The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published “Minimum Information for the publication of real-time Quantitative PCR Experiments” (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95–107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.

Objectives. Clinical experience of the impact of serum biomarkers for invasive fungal disease (IFD) varies markedly in haemato-oncology. Invasive pulmonary aspergillosis (IPA) is the commonest manifestation, so we evaluated biomarkers in broncho-alveolar lavage fluid (BAL). Methods. Aspergillus-specific lateral flow device (LFD), qPCR and galactomannan (GM) tests were used with 32 BAL fluid samples from 32 patients at risk of IPA. Eight patients had proven, 3 probable, 6 possible and 15 patients no IPA by EORTC/MSG criteria. Diagnostic accuracies of the tests were evaluated and pair-wise agreement between biomarkers was calculated. The diagnostic performance of the EORTC/MSG criteria was evaluated against the test(s) identified as most useful in IPA diagnosis. Results. Using EORTC/MSG criteria, sensitivities of qPCR and LFD were 100%, GM was 87.5% (cut-off, GM > 0.8), with specificities between 66.7 and 86.7%. The agreement between the qPCR and LFD was almost perfect (Cohen's kappa = 0.93, 95% CI: 0.81 – 1.00). Combined LFD+qPCR had a sensitivity of 100% and specificity of 85.7%. Calcofluor staining and culture of all BAL samples was negative for fungal infection. The median time from starting mould-active antifungal therapy to obtaining BAL was 6 days. Reversing roles, and using LFD+qPCR dual testing to classify cases, the EORTC/MSG criteria had a sensitivity of 83.3%. Conclusions. All three tests are useful for diagnosis of IPA in BAL samples. Despite the significant delays between starting antifungal therapy and bronchoscopy, unlike microscopy and culture, the biomarkers remain informative. In particular, the combination of LFD+qPCR allows sensitive and specific detection of IPA.

Galactomannan (GM) is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer’s recommendations. Short-term (ST) storage of samples was at +4 °C while long-term (LT) storage was at −80 °C. Bronchoalveolar (BAL) fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA) pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001) and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001) of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64%) became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable.