Loop-mediated Isothermal Amplification and nested PCR of the Internal Transcribed Spacer (ITS) for Histoplasma capsulatum detection.

• Published in: PLOS Neglected Tropical Diseases
• Main Authors: Matheus da Silva Zatti, Thales Domingos Arantes, José Alex Lourenço Fernandes, Mônica Baumgardt Bay, Eveline Pipolo Milan, Georggia Fatima Silva Naliato, Raquel Cordeiro Theodoro
• Format: Article in Journal/Newspaper
• Language: English
• Published: Public Library of Science (PLoS) 2019
• Subjects: Arctic medicine. Tropical medicine; RC955-962; Public aspects of medicine; RA1-1270; Arctic
• Online Access: https://doi.org/10.1371/journal.pntd.0007692; https://doaj.org/article/46abc95c354341e4b54de37835d478c5

Background Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis. Methodology Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction. Results Both assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL. Conclusion ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment ...