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

Autor: Matheus da Silva Zatti, Mônica Baumgardt Bay, Raquel Cordeiro Theodoro, Eveline Pipolo Milan, Thales Domingos Arantes, Georggia Fatima Silva Naliato, José A. L. Fernandes
Jazyk: angličtina
Rok vydání: 2019
Předmět:
RNA viruses
0301 basic medicine
Physiology
RC955-962
Artificial Gene Amplification and Extension
Pathology and Laboratory Medicine
Polymerase Chain Reaction
law.invention
0302 clinical medicine
Immunodeficiency Viruses
Bone Marrow
law
Arctic medicine. Tropical medicine
Medicine and Health Sciences
Prospective Studies
Histoplasmosis
DNA extraction
Polymerase chain reaction
Whole blood
Fungal Pathogens
Fungal Diseases
Progressive disseminated histoplasmosis
Genomics
Body Fluids
Blood
Infectious Diseases
Molecular Diagnostic Techniques
Medical Microbiology
Viral Pathogens
Viruses
Histoplasma Capsulatum
Pathogens
Anatomy
Public aspects of medicine
RA1-1270
Nucleic Acid Amplification Techniques
Research Article
Histoplasma
030231 tropical medicine
Loop-mediated isothermal amplification
Mycology
Biology
Microbiology
Sensitivity and Specificity
03 medical and health sciences
Extraction techniques
Retroviruses
DNA
Ribosomal Spacer
Genetics
medicine
Humans
Fungal Genetics
Internal transcribed spacer
Molecular Biology Techniques
Microbial Pathogens
Molecular Biology
Fungal Genomics
Lentivirus
Organisms
Public Health
Environmental and Occupational Health
Biology and Life Sciences
HIV
medicine.disease
Virology
Research and analysis methods
030104 developmental biology
Nested polymerase chain reaction
Zdroj: PLoS Neglected Tropical Diseases, Vol 13, Iss 8, p e0007692 (2019)
PLoS Neglected Tropical Diseases
ISSN: 1935-2735
1935-2727
Popis: 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 of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.
Author summary Histoplasmosis is a worldwide neglected disease with a high mortality rate associated with HIV/AIDS patients, killing more than tuberculosis in some endemic countries in Latin America. Part of this elevated mortality rate is due to delayed diagnosis and treatment. Here we present two novel methods, one based on Loop-mediated Isothermal Amplification (LAMP) and another on nested Polymerase Chain Reaction (nPCR), for fast, sensitive and specific diagnosis of histoplasmosis. Tests of blood samples spiked with Histoplasma capsulatum suggest the possibility of direct application of the LAMP assay proposed herein to clinical specimens without the need for previous DNA extraction and with the added advantage of naked-eye evaluation of the reaction results. Once the assay has been validated in different clinical specimens, it may be a promising tool for fast histoplasmosis screening.
Databáze: OpenAIRE
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