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.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region.

The rapid identification of fungal pathogens in clinical specimens is a prerequisite for timely onset of the most appropriate treatment. The aim of the present study was to develop a sensitive and rapid method for the species-specific identification of clinically relevant fungi. We employed fluorescent polymerase chain reaction (PCR)-fragment length analysis of the highly variable internally transcribed spacer 2 (ITS2) region to identify individual fungal species by their specific amplicon sizes. The specificity of the technique was ascertained by the detailed analysis of 96 strains derived from 60 different human-pathogenic fungal species. To achieve adequate sensitivity for species identification in patients with invasive fungal infection, who often display very low pathogen loads in peripheral blood, the ITS2 region was amplified by semi-nested PCR prior to amplicon-length analysis. Serial specimens from 26 patients with documented fungal infections were investigated. The fungal pathogens identified included different Aspergillus and Candida species, Rhizopus oryzae and Fusarium oxysporum. Fragment length analysis of the ITS2 region upon amplification by semi-nested PCR permits the sensitive identification of fungal species. The technique can be readily implemented in routine diagnostics.

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.

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.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Importance of allogeneic T-cells for disease control after stem cell transplantation for high-risk Langerhans cell histiocytosis.

Reduced intensity conditioning followed by allogeneic SCT (RIC-SCT) has recently emerged as promising new salvage option for children suffering from Langerhans cell histiocytosis (LCH) with risk organ involvement and failure to conventional therapy. We report on the posttransplant course of female toddler with high-risk LCH, who achieved complete remission after RIC-SCT, despite a posttransplant chimerism constellation, in which only the T-cell subset proved to be of donor origin in the long-term. We therefore suggest that allogeneic T-cells have played a crucial role in controlling disease activity in this patient and may exert the major curative effect after RIC-SCT for LCH.

Detection and monitoring of virus infections by real-time PCR.

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site.

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.

Absence of N-ras mutations in myeloid and lymphoid blast crisis of chronic myeloid leukemia.

Mutations within N-ras oncogene codons 12, 13, and 61 occur in approximately 25-30% of patients with acute nonlymphocytic leukemia and at a lower frequency (6-20%) in patients with acute lymphocytic leukemia. Moreover, N-ras mutations have been described in patients with chronic myeloid leukemia (CML) in blast crisis but have not been observed during the chronic phase of the disease. In view of the morphological and clinical similarities between acute leukemia and the blast crisis of CML, the question was raised whether the presence of N-ras mutations is associated with the phenotype of acute leukemia. We investigated leukemic cells from 100 patients with CML for the presence of N-ras mutations in the mutational hot spot codons. The cases analyzed included 87 diagnosed with different types of blast crisis and 13 cases in accelerated or chronic phase of the disease. Fragments from N-ras exons I and II containing the codons of interest were amplified by polymerase chain reaction and analyzed for the presence of point mutations by three different technical approaches, including specific oligonucleotide hybridization, direct sequencing, and single-strand conformation polymorphism analysis. N-ras mutations were not detected in any of the CML patients investigated. Only one patient, in whom the initial diagnosis of CML-blast crisis had been revised to chronic myelomonocytic leukemia, displayed an N-ras mutation within codon 13. Our data strongly suggest that N-ras mutations do not play a role in myeloid or lymphoid blast crisis of CML.

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.

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.

Current recommendations for positive controls in RT-PCR assays.

The choice of adequate controls for reverse transcriptase (RT-) PCR analysis has been the focus of a debate pursued in Leukemia over the past 3 years. Twenty-six authors from 15 different centers contributed to the Debate, and the points presented have been carefully evaluated. This survey reviews the issues discussed, and presents current options for appropriate positive controls in RT-PCR assays which are based on the views shared by the majority of participants in the Debate. It is understood, however, that the recommendations presented cannot be regarded as definitive guidelines. They reflect the present state of knowledge, and certainly need to be revisited.

IL-2 inhibits proliferation of K562 cells and reduces accumulation of bcr/abl mRNA and oncoprotein.

Cell lines of myeloid origin have been shown to express interleukin-2 receptors (IL-2R). Here, we demonstrate the expression of IL-2R alpha and IL-R beta on the CML blast cell line K562 by FACS analysis and cross-linking assay. Furthermore, we examined the effect of IL-2 on leukemic progenitor growth, employing K562 as a model. Clonogenic growth was assessed after 3 days of culture by colony formation in a serum-free, semi-solid assay system. IL-2 was found to exhibit a dose-dependent suppressive effect on K562 clonogenicity with 48% inhibition of colony formation at 250 U IL-2 and 60% inhibition at 1000 U IL-2. Philadelphia chromosome (Ph)-positive K562 cells possess multiple copies of the bcr/abl fusion gene whose transcript and protein product (p210) is thought to confer growth advantage to CML cells. We therefore investigated IL-2-dependent modulation of bcr/abl mRNA accumulation and p210 protein levels in K562 cells. After 4 h of culture in the presence of IL-2, a 3-15-fold reduction of bcr/abl mRNA accumulation was demonstrated by competitive reverse PCR. Reduction of bcr/abl fusion protein levels was demonstrated at 24 h of IL-2-supplemented cell culture, employing p210 recognizing monoclonal antibodies (mAbs) in FACS analysis. Levels of proliferation marker Ki67 were only marginally affected. We conclude: (1) K562 cells express both IL-2R alpha and IL-R beta; (2) IL-2 inhibits clonogenic growth of K562 in a dose-dependent manner; and (3) IL-2-mediated inhibition of K562 proliferation is preceded by a reduction of bcr/abl mRNA accumulation and p210 protein levels.

