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.

Laboratory Diagnosis of Histoplasmosis: An Update

“…Loop-mediated isothermal amplification (LAMP) rests on the use of a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA [52]. Some reports described the implementation of this technique for the molecular diagnosis of histoplasmosis showing great results [53,54]. The high efficiency, sensitivity, specificity and costeffectiveness of these assays make them good candidates to be implemented in the diagnostic routine of resource-limited laboratories.…”

Current and New Perspectives in the Diagnosis of Blastomycosis and Histoplasmosis

“…This assay can be modified with the addition of fluorescent markers or dyes to allow easy interpretation [ 77 , 78 ]. LAMP methods have been developed for the detection of the Hcp100 locus of H. capsulatum and for ITS [ 79 , 80 ]. LAMP of the Hcp100 locus is very specific in laboratory conditions, but less so with clinical specimens [ 79 ].…”

“…LAMP of the Hcp100 locus is very specific in laboratory conditions, but less so with clinical specimens [ 79 ]. When a positive culture for H. capsulatum is used as the gold standard, LAMP of clinical specimens (bone marrow) had low sensitivity and specificity (54% and 64%, respectively) [ 80 ]. LAMP has the potential to be useful in a low-resource setting, but further data are needed before use of this technique becomes mainstream.…”

Timely Diagnosis of Histoplasmosis in Non-endemic Countries: A Laboratory Challenge

“…All of them dealt with progressive disseminated histoplasmosis samples of HIV + patients from endemic countries, with reasonably good results (Scheel et al, 2014;Zatti et al, 2019). Their diagnostic yield varied greatly depending on the type of sample tested: an 83% sensitivity was reported for blood and bone marrow as compared to a nested PCR targeting the Hcp100 gene (Zatti et al, 2019), whereas a more modest 67% sensitivity was achieved in antigen positive urine samples. Altogether, isothermal assays could become suitable for use as a complementary diagnostic test in low-income countries, and potentially useful techniques for implementation in non-endemic areas.…”