We recently described a mass spectrometry (MS) method based on the detection and quantification of a serum disaccharide (MSDS) in patients with invasive fungal infections (IFIs). Investigations concerned patients from our tertiary hospital with Candidiasis (IC), Aspergillosis (IA) and Mucormycosis (MM). As MSDS seemed to provide a new contribution for a pan fungal diagnosis, we aimed here to confirm these results through a blind study involving European expert centers in the clinical management of IC, IA and MM. 
  
IFI patients were selected in each center according to availability of sera drawn around the time that clinical/mycological/imaging evidence of IFI was obtained. IC patients came from Genoa and Roma (26 patients, 57 sera), IA patients rom Strasbourg (19 patients, 52 sera), MM patients from Paris and Lille (23 patients, 72 sera). Hospitalized controls consisted in 20 neutropenic patients from Genoa (40 sera) and 20 bacteraemic patients (20 sera) from Lausanne. All sera were received blindly. 
Detection of MSDS was performed according to the procedure previously published. Detection of glucans (Fungitell test) was used for the diagnosis of IC and IA in parallel with detection of mannan (Mnn) and Galactomannan (GM) respectively (Platelia Candida and Aspergillus Ag tests). For mucormycosis q-PCR was performed.  MS-DS results were sent to the different centers for lift of blind and analysis which comprised clinical information, results from diagnostic tests. 

For I.C all tests significantly discriminated sera from IC patients from controls with bacteraemia (p ≤ 0.0009). MS-DS Se/Sp (%) per serum were 51/87 providing intermediate values when compared to BDG (70/76) and Mnn tests (45/100). The analysis per patient reached sensitivities of 79; 85; 64 without altering the specificity. MS-DS complemented more positively high specificity of Mnn monitoring than BDG. For IA, all tests significantly discriminated sera from IA patients from control neutropenic patients (p ≤ 0.0009). MS-DS Se/Sp (%) per serum were of 64/90 providing intermediate values when compared to BDG (50/100) and GM tests (44/100). The analysis per patient reached sensitivities of 63, 50 and 78 without altering specificity. Interestingly the lower specificity attributed to MS-DS was related to 2 two neutropenic controls which developed later IA. For both IC and IA individual patients follow up of the 3 glycan biomarkers evidenced complex figures of release and degradation with independent or simultaneous circulation. For MM only 13 sera over 36 were concordant for MSDS and q-PCR (6 positive and 7 negative), 23 sera were discordant, among which 13 were positive for MSDS alone. q-PCR and MSDS contributed similarly to the establishment of MM diagnosis before mycological evidence was gained. For patients having long term monitoring persistent MSDS circulation could be observed whereas DNA was only be detected during a short period after initiation of treatment.  

This study confirms the contributions of MSDS to early diagnosis of IFI with sensitivities and specificities similar to those of tests recommended by EORTC and IDSA. Its pan fungal nature and complementarity with other tests may justify its use in combination for a better management of IFI. 


3 / Diagnosis of invasive fungal infections through detection of a circulating pan fungal disaccharide by mass spectrometry. A european multicenter study.