Molecular and nonmolecular diagnostic methods for invasive fungal infections

Abstract

Invasive fungal infections constitute a serious threat to an ever-growing population of immunocompromised individuals and other individuals at risk. Traditional diagnostic methods, such as histopathology and culture, which are still considered the gold standards, have low sensitivity, which underscores the need for the development of new means of detecting fungal infectious agents. Indeed, novel serologic and molecular techniques have been developed and are currently under clinical evaluation. Tests like the galactomannan antigen test for aspergillosis and the β-glucan test for invasive Candida spp. and molds, as well as other antigen and antibody tests, for Cryptococcus spp., Pneumocystis spp., and dimorphic fungi, have already been established as important diagnostic approaches and are implemented in routine clinical practice. On the other hand, PCR and other molecular approaches, such as matrix-assisted laser desorption ionization (MALDI) and fluorescence in situ hybridization (FISH), have proved promising in clinical trials but still need to undergo standardization before their clinical use can become widespread. The purpose of this review is to highlight the different diagnostic approaches that are currently utilized or under development for invasive fungal infections and to identify their performance characteristics and the challenges associated with their use.

FIG 1

Technology behind novel diagnostic methods for fungal infections. (A) Fluorescence in situ hybridization. Fluorescent probes against a specific target sequence are mixed with the tested sample and allowed to bind to their complementary DNA sequence, if present in the sample. The excess probes are then washed off, while the bound probes are detected via their fluorescence under a fluorescence microscope. (B) Matrix-assisted laser desorption ionization–time of flight mass spectrometry. The tested sample is added to a well that contains matrix material which has the ability to absorb UV light and transform it into heat. A laser beam is targeted to the mix. The laser beam is absorbed by the matrix, and part of the analyte-matrix mix is vaporized and ionized, creating a cloud of ionized proteins and matrix. This cloud subsequently is subjected to an electric field, which leads the particles to accelerate toward a detector. The mass and charge of each particle determine the time needed to reach the detector. This allows the mass spectrometer to determine the characteristics of the particles within the tested sample. Comparison of the produced spectral pattern against a standard database allows for identification of the microorganism in the sample. (C) Surface enhanced resonance Raman spectroscopy. The tested sample is placed on a rough surface, which helps to create scattered light. DNA probes coupled with specialized dyes that emit light are added to the sample. The probes then bind to the target DNA in the sample, and subsequently, a double-stranded DNA exonuclease is added to the mix and digests all bound probes, while the unbound probes, which are single stranded, are left indigested. Finally, the scattered light from the undigested probe is detected by a sensor and analyzed, thus identifying the DNA sequence of the digested probes. (D) T2 nuclear magnetic resonance. First, the target sequence of a microorganism that is found in the sample is amplified. Subsequently, DNA probes coupled with paramagnetic nanoparticles are added to the amplicon and are allowed to hybridize with the amplified sequence. This changes the T2 relaxation time of the nanoparticles, and the change is detected via magnetic resonance imaging, thus identifying the target.