Performance of the Bronchoalveolar Lavage Fluid Aspergillus Galactomannan Lateral Flow Assay With Cube Reader for Diagnosis of Invasive Pulmonary Aspergillosis: A Multicenter Cohort Study.

BACKGROUND: The Aspergillus Galactomannan Lateral Flow Assay (LFA) is a rapid test for the diagnosis of invasive aspergillosis (IA) that has been almost exclusively evaluated in patients with hematologic malignancies. An automated digital cube reader that allows for quantification of results has recently been added to the test kits. METHODS: We performed a retrospective multicenter study on bronchoalveolar lavage fluid (BALF) samples obtained from 296 patients with various underlying diseases (65% without underlying hematological malignancy) who had BALF galactomannan (GM) ordered between 2013 and 2019 at the University of California, San Diego, the Medical University of Graz, Austria, and the Mannheim University Hospital, Germany. RESULTS: Cases were classified as proven (n = 2), probable (n = 56), putative (n = 30), possible (n = 45), and no IA (n = 162). The LFA showed an area under the curve (AUC) of 0.865 (95% confidence interval [CI] .815-.916) for differentiating proven/probable or putative IA versus no IA, with a sensitivity of 74% and a specificity of 83% at an optical density index cutoff of 1.5. After exclusion of GM as mycological criterion for case classification, diagnostic performance of the LFA was highly similar to GM testing (AUC 0.892 vs 0.893, respectively). LFA performance was consistent across different patient cohorts and centers. CONCLUSIONS: In this multicenter study the LFA assay from BALF demonstrated good diagnostic performance for IA that was consistent across patient cohorts and locations. The LFA may serve a role as a rapid test that may replace conventional GM testing in settings where GM results are not rapidly available.

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Aspergillus specific nested PCR from the site of infection is superior to testing concurrent blood samples in immunocompromised patients with suspected invasive aspergillosis.

Invasive aspergillosis (IA) is a severe complication in immunocompromised patients. Early diagnosis is crucial to decrease its high mortality, yet the diagnostic gold standard (histopathology and culture) is time-consuming and cannot offer early confirmation of IA. Detection of IA by polymerase chain reaction (PCR) shows promising potential. Various studies have analysed its diagnostic performance in different clinical settings, especially addressing optimal specimen selection. However, direct comparison of different types of specimens in individual patients though essential, is rarely reported. We systematically assessed the diagnostic performance of an Aspergillus-specific nested PCR by investigating specimens from the site of infection and comparing it with concurrent blood samples in individual patients (pts) with IA. In a retrospective multicenter analysis PCR was performed on clinical specimens (n = 138) of immunocompromised high-risk pts (n = 133) from the site of infection together with concurrent blood samples. 38 pts were classified as proven/probable, 67 as possible and 28 as no IA according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions. A considerably superior performance of PCR from the site of infection was observed particularly in pts during antifungal prophylaxis (AFP)/antifungal therapy (AFT). Besides a specificity of 85%, sensitivity varied markedly in BAL (64%), CSF (100%), tissue samples (67%) as opposed to concurrent blood samples (8%). Our results further emphasise the need for investigating clinical samples from the site of infection in case of suspected IA to further establish or rule out the diagnosis.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

FunResDB-A web resource for genotypic susceptibility testing of Aspergillus fumigatus.

Therapy of invasive aspergillosis is becoming more difficult due to the emergence of azole resistance in Aspergillus fumigatus. A majority of resistant strains carries mutations in the CYP51A gene. Due to a lack of sensitivity of culture-based methods, molecular detection of A. fumigatus has become an important diagnostic tool. We set up the database FunResDB (www.nrz-myk.de/funresdb) to gather all available information about CYP51A-dependent azole resistance from published literature. In summary, the screening resulted in 79 CYP51A variants, which are linked to 59 nonsynonymous mutations. A tailor-made online sequence analysis tool allows for genotypic susceptibility testing of A. fumigatus.

Progressive Dispersion of Azole Resistance in Aspergillus fumigatus: Fatal Invasive Aspergillosis in a Patient with Acute Myeloid Leukemia Infected with an A. fumigatus Strain with a cyp51A TR46 Y121F M172I T289A Allele.

