Aplicación de ensayos de PCR en tiempo real para el diagnóstico de la histoplasmosis utilizando tres dianas moleculares en tejidos humanos de FFPE y sangre entera.

Luisa F. López, Oliver Keatinge Clay, Diego H. Cáceres, Beatriz L. Gómez

Resultado de la investigación: Tipos de Contribuciónes en ConferenciaAfiche

Resumen

Objetivo: La histoplasmosis es una infección fúngica que causa morbilidad y mortalidad significativas en las personas que viven con el VIH/sida, especialmente en países con recursos limitados. Las pruebas diagnósticas utilizadas actualmente se basan en el cultivo y la serología, carecen de sensibilidad y a menudo requieren semanas para obtener resultados que causan retrasos significativos en el diagnóstico; los ensayos moleculares no están disponibles comercialmente. Métodos: se intentó aplicar la PCR cuantitativa en tiempo real (qPCR) dirigida a tres genes codificadores de proteínas de Histoplasma capsulatum (antígenos 100-kDa, H y M) para la detección de la infección por H. capsulatum en muestras incrustadas de parafina fijada con formalina (FFPE, por sus siglas en inglés) y de sangre entera (WB, por sus siglas en inglés) de pacientes con histoplasmosis comprobada. Resultados: Para las muestras FFPE, la sensibilidad de los ensayos de qPCR de 100 kDa, H y M fue de 93,9%, 91% y 57%, respectivamente; sin embargo, los mismos ensayos de qPCR mostraron sólo 23%, 19% y 11,5% de sensibilidad para los ensayos de qPCR de 100 kDa, H y M cuando se usaron con las muestras de WB. La especificidad de la qPCR se determinó analizando muestras de pacientes con otras infecciones clínicas y controles sanos y fue del 93%-100% dependiendo del ensayo y del tipo de muestra. Conclusión: se aplicaron tres ensayos de qPCR para la detección del ADN de H. capsulatum en muestras humanas, y se demostró que los protocolos moleculares basados en la amplificación de antígenos 100-kDa y H pueden ser utilizados con éxito para el diagnóstico de esta micosis cuando se utilizan muestras de FFPE; sin embargo, no se recomienda el uso de WB para el diagnóstico rutinario de la histoplasmosis por qPCR en pacientes con histoplasmosis diseminada progresiva.
Título traducido de la contribuciónAplicación de ensayos de PCR en tiempo real para el diagnóstico de la histoplasmosis utilizando tres dianas moleculares en tejidos humanos de FFPE y sangre entera.
IdiomaEnglish (US)
PáginasS63
Número de páginas1
DOI
EstadoPublished - jun 2018
Evento20th Congress of the International Society for Human and Animal Mycology - Amsterdam
Duración: jun 30 2018jul 4 2018
https://www.isham2018.org/en/Home_10_6_12.html

Conference

Conference20th Congress of the International Society for Human and Animal Mycology
Título abreviado20th Congress ISHAM
PaísNetherlands
CiudadAmsterdam
Período6/30/187/4/18
Dirección de internet

Citar esto

López, L. F., Clay, O. K., Cáceres, D. H., & Gómez, B. L. (2018). Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood. S63. Sesión de cárteles presentada en 20th Congress of the International Society for Human and Animal Mycology, Amsterdam, . https://doi.org/10.1093/mmy/myy036
López, Luisa F. ; Clay, Oliver Keatinge ; Cáceres, Diego H. ; Gómez, Beatriz L. / Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood. Sesión de cárteles presentada en 20th Congress of the International Society for Human and Animal Mycology, Amsterdam, .1 p.
@conference{65dbc97adbaa4ca8ba253549bdfeb23a,
title = "Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood",
abstract = "Objective: Histoplasmosis is a fungal infection that causes significantmorbidity andmortality in persons living withHIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology, lack sensitivity and often require weeks to obtain results causing significant diagnosis delays; molecular assays are not commercially available. Methods: we aimed to apply quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of H. capsulatum infection in formalin-fixed paraffin-embedded (FFPE) and whole blood (WB) samples from patients with proven histoplasmosis. Results: For FFPE samples, the sensitivity of 100-kDa, H and M qPCR assays were 93.9{\%}, 91{\%} and 57{\%}, respectively; however, the same qPCR assays showed only 23{\%}, 19{\%} and 11.5{\%} of sensitivity for 100-kDa, H y M qPCR assays when used with the WB samples. The specificity of qPCR was determined by testing samples from patients with other clinical infections and healthy controls and was 93{\%}-100{\%} depending upon the assay and the specimen type. Conclusion: we applied three qPCR assays for detecting H. capsulatum DNA in human samples, and demonstrated that the molecular protocols based on amplification of 100-kDa and H antigen can be successfully used for diagnosing this mycosis when using FFPE samples; however, we do not recommend WB for routine diagnosis of histoplasmosis by qPCR in patients with progressive disseminated histoplasmosis.",
author = "L{\'o}pez, {Luisa F.} and Clay, {Oliver Keatinge} and C{\'a}ceres, {Diego H.} and G{\'o}mez, {Beatriz L.}",
year = "2018",
month = "6",
doi = "10.1093/mmy/myy036",
language = "English (US)",
pages = "S63",
note = "20th Congress of the International Society for Human and Animal Mycology, 20th Congress ISHAM ; Conference date: 30-06-2018 Through 04-07-2018",
url = "https://www.isham2018.org/en/Home_10_6_12.html",

