We have evaluated the contribution of the 1,3-b-D-glucan (BG) assay for the screening of invasive fungal infections (IFIs) in patients with haematological malignancies. Serum samples from patients at risk of IFI were collected twice a week and retrospectively tested using the BG assay. BG screening was performed on 1143 samples from 91 patients during 104 anticancer treatment cycles. Proven and probable cases of IFI occurred in 9 (8.7 %) treatment cycles. Depending on the criterion of positivity used (1¾ .60 pg ml "1 , 1¾ .80 pg ml "1 , 2¾ .60 pg ml "1 or 2¾ .80 pg ml "1 ) the sensitivity and specificity were 89, 89, 67 and 44 %, and 20, 48, 33 and 56 %, respectively. Although the test was marked as positive in 82, 68, 54 and 45 % of all the treatment cycles, in the majority of cases, these positivities were probably false. The major limit of the BG test was an extremely low positive predictive value (10 to 12 %). We have analysed mucositis, candida colonization, bacteraemia, use of antimicrobials, erythrocyte and thrombocyte filtered blood products, collecting tubes or sampling via venous catheters. Even though no factor is a major source of BG, it could at least partially influence BG assay performance. Thus, BG detection has a limited usefulness as a screening method for IFIs in patients with haematological malignancies.

In contrast to recently published data, we found only moderate sensitivity, but high specificity and high positive predictive value of the detection of GM in BAL fluid. In addition, neutropenia, antifungal therapy, and BAL standardization affected GM assay performance.

The objective of this retrospective study was to evaluate results from voriconazole therapeutic drug monitoring (TDM) in haematological patients in routine clinical practice. Between 2005 and 2010, 1228 blood samples were obtained from 264 haematological patients (median 3 samples/patient; range 1-27) receiving voriconazole for targeted/preemptive treatment of invasive aspergillosis (IA) (46.3% of samples), empirical therapy (12.9%) or prophylaxis (40.8%). A high-pressure liquid chromatography assay was used to analyse voriconazole concentrations. Clinical and laboratory data were analysed retrospectively. The median of the detected voriconazole plasma concentration was 1.00 μg ml(-1) (range <0.20-13.47 μg ml(-1)). Significant inter- and intra-patients variability of measured concentrations (81.9% and 50.5%) were identified. With the exception of omeprazole administration, there was no relevant relationship between measured voriconazole concentrations and drug dose, route administration, age, gender, CYP2C19*2 genotype, gastrointestinal tract abnormality, administration via nasogastric tube, serum creatinine, and liver enzymes. However, per patient analysis identified significant role of individual voriconazole dose and drug form change on measured plasma concentration. Measured voriconazole concentrations did not correlate with the treatment outcome of patients with IA. We only identified a limited number of adverse events related to voriconazole therapy; however, the median plasma concentration was not different from concentrations measured in samples without reported toxicity. Our retrospective study has suggested that routine monitoring of voriconazole plasma concentrations has probably only a limited role in daily haematological practice.

e Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by highresolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/ HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2,9,11,16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1,5,10,14,15,17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by highresolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 l of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer's protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 m each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 l of ATL buffer (Qiagen) and 20 l of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folinpositive amin...