Patients with hematologic malignancies as well as allogeneic hematopoietic stem cell transplantation (HSCT) patients are at high risk for invasive aspergillosis. Here, we report a culture- and autopsy-proven fatal invasive aspergillosis in an allogeneic HSTC patient which he developed despite posaconazole prophylaxis. The agent was determined to be an azole-resistant Aspergillus fumigatus strain bearing the cyp51A mutation combination TR46 Y121F M172I T289A. At increasing frequency, the azole resistance of A. fumigatus is being reported globally, limiting treatment options and complicating regimens.

The evolving landscape of new diagnostic tests for invasive aspergillosis in hematology patients: strengths and weaknesses.

PURPOSE OF REVIEW: The diagnosis of invasive aspergillosis in hematologic patients is a complex composite of clinical preconditions and features, imaging findings, biomarker combinations from appropriate clinical samples and microbiological and/or histological findings. RECENT FINDINGS: Recent developments in the evolving landscape of diagnostic tests for invasive aspergillosis in adult hematology patients are highlighted. SUMMARY: Novel approaches and tools are currently under development. Focusing optimized diagnostic performance, in particular the combination of biomarkers from appropriate clinical samples, improved diagnostic performance distinctly.

Galactomannan testing and Aspergillus PCR in same-day bronchoalveolar lavage and blood samples for diagnosis of invasive aspergillosis.

In recent years galactomannan antigen testing (GM) and also Aspergillus PCR have become increasingly important for diagnosis of invasive aspergillosis (IA). Whether or not these tests need to be performed with bronchoalveolar lavage fluid (BALF; i.e., primary site of infection), or testing of blood samples is sufficient, remains, however, a matter of debate. We evaluated the diagnostic performance of GM ELISA, and Aspergillus PCR by using BALF samples and blood samples obtained at the same day from a total of 53 immunocompromised patients (16 with probable/proven IA and 37 with no evidence of IA according to the revised EORTC/MSG criteria; 38 patients with hematological malignancies were prospectively enrolled at the Medical University of Graz, Austria, 15 patients with mixed underlying diseases at the Mannheim University Hospital). Patients with possible IA were excluded from this analysis. A total of 34/53 (64%) of all patients and 12/16 (75%) of patients with probable/proven IA received mold-active antifungal prophylaxis/therapy at the time of the BALF procedure. Sensitivities of GM and Aspergillus PCR were 38% and 44% in BALF, and 31% and 0% in blood, respectively. Best sensitivity (75%) for detecting proven/probable IA was achieved when BALF Aspergillus PCR, BALF GM (>1.0 ODI), BALF-culture and serum-GM (>0.5 ODI) were combined (specificity 95%). In conclusion, sensitivities of the evaluated diagnostic tests-when interpreted on their own-were low in BALF and even lower in blood, sensitivities increased markedly when diagnostic tests were combined.

Direct comparison of galactomannan performance in concurrent serum and bronchoalveolar lavage samples in immunocompromised patients at risk for invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection mainly affecting immunocompromised patients. Early diagnosis is critical, but the diagnostic gold standard (histopathology and culture) is time consuming and cannot offer early confirmation of IPA. Fungal biomarkers like galactomannan (GM) are a promising extension to the diagnostic repertoire. However, it still remains under discussion if biomarker analysis from the site of the infection is superior to testing blood samples. We retrospectively evaluated the diagnostic performance of concurrent serum GM and bronchoalveolar lavage (BAL) GM (obtained within 24 h) of immunocompromised patients at high risk of IPA. Twenty-six proven/probable patients and eight patients with no IPA according to the EORTC/MSG 2008 criteria were included in this study. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were for BAL GM: 85%, 88%, 96%, 64% and 38.5, and for serum GM: 23%, 88%, 88%, 26% and 2.1 respectively. BAL GM proved to be significantly more sensitive for the detection of IPA compared to same-day serum GM in patients at high risk of IPA (P < 0.0001). Our data show that BAL GM testing is significantly superior to serum GM implying that diagnostic efforts should focus on specimens from the site of infection.

Biomarker-based diagnostic work-up of invasive pulmonary aspergillosis in immunocompromised paediatric patients--is Aspergillus PCR appropriate?

Invasive aspergillosis (IA) is an important cause of morbidity and mortality in children and adults with haematologic malignancies or undergoing allogeneic haematopoietic stem cell transplantation, and early diagnosis and adequate antifungal treatment improve outcome. However, important differences exist between children and adults regarding epidemiology, underlying disease, and comorbidities, and the value of diagnostic tools to detect IA may also differ between these patient populations. Imaging studies are important to detect IA early, but typical findings of IA in chest computed tomography of adults are not detected in the majority of children. Whereas the value of the serum marker galactomannan seems to be comparable in children and adults, data on the performance of beta-d-glucan in children are too limited for firm conclusions. PCR-based assays are a promising diagnostic approach to rapidly and reliably detect and identify Aspergillus species in various clinical samples. However, as the majority of data on PCR-based approaches has been obtained in adult patients, the value of this method in paediatric patients has not been defined to date. The present review focuses on studies of PCR-based methods to diagnose IA in immunocompromised paediatric patients.

Multicenter evaluation of a lateral-flow device test for diagnosing invasive pulmonary aspergillosis in ICU patients.

INTRODUCTION: The incidence of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is increasing, and early diagnosis of the disease and treatment with antifungal drugs is critical for patient survival. Serum biomarker tests for IPA typically give false-negative results in non-neutropenic patients, and galactomannan (GM) detection, the preferred diagnostic test for IPA using bronchoalveolar lavage (BAL), is often not readily available. Novel approaches to IPA detection in ICU patients are needed. In this multicenter study, we evaluated the performance of an Aspergillus lateral-flow device (LFD) test for BAL IPA detection in critically ill patients. METHODS: A total of 149 BAL samples from 133 ICU patients were included in this semiprospective study. Participating centers were the medical university hospitals of Graz, Vienna and Innsbruck in Austria and the University Hospital of Mannheim, Germany. Fungal infections were classified according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. RESULTS: Two patients (four BALs) had proven IPA, fourteen patients (sixteen BALs) had probable IPA, twenty patients (twenty-one BALs) had possible IPA and ninety-seven patients (one hundred eight BALs) did not fulfill IPA criteria. Sensitivity, specificity, negative predictive value, positive predictive value and diagnostic odds ratios for diagnosing proven and probable IPA using LFD tests of BAL were 80%, 81%, 96%, 44% and 17.6, respectively. Fungal BAL culture exhibited a sensitivity of 50% and a specificity of 85%. CONCLUSION: LFD tests of BAL showed promising results for IPA diagnosis in ICU patients. Furthermore, the LFD test can be performed easily and provides rapid results. Therefore, it may be a reliable alternative for IPA diagnosis in ICU patients if GM results are not rapidly available. TRIAL REGISTRATION: ClinicalTrials.gov NCT02058316. Registered 20 January 2014.

The SNP rs460089 in the gene promoter of the drug transporter OCTN1 has prognostic value for treatment-free remission in chronic myeloid leukemia patients treated with imatinib.

Membrane transporters are important determinants of drug bioavailability. Their expression and activity affect the intracellular drug concentration in leukemic cells impacting response to therapy. Pharmacogenomics represents genetic markers that reflect allele arrangement of genes encoding drug transporters associated with treatment response. In previous work, we identified SNP rs460089 located in the promotor of SLC22A4 gene encoding imatinib transporter OCTN1 as influential on response of patients with chronic myeloid leukemia treated with imatinib. Patients with rs460089-GC pharmacogenotype had significantly superior response to first-line imatinib treatment compared to patients with rs460089-GG. This study investigated whether pharmacogenotypes of rs460089 are associated with sustainability of treatment-free remission (TFR) in patients from the EUROpean Stop Kinase Inhibitor (EURO-SKI) trial. In the learning sample, 176 patients showed a significantly higher 6-month probability of molecular relapse free survival (MRFS) in patients with GC genotype (73%, 95% CI: 60-82%) compared to patients with GG (51%, 95% CI: 41-61%). Also over time, patients with GC genotype had significantly higher MRFS probabilities compared with patients with GG (HR: 0.474, 95% CI: 0.280-0.802, p = 0.0054). Both results were validated with data on 93 patients from the Polish STOP imatinib study. In multiple regression models, in addition to the investigated genotype, duration of TKI therapy (EURO-SKI trial) and duration of deep molecular response (Polish study) were identified as independent prognostic factors. The SNP rs460089 was found as an independent predictor of TFR.