Non-radioactive detection of the rearranged BCR/ABL sequences amplified by polymerase chain reaction.

The polymerase chain reaction (PCR) is a powerful technique for the detection of the bcr/abl rearrangement in chronic myelogenous leukemia (CML). It allows the exponential amplification of the rearranged region, thus facilitating its detection. The specificity of the bcr/abl cDNA sequence amplified by PCR is most commonly verified by hybridization to a 32P-labeled probe. In this paper, an assay for the colorimetric detection of the amplified bcr/abl fragment is described, which offers several advantages over the use of radioactive probes. We adapted the PCR for synthesis and simultaneous labeling of DNA fragments with a non-radioactive steroid compound called digoxigenin. This labeling procedure was used to generate a digoxigenin-labeled internal probe for the chimeric bcr/abl mRNA. The assay described is based on the hybridization of the amplified bcr/abl sequence to the non-radioactively labeled probe and on the subsequent detection by an enzyme-linked immunoassay and enzyme-catalyzed color reaction. Using this protocol, we investigated 20 patients with CML along with six healthy individuals and two cell lines derived from patients with CML for the presence of the bcr/abl rearrangement. It is shown that the assay is both highly sensitive and specific and that it is readily applicable to the routine diagnosis of CML. In addition, the assay could be adapted to a number of clinical diagnostic uses.

Use of quantitative polymerase chain reaction to monitor residual disease in chronic myelogenous leukemia during treatment with interferon.

Interferon-alpha (IFN-alpha) has become a widely used treatment modality in chronic myelogenous leukemia (CML) and was shown to induce complete hematologic responses in about 70% of the patients. In a minority of cases, complete suppression of the Philadelphia (Ph)-positive clone has been observed by cytogenetic investigation or by Southern blot analysis. In most instances, however, analyses by the highly sensitive two-step polymerase chain reaction (PCR) reveal the presence of residual leukemic cells despite continuous treatment. Since PCR positivity has not been associated with an increased risk of disease recurrence, the monitoring of cells carrying the characteristic BCR/ABL rearrangement by qualitative PCR may not facilitate early identification of patients who are likely to relapse. We have therefore employed a quantitative PCR technique to monitor the BCR/ABL mRNA expression levels during the course of treatment in an attempt to assess the response to IFN-alpha at the molecular level and to provide a basis for early detection of progressive disease. Twenty CML patients who received therapy with IFN-alpha in first chronic phase of the disease were enrolled in the study. In addition, we have monitored two CML patients treated with IFN-alpha for relapse after bone marrow transplantation. Thirteen patients who displayed decreasing, constant or fluctuating levels of BCR/ABL expression during an observation period of up to 4 years (mean 25 months) have remained in hematologic remission. Two patients showed an elevation in the marker gene expression upon discontinuation of treatment, but no further increase in BCR/ABL mRNA has been observed after reinitiation of therapy with IFN, and the patients have remained in hematologic remission. In seven patients, quantitative PCR (Q-PCR) analyses revealed increasing expression of the chimeric gene during treatment with IFN-alpha. In all seven cases, the detection of elevated BCR/ABL transcripts by quantitative PCR preceded signs of hematologic or cytogenetic disease progression by up to 8 months (median 6 months). Our data show that quantitative PCR analysis facilitates the monitoring of the response to IFN-alpha therapy and provides an effective diagnostic tool for the timely detection of recurrent disease. The employment of this technique greatly enhances the diagnostic possibilities in the management of chronic myelogenous leukemia.

Monitoring of residual disease in chronic myelogenous leukemia by quantitative polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard method for highly sensitive detection of the bcr/abl rearrangement in patients with chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). The exquisite sensitivity of the PCR facilitates the detection of residual leukemic cells after chemotherapy or after bone marrow transplantation. However, the detection of minimal residual disease does not yield any information on the malignant potential of the bcr/abl-rearranged cells. Qualitative PCR is therefore of limited prognostic value in the monitoring of residual leukemia. We have adapted the PCR for quantitative evaluation of cells carrying the bcr/abl rearrangement and by means of two exemplary cases of CML patients after bone marrow transplantation and under treatment with alpha-interferon, respectively, we show that this new technique is suitable for the long term follow-up of the activity of the residual bcr/abl rearranged clone. Longitudinal monitoring of residual disease by the technique presented provides a novel tool for detection of incipient relapse at a very early stage.

Frequent detection of BCR-ABL specific mRNA in patients with chronic myeloid leukemia (CML) following allogeneic and syngeneic bone marrow transplantation (BMT).

We performed a two-step polymerase chain reaction (PCR) to detect bcr-abl-specific mRNA in 440 peripheral blood and/or bone marrow samples of 30 chronic myeloid leukemia (CML) patients (mean 15, range 2-50 samples) following non T-cell-depleted allogeneic (n = 28) or syngeneic (n = 2) bone marrow transplantation (BMT). Median follow-up after BMT is 40 months (range 2-116 months), the median observation time 29 months (range 2-40 months). In 15 patients (50%), bcr-abl-specific mRNA could be detected following BMT. Bcr-abl positivity was rare in patients who were in hematological remission for at least 40 months (2/11). In five patients, PCR positivity was observed only once; all five patients are in complete hematological remission. Ten patients showed bcr-abl specific mRNA in two or more consecutive samples. Hematological relapse occurred in five of the latter patients. Bcr-abl positivity preceded hematological relapse in all cases. Bcr-abl positivity was detected more frequently in patients without graft-versus-host disease (GVHD) (11/15), than in patients with GVHD (4/15) (p < 0.02). Our data indicate that transient bcr-abl positivity is not usually followed by hematological relapse, while patients, who are positive in serial samples have a high risk of relapse.