Abstract
The European Aspergillus PCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated “in vivo” specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and β-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and β-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and β-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and β-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.
Highlights
The diagnosis of invasive aspergillosis (IA) remains difficult, resulting in the widespread use of empirical therapy or posaconazole prophylaxis in high-risk patients
Direct detection of Aspergillus in clinical specimens using molecular methods is yet to be included in criteria for defining invasive fungal disease (IFD), which is a result of limited standardization [1]
The European Aspergillus PCR Initiative (EAPCRI) provided recommendations based on simulated samples to standardize procedures for whole blood (WB) and serum/plasma [2, 3]
Summary
The diagnosis of invasive aspergillosis (IA) remains difficult, resulting in the widespread use of empirical therapy or posaconazole prophylaxis in high-risk patients. Most patients are diagnosed with probable or possible IA based on specific radiological signs with or without the detection of biomarkers (galactomannan [GM] or -D-glucan [BDG]) or culture of Aspergillus [1]. Direct detection of Aspergillus in clinical specimens using molecular methods is yet to be included in criteria for defining invasive fungal disease (IFD), which is a result of limited standardization [1]. In order to continue with the PCR standardization process, the EAPCRI formed an international collaboration with the NIHfounded Invasive Aspergillosis Animal Models (IAAM) group to evaluate the performance of the Aspergillus PCR protocols recommended by the EAPCRI when testing blood samples derived from a guinea pig model of IA. In addition to the performance of PCR, the detection of other circulating biomarkers, galactomannan (GM) and -D-glucan (BDG), was evaluated with particular focus given to the timing of positivity and the concept of combination testing, and the findings are described
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