Reverse Transcription Can Critically Impact the Diagnostic Outcome of BCR::ABL1 Quantitative Real-Time RT-PCR.

Reverse transcriptases (RT) are essential tools in BCR::ABL1 fusion transcript monitoring in chronic myeloid leukemia (CML). The RT type and cDNA priming method may impair the stoichiometry of cDNA synthesis, thereby potentially introducing a bias in BCR::ABL1 qRT-PCR data. Using the Acrometrix™ BCR::ABL1 reference panel and 37 clinical specimens, we have comparatively investigated the performance of the RTs MLV and SuperScript IV with random hexamer vs. target-specific priming. Quantitative RT-PCR results identified the priming type and RT type as major factors for diagnostic data variation, mainly due to the different efficacies of processing BCR::ABL1 low-copy-numbers (<50) compared to GUSB or ABL1 high-copy targets. The impairment of SuperScript IV in processing low- and high-copy-number RNA targets equally was not reflected by the diagnostically relevant Log (BCR::ABL1/GUSB%) values. Therefore, the correct representation of housekeeping and BCR::ABL1 target genes should have priority when aiming at as high a number of housekeeping gene copies as possible. Our data suggest that for improving BCR::ABL1 assay sensitivity, increased RNA/cDNA amounts and the use of distinct RT/priming combinations are advantageous. However, for inter-laboratory harmonization, the proper conversion factor according to the CML international standard (IS) has to be reevaluated each time the grade of RT is changed.

Gene Expression Pattern of ESPL1, PTTG1 and PTTG1IP Can Potentially Predict Response to TKI First-Line Treatment of Patients with Newly Diagnosed CML.

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1/Separase, PTTG1/Securin and PTTG1IP/Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB) of ESPL1, PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort (p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months (p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1, PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment.

Development of novel PCR assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients.

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

Therapy with antifungals decreases the diagnostic performance of PCR for diagnosing invasive aspergillosis in bronchoalveolar lavage samples of patients with haematological malignancies.

OBJECTIVES: Invasive aspergillosis (IA) is a life-threatening infection in severely immunocompromised patients, especially those receiving intensive chemotherapy or undergoing haematopoietic stem cell transplantation. As the clinical diagnosis of IA is mostly based on biomarkers (galactomannan, ß-d-glucan, PCR assays) indicating Aspergillus as the underlying pathogen, the effect of antifungal treatment on the performance of these parameters is still controversial. We evaluated the effect of antifungal treatment on the performance of an Aspergillus-specific PCR assay in bronchoalveolar lavage (BAL) samples. PATIENTS AND METHODS: Two-hundred-and-twenty-six BAL samples from 226 patients with haematological malignancies at high risk for IA classified according to the 2008 European Organization for the Research and Treatment of Cancer criteria were analysed retrospectively for the diagnostic performance of a nested Aspergillus PCR assay in relation to the number and type of mould-active antifungals received prior to BAL sampling. RESULTS: Sensitivity of BAL PCR for patients without antifungal treatment prior to BAL sampling was 0.69, whereas specificity was 0.87. While no significant change in diagnostic performance by the addition of one antifungal was observed, receiving two or more antifungals prior to BAL sampling led to a significant decrease in the diagnostic performance of BAL PCR testing (P < 0.009). CONCLUSIONS: Treatment with mould-active antifungals prior to BAL sampling significantly decreases the performance of the Aspergillus PCR assay in haematological patients if BAL was performed after administration of more than one antifungal agent. Performing BAL sampling for Aspergillus PCR diagnostic despite pre-treatment with one antifungal or while on prophylaxis is feasible.