}

Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood. / López, Luisa F.; Clay, Oliver Keatinge; Cáceres, Diego H.; Gómez, Beatriz L.

2018. S63 Sesión de cárteles presentada en 20th Congress of the International Society for Human and Animal Mycology, Amsterdam, .

Resultado de la investigación: Tipos de Contribuciónes en ConferenciaAfiche

TY - CONF

T1 - Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood

AU - López, Luisa F.

AU - Clay, Oliver Keatinge

AU - Cáceres, Diego H.

AU - Gómez, Beatriz L.

PY - 2018/6

Y1 - 2018/6

N2 - Objective: Histoplasmosis is a fungal infection that causes significantmorbidity andmortality in persons living withHIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology, lack sensitivity and often require weeks to obtain results causing significant diagnosis delays; molecular assays are not commercially available. Methods: we aimed to apply quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of H. capsulatum infection in formalin-fixed paraffin-embedded (FFPE) and whole blood (WB) samples from patients with proven histoplasmosis. Results: For FFPE samples, the sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively; however, the same qPCR assays showed only 23%, 19% and 11.5% of sensitivity for 100-kDa, H y M qPCR assays when used with the WB samples. The specificity of qPCR was determined by testing samples from patients with other clinical infections and healthy controls and was 93%-100% depending upon the assay and the specimen type. Conclusion: we applied three qPCR assays for detecting H. capsulatum DNA in human samples, and demonstrated that the molecular protocols based on amplification of 100-kDa and H antigen can be successfully used for diagnosing this mycosis when using FFPE samples; however, we do not recommend WB for routine diagnosis of histoplasmosis by qPCR in patients with progressive disseminated histoplasmosis.

AB - Objective: Histoplasmosis is a fungal infection that causes significantmorbidity andmortality in persons living withHIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology, lack sensitivity and often require weeks to obtain results causing significant diagnosis delays; molecular assays are not commercially available. Methods: we aimed to apply quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of H. capsulatum infection in formalin-fixed paraffin-embedded (FFPE) and whole blood (WB) samples from patients with proven histoplasmosis. Results: For FFPE samples, the sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively; however, the same qPCR assays showed only 23%, 19% and 11.5% of sensitivity for 100-kDa, H y M qPCR assays when used with the WB samples. The specificity of qPCR was determined by testing samples from patients with other clinical infections and healthy controls and was 93%-100% depending upon the assay and the specimen type. Conclusion: we applied three qPCR assays for detecting H. capsulatum DNA in human samples, and demonstrated that the molecular protocols based on amplification of 100-kDa and H antigen can be successfully used for diagnosing this mycosis when using FFPE samples; however, we do not recommend WB for routine diagnosis of histoplasmosis by qPCR in patients with progressive disseminated histoplasmosis.

U2 - 10.1093/mmy/myy036

DO - 10.1093/mmy/myy036

M3 - Poster

SP - S63

ER -

López LF, Clay OK, Cáceres DH, Gómez BL. Application of real-time PCR assays for the diagnosis of histoplasmosis using three molecular targets in human FFPE tissues and whole-blood. 2018. Sesión de cárteles presentada en 20th Congress of the International Society for Human and Animal Mycology, Amsterdam, . https://doi.org/10.1093/mmy/myy036