**4. Molecular methods**

For more rapid and accurate detection of *H*. *capsulatum* in tissues and body fluids, a number of polymerase chain reaction (PCR) methods have been developed: conventional, nested, and real-time PCR (targeting different regions of the *H*. *capsulatum* genome) with higher sensitivity and specificity, but many of these have been developed in-house. Although these molecular methods are more sensitive and more specific than antigen detection or serologic testing, there is no FDA-approved commercial PCR-based test.

Currently, the main molecular method for diagnosing HPM involves the use of a rapid DNA probe to identify *H*. *capsulatum* isolated from a variety of culture extracts. A real-time PCR assay has been used to identify *H*. *capsulatum* in tissue biopsies or at BAL. Other semi-nested PCR assays showed promising results in identifying *H*. *capsulatum* in blood or tissue from patients in whom HPM was detected.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used in laboratories with limited resources but has some limitations.

Nucleic acid amplification tests (NAAT) such as PCR or LAMP have a lower probability of false-positive results due to other fungi compared to conventional tests.

A reference database has been established for the identification of *H*. *capsulatum* using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), but limited data are available [31].

Metagenomic next-generation sequencing (mNGS) has been used on BAL to diagnose chronic progressive pulmonary lesions; panfungal PCR can also be used to diagnose HPM, but so far they have only been used for research purposes [32].

Although culture is considered the gold standard test for the diagnosis of HPM, molecular methods may be more sensitive. In the future, molecular methods will play an increasingly important role in the diagnosis of HPM to assist clinicians. Their results are currently limited by the heterogeneity of molecular assays, the targets used, the small number of subjects included in the studies, the different types of specimens tested, and the lack of a standard comparative method, as well as the limited presence of molecular methods in international diagnostic guidelines [33].

More robust multicenter studies including studies in large populations are needed for performance evaluation and validation of these assays in different types of patients or samples.
