Jana Winterová

Difficulties in using 1,3-β-d-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies – high frequency of false-positive results and their analysis

We have evaluated the contribution of the 1,3-beta-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 (1x >60 pg ml(-1), 1x >80 pg ml(-1), 2x >60 pg ml(-1) or 2x >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.

Monitoring trough voriconazole plasma concentrations in haematological patients: real life multicentre experience.

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.

Intravenous PLASMA-LYTE as a major cause of false-positive results of platelia Aspergillus test for galactomannan detection in serum

Galactomannan (GM) detection by the Platelia Aspergillus (PA) enzyme immunoassay (Bio-Rad, France) is a test widely used for the early diagnosis of invasive aspergillosis (IA) in hematooncological patients. False-positive results for this test were reported by several authors, associated mainly with

Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

ABSTRACT 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.

Reactivity of the 1,3-β-D-glucan assay during bacteraemia: limited evidence from a prospective study

There are discrepancies in the retrospective studies published in literature of whether or not bacteraemia could lead to false positivity of 1,3‐β‐D (BG) glucan assay. We performed, for the first time, a prospective study evaluating the role of bacterial bloodstream infection to the reactivity of BG assay. Twenty‐six episodes of bacteraemia that occurred in high‐risk haematological patients were included in our study. Consecutive BG levels >80 pg ml−1 were required for test positivity. Only 2 of 26 patients were BG positive – both with IFDs. Thus, we prospectively did not prove bacteraemia as the source of cross reactivity of BG assay in haematological patients.

Mucositis does not lead to false-positivity of the Platelia Aspergillus enzyme-linked immunosorbent assay

We are reporting a study evaluating the crossover of antigens reacting in Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) from faeces to vessels during mucositis as a possible cause of false-positivity of this test. In our series of 102 episodes of different grades of mucositis, we found strong reactivity of faeces in the PA ELISA test irrespective of the grade of mucositis, the percentage of oral food intake or the presence of total parenteral nutrition. However, none of the patients included in the study were positive in the serum (when the criterion of two samples with cut-off index of positivity [IP] > 0.5 was used).

Monitoring of Epstein–Barr virus load in patients after allogeneic hematopoietic stem cell transplantation

Based on our results, we recommend the weekly monitoring of patients for at least 3 months after HSCT (2 out of 4 patients with EBV-associated disease passed the cut-off at week 11) and longer in patients with GVHD, due to the rapid progression of EBV infection, particularly in patients who develop clinical symptoms (lymphadenopathy, hepatosplenomegaly, or organ manifestations) related to EBV disease according to the European Conference on Infections in Leukemia (ECIL) 2011 guidelines. EBV reactivation occurred in some of the patients suffering fromGVHDin this cohort, but did not require clinical intervention unless the copy numbers were steadily increasing. A stable EBV viral load\1,000 copies/lg DNA temporarily occurred in some patients after HSCT; however, clinical symptoms of EBV infection did not develop and they did not require therapeutic intervention. Therefore, to improve the diagnostic value of EBV monitoring, the load measurement and quantitative monitoring of viral load kinetics is necessary.

Monitoring of Mannan Antigenemia (Mn) and Antimannan Antibodies (anti-Mn) for Screening of Invasive Candidiasis in High Risk Hematooncological Patients

Introduction and Methods: Between January 2005 and July 2007 we conducted a retrospective study to assess the diagnostic utility of regular Mn and anti-Mn monitoring in neutropenic patients with hematological malignancy at high risk for invasive fungal infection, including invasive candidiasis (IC). Blood samples were obtained twice weekly and Platelia Candida Ag and Platelia Candida Ab/Ac/Ak test were used for Mn and anti-Mn detection. Classification of IC cases was performed using EORTC/MSG criteria. All patients received antifungal prophylaxis. Results: Ninety-one high risk patients were screened for Mn and anti-Mn during 104 treatment cycles. Detection of Mn and anti-Mn was performed in 1199 (mean 11.5/patient) blood samples. Only one patient fulfilled criteria for proven and probable IC (candidaemia caused by C. krusei) and thus incidence of IC was only 0.95% in our patient group. At least 1 sample with Mn > 0.25ng/ml (intermediate) was detected in 35 (33.7%) and > 0.5ng/ml (positive) in 6 (5.8%) treatment cycles respectively. Only in 1 patient Mn > 0.5ng/ml was detected in more than 1 sample this was the patient with serious typhlytis on voriconazole prophylaxis where transient passage of Candida spp. or candida antigens could not be excluded. Even more, simultaneously performed 1,3-ß-D glucan was also positive. All other Mn positive or intermediate results were not associated with proven/probable IC. The only patient with proven IC was Mn negative in repeat samples. AntiMn was detected at least in 1 sample at intermediate range (>5 A.U./ml) in 56 (53.8%) treatment cycles and in 31 (29.8%) at positive range (>10 A.U./ml). Positive anti-Mn levels in repeat samples were found in 20 (19.2%) cycles. None of anti-Mn intermediate or positive patients were classified as having proven/probable IC. The only patient with candidaemia caused by C. krusei was anti-Mn negative. There was no correlation between Mn/anti-Mn positivity and empirical antifungal treatment or Candida spp. colonisation. Conclusions: Contribution of Mn and anti-Mn detection in routing screening of hemato-oncological patients in high risk of IFI is limited. Our analysis showed that only when the Mn antigen is detected (>0.5ng/ml) in repeat samples, IC should be considering. On the other hand, the positivity (>10 A.U./ml) of anti-Mn was very frequent (20–30% of treatment cycles) but never associated with IC.