PubTransformer

A site to transform Pubmed publications into these bibliographic reference formats: ADS, BibTeX, EndNote, ISI used by the Web of Knowledge, RIS, MEDLINE, Microsoft's Word 2007 XML.

Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model.

Abstract Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.
PMID
Related Publications

Applying a real-time PCR assay for Histoplasma capsulatum to clinically relevant formalin-fixed paraffin-embedded human tissue.

Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue.

Multilocus sequence typing of Histoplasma capsulatum in formalin-fixed paraffin-embedded tissues from cats living in non-endemic regions reveals a new phylogenetic clade.

Validation and clinical application of a molecular method for identification of Histoplasma capsulatum in human specimens in Colombia, South America.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

Authors

Mayor MeshTerms
Keywords
Journal Title plos one
Publication Year Start




PMID- 29287097
OWN - NLM
STAT- In-Process
LR  - 20180117
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 12
IP  - 12
DP  - 2017
TI  - Standardization and validation of real time PCR assays for the diagnosis of
      histoplasmosis using three molecular targets in an animal model.
PG  - e0190311
LID - 10.1371/journal.pone.0190311 [doi]
AB  - Histoplasmosis is considered one of the most important endemic and systemic
      mycoses worldwide. Until now few molecular techniques have been developed for its
      diagnosis. The aim of this study was to develop and evaluate three real time PCR 
      (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens)
      using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung
      tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum
      yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A
      collection of DNA from cultures representing different clades of H. capsulatum
      (30 strains) and other medically relevant pathogens (36 strains of related fungi 
      and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity.
      Analytical sensitivity and specificity were 100% when DNAs from the different
      strains were tested. The highest fungal burden occurred at first week
      post-infection and complete fungal clearance was observed after the third week;
      similar results were obtained when the presence of H. capsulatum yeast cells was 
      demonstrated in histopathological analysis. In the first week post-infection, all
      fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for
      the qPCR protocols tested except for the M antigen protocol, which gave variable 
      results when fresh lung tissue samples were analyzed. In the second week, all
      qPCR protocols showed variable results for both fresh and FFPE tissues. Samples
      from the infected mice at the remaining times post-infection and uninfected mice 
      (controls) were negative for all protocols. Good agreement was observed between
      CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen
      protocols. We successfully standardized and validated three qPCR assays for
      detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the
      100-kDa and H antigen molecular assays are promising tests for diagnosing this
      mycosis.
FAU - Lopez, Luisa F
AU  - Lopez LF
AD  - Medical and Experimental Mycology Group, Corporacion para Investigaciones
      Biologicas (CIB), Medellin, Colombia.
FAU - Munoz, Cesar O
AU  - Munoz CO
AD  - Medical and Experimental Mycology Group, Corporacion para Investigaciones
      Biologicas (CIB), Medellin, Colombia.
FAU - Caceres, Diego H
AU  - Caceres DH
AD  - Medical and Experimental Mycology Group, Corporacion para Investigaciones
      Biologicas (CIB), Medellin, Colombia.
AD  - Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA,
      United States of America.
FAU - Tobon, Angela M
AU  - Tobon AM
AD  - Medical and Experimental Mycology Group, Corporacion para Investigaciones
      Biologicas (CIB), Medellin, Colombia.
FAU - Loparev, Vladimir
AU  - Loparev V
AD  - Biotechnology Core Facility Branch, Centers for Disease Control and Prevention,
      Atlanta, GA, United States of America.
FAU - Clay, Oliver
AU  - Clay O
AD  - School of Medicine and Health Sciences, Universidad del Rosario, Bogota,
      Colombia.
AD  - Cell and Molecular Biology Group, Corporacion para Investigaciones Biologicas
      (CIB), Medellin, Colombia.
FAU - Chiller, Tom
AU  - Chiller T
AD  - Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA,
      United States of America.
FAU - Litvintseva, Anastasia
AU  - Litvintseva A
AD  - Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA,
      United States of America.
FAU - Gade, Lalitha
AU  - Gade L
AD  - Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA,
      United States of America.
FAU - Gonzalez, Angel
AU  - Gonzalez A
AD  - Basic and Applied Microbiology Research Group (MICROBA), School of Microbiology, 
      Universidad de Antioquia, Medellin, Colombia.
FAU - Gomez, Beatriz L
AU  - Gomez BL
AUID- ORCID: 0000-0002-6641-1924
AD  - Medical and Experimental Mycology Group, Corporacion para Investigaciones
      Biologicas (CIB), Medellin, Colombia.
AD  - School of Medicine and Health Sciences, Universidad del Rosario, Bogota,
      Colombia.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20171229
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
EDAT- 2017/12/30 06:00
MHDA- 2017/12/30 06:00
CRDT- 2017/12/30 06:00
PHST- 2017/09/19 00:00 [received]
PHST- 2017/12/12 00:00 [accepted]
PHST- 2017/12/30 06:00 [entrez]
PHST- 2017/12/30 06:00 [pubmed]
PHST- 2017/12/30 06:00 [medline]
AID - 10.1371/journal.pone.0190311 [doi]
AID - PONE-D-17-31650 [pii]
PST - epublish
SO  - PLoS One. 2017 Dec 29;12(12):e0190311. doi: 10.1371/journal.pone.0190311.
      eCollection 2017.