HTS and PCR Methods Are the Most Used in the Diagnosis of Aspergillosis: Advantages over Other Molecular Methods.

Aspergillosis includes a variety of diseases caused by species of the genus Aspergillus, ranging from non-invasive allergic diseases to chronic, invasive pulmonary infections, which are potentially fatal in immunocompromised hosts. Therefore, there is an urgent need for new diagnostic tools and the optimization of existing tests to improve patient care. This work reviews the most commonly used molecular methods for the diagnosis of aspergillosis from clinical samples, emphasizing their advantages. These methods included HTS, NTS, ISH, microarrays, PCR-RFLP, LAMP, and PCR in various modalities (qPCR, multiplex PCR, nested PCR, RT-PCR, endpoint PCR, U-dHRM, and ddPCR). The review showed that the most commonly used methods for diagnosing aspergillosis are NGS and PCR in their different modalities; however, each method has advantages and disadvantages. qPCR is the method that has demonstrated the greatest sensitivity and specificity on clinical samples (such as blood, serum, bronchoalveolar lavage [BAL], tissue, or sputum), since it detects specific sequences, and the validation of this method shows greater progress in achieving this objective. Likewise, NGS showed that BAL is the most suitable sample, with a higher fungal load than sputum or blood. On the other hand, NGS is not a targeted technique, since it sequences all the genetic material present. Additionally, the sensitivity for detecting pathogens decreases when clinical samples are used due to the high background of nucleic acids present in the human host.