New Histoplasma diagnostic assays designed via whole genome comparisons

Juan E Gallo; Isaura Torres; Oscar M Gómez; Lavanya Rishishwar; Fredrik Vannberg; I King Jordan; Juan G McEwen; Oliver K Clay

doi:10.3390/jof7070544

New Histoplasma diagnostic assays designed via whole genome comparisons

Título traducido de la contribución: Nuevos ensayos de diagnóstico de Histoplasma diseñados mediante comparaciones del genoma completo

Juan E Gallo
, Isaura Torres
, Oscar M Gómez
, Lavanya Rishishwar
, Fredrik Vannberg
, I King Jordan
, Juan G McEwen
, Oliver K Clay

Producción científica: Contribución a revista › Artículo de Investigación › revisión exhaustiva

8 Citas (Scopus)

Resumen

La histoplasmosis es una enfermedad fúngica sistémica causada por el patógeno Histoplasma spp. que resulta en una morbilidad y mortalidad significativas en personas con VIH/SIDA y también puede afectar a personas inmunocompetentes. Aunque se han desarrollado algunas pruebas de PCR y de detección de antígenos, el diagnóstico convencional se ha basado en gran medida en el cultivo, que puede llevar semanas. Nuestro objetivo era proporcionar una prueba de principio para diseñar y estandarizar racionalmente ensayos de PCR basados en secuencias genómicas específicas de Histoplasma. A través de comparaciones automatizadas de contigs/andamios del genoma alineados y (sub)secuencias de genes, identificamos genes codificadores de proteínas que están presentes en secuencias existentes de cepas de Histoplasma pero no en otros géneros. Dos de los genes, PPK y CFP4, se utilizaron para diseñar conjuntos de cebadores para ensayos de PCR convencionales y en tiempo real. Ambos dieron como resultado una especificidad analítica del 100 % in vitro y detectaron 62/62 aislados de H. capsulatum utilizando ADN purificado. También obtuvimos detecciones positivas de 2/2 muestras clínicas FFPE (fijadas con formalina e incluidas en parafina) confirmadas de H. capsulatum utilizando ambos conjuntos de cebadores. Las diluciones en serie de 10 veces del plásmido de control positivo confirmaron la sensibilidad analítica de los ensayos. Los hallazgos sugieren que estos nuevos conjuntos de cebadores deberían permitir la sensibilidad de detección y reducir los resultados falsos positivos y las reacciones cruzadas. Nuevos ensayos para detectar hongos patógenos, construidos siguiendo estas líneas, podrían ser sencillos y asequibles de implementar.

Título traducido de la contribución	Nuevos ensayos de diagnóstico de Histoplasma diseñados mediante comparaciones del genoma completo
Idioma original	Inglés estadounidense
Número de artículo	544
Publicación	Journal of Fungi
Volumen	7
N.º	7
DOI	https://doi.org/10.3390/jof7070544 https://doi.org/10.3390/jof7070544
Estado	Publicada - jul. 9 2021

ODS de las Naciones Unidas

Este resultado contribuye a los siguientes Objetivos de Desarrollo Sostenible

ODS 3: Salud y bienestar

Áreas temáticas de ASJC Scopus

Ecología, evolución, comportamiento y sistemática
Botánica
Microbiología (médica)

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title = "New Histoplasma diagnostic assays designed via whole genome comparisons",

abstract = "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.",

author = "Gallo, \{Juan E\} and Isaura Torres and G{\'o}mez, \{Oscar M\} and Lavanya Rishishwar and Fredrik Vannberg and Jordan, \{I King\} and McEwen, \{Juan G\} and Clay, \{Oliver K\}",

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AU - Gallo, Juan E

AU - Torres, Isaura

AU - Gómez, Oscar M

AU - Rishishwar, Lavanya

AU - Vannberg, Fredrik

AU - Jordan, I King

AU - McEwen, Juan G

AU - Clay, Oliver K

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N2 - 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.

AB - 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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