Monitoring of invasive Aspergillosis antifungal therapy by quantitative reverse transcriptase real-time polymerase chain reaction (qRT- PCR)

Hossein Khodadadi*1, Ladan Karimi2, Keyvan Pakshir1, Kamiar Zomorodian1 
1Department of Medical Parasitology and Mycology, Shiraz University of Medical Sciences, shiraz, Iran 
2Clinical Laboratory of Shahid Beheshti Hospital, Social Security Organization, shiraz, Iran

Objectives: Assessment of antifungal therapeutic efficacy of invasive aspergillosis (IA) is essential and reliable methods to quantify response to anti-Aspergillus drugs are desirable. Although serum beta-glucan (BG) and galactomannan (GM) levels were evaluated during antifungal therapy, the levels of these antigens fluctuate irregularly and do not show early therapeutic response correctly. Recently some efforts focused on detection and quantification of Aspergillus RNA in serum as a biomarker. We developed a quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) for following up responses to antifungals in IA patients.
Methods: Total RNA was extracted from 188 blood samples of 36 patients with proven or probable IA during their antifungal therapy regimen.  The loads of Aspergillus 18S rRNA were quantified by a newly designed pan-Aspergillus qRT-PCR. GM levels in serum were measured simultaneously and clinical outcome were assessed. The correlation between the changes of circulating Aspergillus RNA load and serum GM levels and the clinical outcome were statistically compared.
Results: Thirty-six patients with newly diagnosed IA and receiving anti-Aspergillus drugs were evaluated weekly for 10 weeks. 34 out of 188 (18.1%) blood samples belonging to 9 patients had positive qRT-PCR results. Aspergillus RNA load decreasing was seen in 7 patients. Fungal RNA decrease began from third week of antifungal therapy initiation. This decrease associated with an excellent response to antifungals. Although changes of blood Aspergillus RNA load correlates with clinical outcome, the fungal RNA load did not show correlation with GM levels.
Conclusions: Changes of blood Aspergillus RNA load during antifungal therapy correlates with final clinical outcome. The measurement of changes of circulating Aspergillus RNA load in combination with other assays may help for monitoring the response to anti-fungal therapy.