A highly sensitive and specific method to evaluate viable fungal burden of Aspergillus fumigatus in mice by RT-qPCR for 18S ribosomal RNA.

Potent fungicidal activity is one of the key factors of antifungals to overcome invasive pulmonary aspergillosis (IPA). To date, quantification of Aspergillus DNA in the lungs and galactomannan (GM) in serum or bronchoalveolar lavage fluid have been developed as general methods for measuring fungal burden in IPA animal models. However, GM quantification is not supposed to be a suitable method for precise evaluation of the fungicidal effects of antifungals, because killed Aspergillus hyphae can release GM for a certain period until they are eliminated by the host. Therefore, in terms of detecting viable fungal burden of Aspergillus, quantification of Aspergillus DNA has been thought to be a suitable method. Here, to obtain a method with much higher sensitivity, we applied reverse transcription quantitative PCR (RT-qPCR) for A. fumigatus 18S ribosomal RNA to measure the viable fungal burden in murine IPA models. Prior to in vivo tests, we confirmed that the sensitivity of 18S rRNA was nearly 50-fold higher than that of 18S ribosomal DNA in vitro. This highly sensitive method made it possible to evaluate the fungicidal effects of antifungals in a low-inoculation murine IPA model. In this model, single administrations of higher doses of voriconazole and posaconazole, which have fungicidal activity, were able to display fungicidal effects with ≥1 log10 reductions by 18S rRNA quantification, whereas significant reductions in serum GM were not observed. These results suggest that 18S rRNA quantification is a powerful tool for screening novel antifungals with potent fungicidal activity only after a single administration.