Measuring serum beta-d-glucan (BDG) is a useful tool for supporting a quantitative PCR (qPCR)-based diagnosis of suspected Pneumocystis pneumonia (PCP) with bronchoalveolar lavage (BAL) fluid. Since the 2000s, the Fungitell assay was the only BDG assay which was FDA cleared and Conformité Européenne (CE) marked. However, the Wako β-glucan test was also recently CE marked and commercialized. We analyzed archived sera from 116 PCP cases (who were considered to have PCP based on compatible clinical and radiological findings plus a BAL fluid qPCR threshold cycle value of ≤28) and 114 controls (those with a BAL fluid qPCR threshold cycle value of >45 and no invasive fungal infection) using the Fungitell and Wako assays in parallel and assessed their diagnostic performance using the manufacturer’s proposed cutoffs of 80 pg/ml and 11 pg/ml, respectively. We found the Wako assay to be more specific (0.98 versus 0.87, P < 0.001) and the Fungitell assay to be more sensitive (0.78 versus 0.85, P = 0.039) at the proposed cutoffs. Overall performance, as determined by the area under the receiver operating characteristic curve, was similar for both assays. We determined a new Wako assay cutoff (3.616 pg/ml) to match the sensitivity of the Fungitell assay (0.88 at a cutoff of ≥60 pg/ml). Using this newly proposed cutoff, the specificity of the Wako assay was significantly better than that of the Fungitell assay (0.89 versus 0.82, P = 0.011). In conclusion, the Wako assay performed excellently compared to the Fungitell assay for the diagnosis of presumed PCP based on qPCR. In addition, contrary to the Fungitell assay, the Wako assay allows for single-sample testing with lower inter- and intrarun variability. Finally, we propose an optimized cutoff for the Wako assay to reliably exclude PCP.

The primary aim of this study was to collect national epidemiological data on candidaemia and to determine the reporting time of species identification and antifungal susceptibility in clinical practice. During a 1-year period (March 2013 until February 2014), every first Candida isolate from each episode of candidaemia was included prospectively from 30 Belgian hospitals. Identification and susceptibility testing were performed according to local procedures and isolates were sent to the National Reference Center for Mycosis. Species identification was checked by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing in case no reliable identification was obtained by MALDI-TOF MS. Antifungal susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. A total of 355 isolates were retrieved from 338 patients. The mean incidence rate of candidaemia was 0.44 (range: 0.07 to 1.43) per 1000 admissions or 0.65 (range: 0.11 to 2.00) per 10,000 patient days. Candida albicans was most frequently found (50.4 %), followed by C. glabrata (27.3 %) and C. parapsilosis sensu lato (9.8 %). The overall resistance to fluconazole was 7.6 %, ranging from 3.9 % in C. albicans to 20.0 % in C. tropicalis. Only one C. glabrata isolate was resistant to the echinocandins. Four days after blood culture positivity, 99.7 % of the identifications and 90.3 % of the antifungal profiles were reported to the treating clinician. Candidaemia incidence rates differed up to 20-fold among Belgian hospitals; no clear factors explaining this difference were identified. The overall antifungal resistance rates were low but high azole resistance rates were recorded in C. tropicalis.

Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n ϭ 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n ϭ 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR 34 /L98H was the most prevalent mutation (46.7%), followed by TR 46 / Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.KEYWORDS VIPcheck, AsperGenius, cyp51A, cyp51B, hapE A zole resistance in Aspergillus fumigatus is an emerging problem worldwide, with major epidemiological and clinical implications (1-6). Mold active triazoles are commonly used as first line treatment and prophylaxis of invasive aspergillosis (IA) (7). Mutations in the cyp51A gene, which encodes the target of azoles, the lanosterol 14␣-demethylase, represent the most commonly reported mechanism conferring azole resistance and consequently treatment failure in A. fumigatus (8-10). The most prevalent mutation (TR 34 /L98H) involves the insertion of a 34-bp tandem repeat (TR 34 ) in the promoter region of the cyp51A gene and a point mutation within the gene leading to an amino acid substitution (L98H). More recently, an additional cyp51A-mediated resistant genotype (TR 46 /Y121F/T289A) has been reported: a 46-bp tandem repeat (TR 46 ) in the promoter of cyp51A gene and two point mutations within the gene

During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the diagnosis of fungal infections. Recently, a new Conidia ID-fungi plate (IDFP) medium was introduced to facilitate growth and sampling of fungi. This study aimed to evaluate the IDFP for fungal MALDI-TOF MS identification by comparison with a standard fungal growth medium using two reference libraries. A total of 75 filamentous fungal isolates (including 32 dermatophytes) were inoculated on IDFP and Sabouraud-gentamicin-chloramphenicol (SGC) agar and identified by MALDI-TOF MS using formic acid/acetonitrile extraction. Both the commercially available Bruker library (version 2.0) and the public available MSI web application (version 2018) were applied. For 15% of the isolates, a faster growth was noticed on IDFP compared to SGC. IDFP enhanced the performance of fungal identification compared to SGC for both MSI (increase of 16% identifications to genus and 5% to species level) and Bruker library (increase of 22% identifications to genus and 8% to species level). In total, only 73% of the tested isolates were present in the Bruker library compared to 92% for MSI library. No significant difference (P = 0.46) in MALDI score between IDFP and SGC was observed for the MSI library, but scores were significantly (P = 0.03) higher for IDFP when using Bruker library, potentially explained by the prevention of agar contamination by using IDFP since the Bruker database was created from liquid media. IDFP is a promising alternative growth medium for MALDI-TOF MS fungal identification which would strongly benefit from optimizing the Bruker reference library.

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