, Juan E.
Torres, Isaura
Gómez, Oscar M.
Rishishwar, Lavanya
Vannberg, Fredrik
Jordan, I. King
McEwen, Juan G.
Clay, Oliver K.
author_sort Gallo, Juan E.
collection PubMed
description 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.
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spelling pubmed-83055312021-07-25 New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons Gallo, Juan E. Torres, Isaura Gómez, Oscar M. Rishishwar, Lavanya Vannberg, Fredrik Jordan, I. King McEwen, Juan G. Clay, Oliver K. J Fungi (Basel) Article 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. MDPI 2021-07-09 /pmc/articles/PMC8305531/ /pubmed/34356923 http://dx.doi.org/10.3390/jof7070544 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gallo, Juan E.
Torres, Isaura
Gómez, Oscar M.
Rishishwar, Lavanya
Vannberg, Fredrik
Jordan, I. King
McEwen, Juan G.
Clay, Oliver K.
New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title_full New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title_fullStr New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title_full_unstemmed New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title_short New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons
title_sort new histoplasma diagnostic assays designed via whole genome comparisons
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305531/
https://www.ncbi.nlm.nih.gov/pubmed/34356923
http://dx.doi.org/10.3390/jof7070544
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