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

Abstract

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.

Fig 1. Illustrative diagram showing the hybridization regions of ITS LAMP and nPCR primer sets.

SSU, small ribosomal subunit (18S); ITS, Internal Transcribed Spacer; LSU, large ribosomal subunit (28S).

Fig 2. The LOD of ITS LAMP and nPCR for different concentrations (from 10 ng/μL to 0.1 fg/μL) of H. capsulatum DNA.

A: ITS LAMP products in 1.5% agarose gel stained with ethidium bromide; B: ITS LAMP product observed by addition of Sybr Green I in the reaction tube under white light and UV light, respectively; C: ITS nPCR product in 1.5% agarose gel stained with ethidium bromide; NT: No Template control.

Fig 3

Specificity assay for ITS LAMP (A) and ITS nPCR (B). NT, No Template; Af, Aspergillus flavus; An, Aspergillus niger; Ca, Candida spp; Cc, Cladophialophora carrionii; Cg, Cryptococcus gattii; Cn, Cryptococcus neoformans; Hc, Histoplasma capsulatum; Mc, Microsporum canis; Pb, Paracoccidioides brasiliensis; Sb, Sporothrix brasiliensis; Tm, Trichophyton mentagrophytes; Tri, Trichosporon spp.

Fig 4. ITS LAMP on crude samples spiked with different concentrations of alive H. capsulatum yeasts.

In A and B, yeasts were diluted in PBS, heat-treated and assayed. In C and D, yeasts were diluted in heparin-containing whole blood and direct assayed. ITS LAMP products were visualized on an agarose gel stained with ethidium bromide (A and C) and by addition of Sybr Green I, under UV and white lights (B and D).

Fig 5. ITS nPCR on crude samples spiked with different concentrations of live H. capsulatum yeasts.

In A, yeasts were diluted in PBS, heat-treated and assayed. In B, yeasts were diluted in heparin-containing whole blood and direct assayed by ITS nPCR. The amplification of a fragment of 397 bp was considered a positive reaction.

Fig 6

Cost (A) and time (B) consumed for ITS LAMP and nPCR assays.