Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

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Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Klebsiella pneumoniae prevents spore germination and hyphal development of Aspergillus species.

Different bacteria and fungi live as commensal organisms as part of the human microbiota, but shifts to a pathogenic state potentially leading to septic infections commonly occur in immunocompromised individuals. Several studies have reported synergistic or antagonistic interactions between individual bacteria and fungi which might be of clinical relevance. Here, we present first evidence for the interaction between Klebsiella pneumoniae and several Aspergillus species including A. fumigatus, A. terreus, A. niger and A. flavus which cohabit in the lungs and the intestines. Microbiological and molecular methods were employed to investigate the interaction in vitro, and the results indicate that Klebsiella pneumoniae is able to prevent Aspergillus spp. spore germination and hyphal development. The inhibitory effect is reversible, as demonstrated by growth recovery of Aspergillus spp. upon inhibition or elimination of the bacteria, and is apparently dependent on the physical interaction with metabolically active bacteria. Molecular analysis of Klebsiella-Aspergillus interaction has shown upregulation of Aspergillus cell wall-related genes and downregulation of hyphae-related genes, suggesting that Klebsiella induces cell wall stress response mechanisms and suppresses filamentous growth. Characterization of polymicrobial interactions may provide the basis for improved clinical management of mixed infections by setting the stage for appropriate diagnostics and ultimately for optimized treatment strategies.

Comparison of different approaches to quantitative adenovirus detection in stool specimens of hematopoietic stem cell transplant recipients.

BACKGROUND: Adenoviruses almost invariably proliferate in the gastrointestinal tract prior to dissemination, and critical threshold concentrations in stool correlate with the risk of viremia. Monitoring of adenovirus loads in stool may therefore be important for timely initiation of treatment in order to prevent invasive infection. OBJECTIVES: Comparison of a manual DNA extraction kit in combination with a validated in-house PCR assay with automated extraction on the NucliSENS-EasyMAG device coupled with the Adenovirus R-gene kit (bioMérieux) for quantitative adenovirus analysis in stool samples. STUDY DESIGN: Stool specimens spiked with adenovirus concentrations in a range from 10E2-10E11 copies/g and 32 adenovirus-positive clinical stool specimens from pediatric stem cell transplant recipients were tested along with appropriate negative controls. RESULTS: Quantitative analysis of viral load in adenovirus-positive stool specimens revealed a median difference of 0.5 logs (range 0.1-2.2) between the detection systems tested and a difference of 0.3 logs (range 0.0-1.7) when the comparison was restricted to the PCR assays only. Spiking experiments showed a detection limit of 102-103adenovirus copies/g stool revealing a somewhat higher sensitivity offered by the automated extraction. The dynamic range of accurate quantitative analysis by both systems investigated was between 103 and 108 virus copies/g. CONCLUSIONS: The differences in quantitative analysis of adenovirus copy numbers between the systems tested were primarily attributable to the DNA extraction method used, while the qPCR assays revealed a high level of concordance. Both systems showed adequate performance for detection and monitoring of adenoviral load in stool specimens.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia.

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.

Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.

Persistence and reactivation of human adenoviruses in the gastrointestinal tract.

Reactivation of persistent human adenoviruses (HAdVs) is associated with high morbidity and mortality in paediatric haematopoietic stem cell transplant (HSCT) recipients. Although invasive HAdV infections mainly arise from the gastrointestinal (GI) tract, the specific sites of HAdV persistence are not well characterised. We prospectively screened biopsies from 143 non-HSCT paediatric patients undergoing GI endoscopy and monitored serial stool specimens from 148 paediatric HSCT recipients for the presence of HAdV by real-time PCR. Persistence of HAdV in the GI tract was identified in 31% of children, with the highest prevalence in the terminal ileum. In situ hybridisation and immunohistochemistry identified HAdV persistence in lymphoid cells of the lamina propria, whereas biopsies from five transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI tract and reactivations post transplant indicated a correlation of intestinal HAdV shedding pre-transplant with high risk of invasive infection. HAdV persistence in the GI tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV persistence and reactivation, whereas the intestinal epithelium is the main site of viral proliferation preceding dissemination. The findings have important implications for assessing the risk of life-threatening invasive HAdV infections.

Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia.

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Ambulatory management of gastrointestinal emergencies: what are the current literature data?

INTRODUCTION: Ambulatory management is a modality of care defined in France by a hospitalization of less than 12h without an overnight stay. Currently, few data are available on its role in the management of gastrointestinal emergencies, such as appendectomy for acute appendicitis, cholecystectomy for acute cholecystitis or emergency proctologic surgery. The aim of this systematic review was to study the published data regarding the feasibility of ambulatory management of emergency visceral surgery and to enquire about the possibilities of further development of this form of management. MATERIALS AND METHODS: A literature search was conducted from the PubMed(®) databank taking into account all published data up to July 2013. RESULTS: For acute appendicitis, the success rate of short-stay hospitalization was 72% with unplanned read-mission rates ranging from 0 to 53%, a rate of unscheduled consultations ranging from 0 to 11%, and unplanned inpatient hospitalization rates ranging from 0% to 5%. For acute cholecystitis and proctology, there are few published data. CONCLUSION: Ambulatory management has been sparingly studied in the setting of gastrointestinal surgical emergencies. However, there is probably a place for development of this form of management.

Rapid identification of compound mutations in patients with Philadelphia-positive leukaemias by long-range next generation sequencing.

An emerging problem in patients with Philadelphia (Ph)-positive leukaemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukaemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukaemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. Polymerase chain reaction (PCR) amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease.

Species-specific identification of a wide range of clinically relevant fungal pathogens by the Luminex(®) xMAP technology.

Invasive fungal infections (IFI) are a common cause of life-threatening events in immunocompromised patients. Early detection and identification of the fungal pathogen is an important prerequisite for timely onset of the most appropriate treatment. Methods based on fungal culture are often too slow to be clinically useful. Other approaches to the identification of fungal species, including molecular techniques, are often restricted to a small number of the most commonly occurring pathogens and are therefore of limited use in the clinical setting. The development of assays for the detection and identification of a broad-range of clinically relevant fungal species is therefore an urgently needed step towards optimized diagnostics of IFI.The Luminex(®) xMAP technology offers a platform for the establishment of multiplex assays permitting high-throughput analysis of up to 100 different target molecules in a single test. Here we describe a Luminex(®)-based multiplex assay permitting rapid detection and identification of 10 fungal genera and 29 different species, including both commonly occurring and emerging fungal pathogens.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Quantitative monitoring of BCR/ABL1 mutants for surveillance of subclone-evolution, -expansion, and -depletion in chronic myeloid leukaemia.

BACKGROUND: In chronic myeloid leukaemia (CML), clonal evolution with resistance to tyrosine kinase inhibitors (TKIs) is often triggered by BCR/ABL1 mutations. However, in the context of the complex pro-oncogenic signalling networks which ultimately lead to clonal expansion and disease progression, the exact contribution of BCR/ABL1 mutants remains uncertain. Recent data indicate that detection of BCR/ABL1 mutant subclones does not permit prediction of their expansion dynamics and their potential to become drivers of resistant disease. METHODS: To determine the patterns of clonal evolution and the distinct proliferation kinetics of individual BCR/ABL1 mutants during treatment, we employed ligase-dependent polymerase chain reaction (LD-PCR) analysis for quantitative surveillance of CML subclones with various tyrosine kinase domain (TKD) mutations including M244V, L248V, G250E, E255K, T315I, F317L-A/G, M351T and F359V. FINDINGS: Inadequate treatment responses were observed in 27 of 100 patients investigated and 16 were found to bear one or more BCR/ABL1 TKD mutations in separate subclones. Rapid subclone expansion upon onset or switch of TKI treatment was common and sometimes preceded corresponding changes in BCR/ABL1 transcript levels. Mutant subclones were found to respond differentially and sometimes unexpectedly to various treatment modalities. Decline and persistent depletion of specific mutation-bearing subclones in response to treatment could be documented by LD-PCR surveillance. INTERPRETATION: The observations show that quantitative monitoring of mutant BCR/ABL1 subclones by LD-PCR is a powerful tool for detection of clonal evolution, subclone-expansion and subclone-depletion and can contribute to optimised management of patients with CML.

Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation.

Timely diagnosis of impending graft rejection is crucial for effective therapeutic intervention after allogeneic hematopoietic stem cell transplantation (SCT). We have investigated the predictive potential of early leukocyte subset-specific chimerism for graft loss in children undergoing SCT. In total, 192 pediatric patients transplanted for the treatment of malignant and non-malignant diseases after reduced-intensity or myeloablative conditioning were investigated. Surveillance of lineage-specific chimerism was initiated upon first appearance of leukocyte counts amenable to cell sorting. Graft rejection occurred in 23 patients between 24 and 492 days post-transplant (median 63 days). The first chimerism analysis of T and NK cells performed at a median of 20 days after SCT identified three different risk groups that were independent from the conditioning regimen: recipient chimerism (RC) levels in T cells below 50% indicated a very low risk of rejection (1.4%), whereas high levels of RC (>90%) both in T and NK cells heralded graft loss in the majority of patients (90%) despite therapeutic interventions. RC >50% in T cells and ≤90% in NK cells defined an intermediate-risk group in which timely immunotherapy frequently prevented rejection. Early assessment of T- and NK-cell chimerism can therefore be instrumental in the risk assessment and therapeutic management of imminent graft rejection.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats.

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients.

Invasive fungal disease (IFD) is a life-threatening event in immunocompromised patients, and there is an urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes. To assess the clinical potential of the assay, more than 600 consecutive specimens from 125 pediatric patients carrying a high risk of IFD were analyzed. An excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the European Organization for Research and Treatment of Cancer criteria was demonstrated, as revealed by the sensitivity of the assay of 96% (95% CI: 82-99%). The negative predictive value of the panfungal PCR assay presented was 98% (95% CI: 90-100%), while the specificity and the positive predictive value were 77% (95% CI: 66-85%) and 62% (95% CI: 47-75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic episodes by the assay presented could help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. Our observations raise the possibility that rapid species identification may be required to increase the positive predictive value for impending fungus-related disease.

Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

Species-specific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology.

In immunocompromised patients suffering from invasive fungal infection, rapid identification of the fungal species is a prerequisite for selection of the most appropriate antifungal treatment. We present an assay permitting reliable identification of a wide range of clinically relevant fungal pathogens based on the high-throughput Luminex microbead hybridization technology. The internal transcribed spacer (ITS2) region, which is highly variable among genomes of individual fungal species, was used to generate oligonucleotide hybridization probes for specific identification. The spectrum of pathogenic fungi covered by the assay includes the most commonly occurring species of the genera Aspergillus and Candida and a number of important emerging fungi, such as Cryptococcus, Fusarium, Trichosporon, Mucor, Rhizopus, Penicillium, Absidia, and Acremonium. Up to three different probes are employed for the detection of each fungal species. The redundancy in the design of the assay should ensure unambiguous fungus identification even in the presence of mutations in individual target regions. The current set of hybridization oligonucleotides includes 75 species- and genus-specific probes which had been carefully tested for specificity by repeated analysis of multiple reference strains. To provide adequate sensitivity for clinical application, the assay includes amplification of the ITS2 region by a seminested PCR approach prior to hybridization of the amplicons to the probe panel using the Luminex technology. A variety of fungal pathogens were successfully identified in clinical specimens that included peripheral blood, samples from biopsies of pulmonary infiltrations, and bronchotracheal secretions derived from patients with documented invasive fungal infections. Our observations demonstrate that the Luminex-based technology presented permits rapid and reliable identification of fungal species and may therefore be instrumental in routine clinical diagnostics.