{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T05:02:25Z","timestamp":1773378145160,"version":"3.50.1"},"reference-count":29,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T00:00:00Z","timestamp":1625788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007637","name":"Departamento Administrativo de Ciencia, Tecnolog\u00eda e Innovaci\u00f3n (COLCIENCIAS)","doi-asserted-by":"publisher","award":["1222-569-34875"],"award-info":[{"award-number":["1222-569-34875"]}],"id":[{"id":"10.13039\/100007637","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005278","name":"Universidad de Antioquia","doi-asserted-by":"publisher","award":["Sostenibilidad 2016\/2017"],"award-info":[{"award-number":["Sostenibilidad 2016\/2017"]}],"id":[{"id":"10.13039\/501100005278","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/jof7070544","type":"journal-article","created":{"date-parts":[[2021,7,9]],"date-time":"2021-07-09T04:14:57Z","timestamp":1625804097000},"page":"544","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons"],"prefix":"10.3390","volume":"7","author":[{"given":"Juan E.","family":"Gallo","sequence":"first","affiliation":[{"name":"Cellular and Molecular Biology Unit, Corporaci\u00f3n para Investigaciones Biol\u00f3gicas (CIB), Medell\u00edn 05534, Colombia"},{"name":"Doctoral Program in Biomedical Sciences, Universidad del Rosario, Bogot\u00e1 111221, Colombia"},{"name":"GenomaCES, Universidad CES, Medellin 050021, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4022-8072","authenticated-orcid":false,"given":"Isaura","family":"Torres","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Biology Unit, Corporaci\u00f3n para Investigaciones Biol\u00f3gicas (CIB), Medell\u00edn 05534, Colombia"},{"name":"GenomaCES, Universidad CES, Medellin 050021, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1714-9672","authenticated-orcid":false,"given":"Oscar M.","family":"G\u00f3mez","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Biology Unit, Corporaci\u00f3n para Investigaciones Biol\u00f3gicas (CIB), Medell\u00edn 05534, Colombia"},{"name":"GenomaCES, Universidad CES, Medellin 050021, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lavanya","family":"Rishishwar","sequence":"additional","affiliation":[{"name":"School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA"},{"name":"Applied Bioinformatics Laboratory, Atlanta, GA 30332, USA"},{"name":"PanAmerican Bioinformatics Institute, Cali, Valle del Cauca 760043, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fredrik","family":"Vannberg","sequence":"additional","affiliation":[{"name":"School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4996-2203","authenticated-orcid":false,"given":"I. King","family":"Jordan","sequence":"additional","affiliation":[{"name":"School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA"},{"name":"Applied Bioinformatics Laboratory, Atlanta, GA 30332, USA"},{"name":"PanAmerican Bioinformatics Institute, Cali, Valle del Cauca 760043, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan G.","family":"McEwen","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Biology Unit, Corporaci\u00f3n para Investigaciones Biol\u00f3gicas (CIB), Medell\u00edn 05534, Colombia"},{"name":"School of Medicine, Universidad de Antioquia, Medell\u00edn 050010, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Oliver K.","family":"Clay","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Biology Unit, Corporaci\u00f3n para Investigaciones Biol\u00f3gicas (CIB), Medell\u00edn 05534, Colombia"},{"name":"Translational Microbiology and Emerging Diseases (MICROS), School of Medicine and Health Sciences, Universidad del Rosario, Bogot\u00e1 111221, Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.ccm.2009.02.002","article-title":"Histoplasmosis","volume":"30","author":"Kauffman","year":"2009","journal-title":"Clin. Chest Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1128\/CVI.00066-09","article-title":"Development and Evaluation of an Enzyme-Linked Immunosorbent Assay To Detect Histoplasma capsulatum Antigenuria in Immunocompromised Patients","volume":"16","author":"Scheel","year":"2009","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.diagmicrobio.2008.05.006","article-title":"Histoplasmosis in HIV-infected patients in a southern regional medical center: Poor prognosis in the era of highly active antiretroviral therapy","volume":"62","author":"Baddley","year":"2008","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_4","first-page":"3162","article-title":"Histoplasma\u00a0capsulatum","volume":"Volume 2","author":"Bennett","year":"2020","journal-title":"Mandell, Douglas, and Bennett\u2019s Principles and Practice of Infectious Diseases"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1128\/CMR.00053-10","article-title":"Histopathologic Diagnosis of Fungal Infections in the 21st Century","volume":"24","author":"Guarner","year":"2011","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_6","first-page":"129","article-title":"Diagnostic methods for histoplasmosis: Focus on endemic countries with variable infrastructure levels","volume":"1","author":"Scheel","year":"2014","journal-title":"Curr. Trop. Med. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1007\/s12281-011-0073-7","article-title":"Histoplasmosis: Epidemiology in Latin America","volume":"5","year":"2011","journal-title":"Curr. Fungal Infect. