Molecular identification of Histoplasma capsulatum using rolling circle amplification
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Published on: June 25, 2020
Jason L Furuie1, Jiufeng Sun2, Mariana M F do Nascimento3
1Bioprocess Engineering and Biotechnology Department, Post-Graduation Program of Bioprocess Engineering and Biotechnology, Federal University of Paraná, Curitiba, Brazil.
Abstract:
Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.
Insights
This study developed a rapid and specific molecular detection method for Histoplasma capsulatum, the fungus causing histoplasmosis. The new padlock probe and rolling circle amplification (RCA) technique offers a faster alternative to traditional diagnostics for this systemic fungal disease.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Histoplasmosis is a global systemic fungal infection, primarily affecting immunocompromised individuals.
- Histoplasma capsulatum, found in soil with bird/bat droppings, is the causative agent.
- Low awareness and similar symptoms to other diseases hinder H. capsulatum diagnosis.
Purpose of the Study:
- To develop and evaluate a padlock probe-based rolling circle amplification (RCA) method.
- To enable rapid and specific detection of Histoplasma capsulatum.
- To target the Internal Transcribed Spacer (ITS) rDNA region of H. capsulatum.
Main Methods:
- Design and testing of two padlock probes targeting H. capsulatum ITS rDNA.
- Utilizing rolling circle amplification (RCA) for DNA detection.
- Evaluating specificity against other fungal species as negative controls.
Main Results:
- One padlock probe (HcPL2) demonstrated specific amplification of H. capsulatum DNA.
- No cross-reactivity was observed with tested negative control fungi.
- The method proved effective for specific H. capsulatum identification.
Conclusions:
- The developed padlock probe-RCA method provides a rapid and specific diagnostic tool for Histoplasma capsulatum.
- This molecular approach is faster than conventional microbiological identification methods.
- Enhanced diagnostic capabilities can improve the management of histoplasmosis, especially in at-risk populations.
