New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons
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, con...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-07-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/7/544 |
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| author | Juan E. Gallo Isaura Torres Oscar M. Gómez Lavanya Rishishwar Fredrik Vannberg I. King Jordan Juan G. McEwen Oliver K. Clay |
| author_facet | Juan E. Gallo Isaura Torres Oscar M. Gómez Lavanya Rishishwar Fredrik Vannberg I. King Jordan Juan G. McEwen Oliver K. Clay |
| author_sort | Juan E. Gallo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T09:35:04Z |
| format | Article |
| id | doaj.art-21e776e0444744b78431270d034ff42f |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T09:35:04Z |
| publishDate | 2021-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-21e776e0444744b78431270d034ff42f2023-11-22T04:09:50ZengMDPI AGJournal of Fungi2309-608X2021-07-017754410.3390/jof7070544New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome ComparisonsJuan E. Gallo0Isaura Torres1Oscar M. Gómez2Lavanya Rishishwar3Fredrik Vannberg4I. King Jordan5Juan G. McEwen6Oliver K. Clay7Cellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB), Medellín 05534, ColombiaCellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB), Medellín 05534, ColombiaCellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB), Medellín 05534, ColombiaSchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USASchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USASchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USACellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB), Medellín 05534, ColombiaCellular and Molecular Biology Unit, Corporación para Investigaciones Biológicas (CIB), Medellín 05534, ColombiaHistoplasmosis 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.https://www.mdpi.com/2309-608X/7/7/544fungal diagnosticsemerging diseasesPCR assayscross-reactionswhole genome sequences |
| spellingShingle | Juan E. Gallo Isaura Torres Oscar M. Gómez Lavanya Rishishwar Fredrik Vannberg I. King Jordan Juan G. McEwen Oliver K. Clay New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons Journal of Fungi fungal diagnostics emerging diseases PCR assays cross-reactions whole genome sequences |
| title | New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons |
| title_full | New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons |
| title_fullStr | New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons |
| title_full_unstemmed | New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons |
| title_short | New <i>Histoplasma</i> Diagnostic Assays Designed via Whole Genome Comparisons |
| title_sort | new i histoplasma i diagnostic assays designed via whole genome comparisons |
| topic | fungal diagnostics emerging diseases PCR assays cross-reactions whole genome sequences |
| url | https://www.mdpi.com/2309-608X/7/7/544 |
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