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"193","DOI":"10.4269\/ajtmh.13-0226","article-title":"How Many Have Died from Undiagnosed Human Immunodeficiency Virus\u2013Associated Histoplasmosis, A Treatable Disease? Time to Act","volume":"90","author":"Nacher","year":"2014","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Carroll, K.C., Pfaller, M.A., Landry, M.L., McAdam, A.J., Patel, R., Richter, S.S., and Warnock, D.W. (2019). Histoplasma, Blastomyces, Coccidioides, and other dimorphic fungi causing systemic mycoses. Manual of Clinical Microbiology, ASM Press. [12th ed.].","DOI":"10.1128\/9781555819842"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"S167","DOI":"10.1016\/S0924-8579(08)70020-7","article-title":"Present situation in the treatment of invasive fungal infection","volume":"32","author":"Picazo","year":"2008","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1644","DOI":"10.1128\/JCM.40.5.1644-1647.2002","article-title":"Evaluation of Two Nested PCR Assays for Detection of Histoplasma capsulatum DNA in Human Tissue","volume":"40","author":"Bialek","year":"2002","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"L\u00f3pez, L.F., Mu\u00f1oz, C.O., C\u00e1ceres, D.H., Tob\u00f3n, \u00c1.M., Loparev, V., Clay, O., Chiller, T., Litvintseva, A., Gade, L., and Gonzalez, A. (2017). Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0190311"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.4269\/ajtmh.17-0214","article-title":"Detection of Histoplasma capsulatum in Organic Fertilizers by Hc100 Nested Polymerase Chain Reaction and Its Correlation with the Physicochemical and Microbiological Characteristics of the Samples","volume":"98","author":"Torres","year":"2018","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Mu\u00f1oz, J.F., Gallo, J.E., Misas, E., Priest, M., Imamovic, A., Young, S., Zeng, Q., Clay, O.K., McEwen, J.G., and Cuomo, C.A. (2014). Genome Update of the Dimorphic Human Pathogenic Fungi Causing Paracoccidioidomycosis. PLOS Neglected Trop. Dis., 8.","DOI":"10.1371\/journal.pntd.0003348"},{"key":"ref_15","unstructured":"Kadir, N. (1998). A Low Cost Approach to PCR: Appropriate Technology Transfer of Biomolecular Techniques, Oxford University Press."},{"key":"ref_16","first-page":"10.3.1","article-title":"Using MUMmer to Identify Similar Regions in Large Sequence Sets","volume":"10","author":"Delcher","year":"2003","journal-title":"Curr. Protoc. Bioinform."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.fgb.2017.05.007","article-title":"Species boundaries in the human pathogen Paracoccidioides","volume":"106","author":"Turissini","year":"2017","journal-title":"Fungal Genet. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2178","DOI":"10.1101\/gr.1224503","article-title":"OrthoMCL: Identification of Ortholog Groups for Eukaryotic Genomes","volume":"13","author":"Li","year":"2003","journal-title":"Genome Res."},{"key":"ref_20","unstructured":"Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3204","DOI":"10.1128\/JCM.00673-11","article-title":"Detection of Blastomyces dermatitidis and Histoplasma capsulatum from Culture Isolates and Clinical Specimens by Use of Real-Time PCR","volume":"49","author":"Babady","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4414","DOI":"10.1128\/IAI.01893-14","article-title":"Glycosylation and Immunoreactivity of the Histoplasma capsulatum Cfp4 Yeast-Phase Exoantigen","volume":"82","author":"Holbrook","year":"2014","journal-title":"Infect. Immun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3","DOI":"10.25518\/1780-4507.13461","article-title":"The influence of amplicon length on real-time PCR results","volume":"21","author":"Debode","year":"2017","journal-title":"Biotechnol. Agron. Soc. Environ."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.bdq.2017.11.001","article-title":"qPCR primer design revisited","volume":"14","author":"Bustin","year":"2017","journal-title":"Biomol. Detect. Quantif."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1007\/s12281-018-0308-y","article-title":"Emergomyces: A New Genus of Dimorphic Fungal Pathogens Causing Disseminated Disease among Immunocompromised Persons Globally","volume":"12","author":"Schwartz","year":"2018","journal-title":"Curr. Fungal Infect. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Maphanga, T.G., Naicker, S.D., G\u00f3mez, B.L., Mhlanga, M., Mpembe, R.S., Schwartz, I.S., Bamford, C., Nel, J., and Govender, N.P. (2020). Cross-reactivity of a Histoplasma capsulatum antigen enzyme immunoassay in urine specimens from persons with emergomycosis in South Africa. Med Mycol.","DOI":"10.1093\/mmy\/myaa100"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3383","DOI":"10.1046\/j.1365-294X.2003.01995.x","article-title":"Phylogeography of the fungal pathogen Histoplasma capsulatum","volume":"12","author":"Kasuga","year":"2003","journal-title":"Mol. Ecol."},{"key":"ref_28","unstructured":"McNabb, S.J.N., Conde, J.M., Ferland, L., and MacWright, W. (2016). Data storms are growing, everywhere, and have to work together. Transforming Public Health Surveillance: Proactive Measures for Prevention, Detection, and Response, Elsevier."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Pappalardo, E., Pardalos, P.M., and Stracquadanio, G. (2013). Optimization Approaches for Solving String Selection Problems, Springer Science and Business Media LLC.","DOI":"10.1007\/978-1-4614-9053-1"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/7\/7\/544\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:28:08Z","timestamp":1760164088000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/7\/7\/544"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,9]]},"references-count":29,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["jof7070544"],"URL":"https:\/\/doi.org\/10.3390\/jof7070544","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,9]]}}}