Real‐time PCR on the first galactomannan‐positive serum sample for diagnosing invasive aspergillosis in liver transplant recipients

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Abstract
Invasive aspergillosis (IA) is a life‐threatening complication of liver transplantation. Detection of circulating galactomannan (GM) in serum samples is a method to improve the microbiological diagnosis in patients at risk for IA. However, the assay is hampered by false‐positive results. The search for circulating Aspergillus DNA in the first GM‐positive sample could improve the specificity of the test. Among 484 liver transplant recipients followed in a single center over 4 years, 25 patients had at least 1 GM‐positive serum sample. The threshold of GM positivity was a ratio ≥1. These 25 patients were classified by the clinicians as probable IA (n=11), possible IA (n=2), and no IA (n=12) using the EORTC/MSG criteria with blinding to the polymerase chain reaction (PCR) results. After 1 mL aliquots of the first GM‐positive serum sample were thawed, 2 independent DNA extractions were performed using the MagNA Pure Compact apparatus. Real‐time amplification targeted at Aspergillus fumigatus mitochondrial DNA was performed on 10 μL of the final eluate in duplicate in the 2 independent DNA extractions using a LightCycler instrument. A sample was considered positive when the crossing point was ≤43 cycles in at least 2 out of the 4 replicates. Among the 13 probable or possible IA, 8 patients were PCR positive. The other 12 patients who had no IA were all PCR negative. Our data suggest that a concomitant real‐time PCR performed on the first GM‐positive sample improves the specificity of the first GM‐positive assay result.

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