Background A mass spectrometry (MS) method that detects a serum disaccharide (MS-DS) was recently described for the diagnosis of invasive fungal infections (IFI). We carried out a European collaborative study to evaluate this assay.Methods Patients with IFI were selected according to the availability of sera obtained around the time that IFI was documented: invasive candidiasis (IC; n=26 patients), invasive aspergillosis (IA; n=19), mucormycosis (MM; n=23). Control sera originated from 20 neutropenic patients and 20 patients with bacteraemia. MS-DS was carried out blind to IFI diagnosis. Diagnosis of IC and IA was confirmed by detection of (1,3)-β-D-glucan (BDG), mannan (Man) and galactomannan (GM), respectively. MM was detected by q-PCR.Results All tests discriminated IC sera from controls with bacteraemia (P≤0.0009). MS-DS sensitivity and specificity were 51% and 87%, respectively. MS-DS complemented the high specificity of Man monitoring. All tests discriminated IA sera from neutropenic controls (P≤0.0009). MS-DS sensitivity and specificity were 64% and 90%, respectively. Only 13/36 MM sera were concordant by MS-DS and q-PCR (six positive and seven negative); 14 were positive by MS-DS alone. q-PCR and MS-DS made a similar contribution to the diagnosis of MM. In patients undergoing long-term monitoring, persistent MS-DS circulation was observed whereas DNA was only detected for a short period after initiation of treatment.Conclusions MS-DS has an important role to play in the early diagnosis of IFI. Its pan fungal nature and complementarity with other tests may justify its use in the management of IFI.