Diagnosing pulmonary aspergillosis in patients with hematological malignancies: a multicenter prospective evaluation of an Aspergillus PCR assay and a galactomannan ELISA in bronchoalveolar lavage samples.

OBJECTIVES: Diagnosing invasive pulmonary aspergillosis (IPA) remains a challenge in patients with hematological malignancies. The clinical significance of testing bronchoalveolar lavage (BAL) samples both with polymerase chain reaction (PCR) and Aspergillus galactomannan ELISA (GM) is unclear, and the BAL cutoff for GM has not been clearly evaluated yet. METHODS: Using a validated nested PCR assay and a GM ELISA, we prospectively examined BAL samples from 87 hematological patients at high risk of IPA. Of 76 (87%) evaluable patients, 29 patients had proven or probable disease. RESULTS: The receiver operating characteristic (ROC) analysis of GM optical density (OD) cutoff levels yielded a BAL OD of 0.5 to be optimal. We identified 29 probable or proven cases based on this OD. Sensitivity and specificity for BAL GM were 0.79 (95% CI, 0.62-0.9) and 0.96 (95% CI, 0.86-0.99), respectively. For BAL PCR, sensitivity and specificity were 0.59 (95% CI, 0.41-0.75) and 0.87 (95% CI, 0.75-0.94), respectively. Combining BAL GM and PCR for diagnosis showed a sensitivity and specificity rate of 0.55 (95% CI, 0.38-0.72) and 1.0 (95% CI, 0.93-1.0), respectively, if positivity was defined by positive results for both tests. If either positive BAL GM or positive BAL PCR results defined test positivity, the sensitivity was 0.83 (95% CI, 0.65-0.92), and the specificity was 0.83 (95% CI, 0.70-0.91) CONCLUSIONS: In terms of optimal sensitivity and specificity, a GM OD cutoff of 0.5 was determined for BAL samples. Positivity for both GM and Aspergillus PCR in BAL makes a pulmonary aspergillosis highly likely.

A new aberrantly spliced BCR-ABL1 transcript variant (e13a1) identified in routine monitoring using different quantitative reverse transcription polymerase chain reaction techniques in a patient with chronic myeloid leukemia.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of BCR-ABL1 transcript level is an essential part of routine disease monitoring in patients with chronic myeloid leukemia. One patient sample (e13a2 transcript detected by nested PCR) attracted attention by revealing an aberrantly spliced BCR-ABL1 transcript variant e13a1. The last 38 base pairs (bp) of BCR exon 13 were replaced by a 37 bp insertion of the ABL1 intron 1-2/exon 1 sequence. The rare aberrant BCR-ABL1 fusion transcript can cause discrepancies in molecular diagnostics. This scenario highlights the importance of an individual characterization of the BCR-ABL1 fusion sequence in case of unclear qRT-PCR results.

Global guideline for the diagnosis and management of rare mould infections: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology and the American Society for Microbiology.

With increasing numbers of patients needing intensive care or who are immunosuppressed, infections caused by moulds other than Aspergillus spp or Mucorales are increasing. Although antifungal prophylaxis has shown effectiveness in preventing many invasive fungal infections, selective pressure has caused an increase of breakthrough infections caused by Fusarium, Lomentospora, and Scedosporium species, as well as by dematiaceous moulds, Rasamsonia, Schizophyllum, Scopulariopsis, Paecilomyces, Penicillium, Talaromyces and Purpureocillium species. Guidance on the complex multidisciplinary management of infections caused by these pathogens has the potential to improve prognosis. Management routes depend on the availability of diagnostic and therapeutic options. The present recommendations are part of the One World-One Guideline initiative to incorporate regional differences in the epidemiology and management of rare mould infections. Experts from 24 countries contributed their knowledge and analysed published evidence on the diagnosis and treatment of rare mould infections. This consensus document intends to provide practical guidance in clinical decision making by engaging physicians and scientists involved in various aspects of clinical management. Moreover, we identify areas of uncertainty and constraints in optimising this management.