Routine Clinical Diagnostics Research Articles

Real-Time Analysis and Visualization of Pathogen Sequence Data.

The rapid development of sequencing technologies has to led to an explosion of pathogen sequence data, which are increasingly collected as part of routine surveillance or clinical diagnostics. In public health, sequence data are used to reconstruct the evolution of pathogens, to anticipate future spread, and to target interventions. In clinical settings, whole-genome sequencing can identify pathogens at the strain level, can be used to predict phenotypes such as drug resistance and virulence, and can inform treatment by linking closely related cases. While sequencing has become cheaper, the analysis of sequence data has become an important bottleneck. Deriving interpretable and actionable results for a large variety of pathogens, each with its own complexity, from continuously updated data is a daunting task that requires flexible bioinformatic workflows and dissemination platforms. Here, we review recent developments in real-time analyses of pathogen sequence data, with a particular focus on the visualization and integration of sequence and phenotype data.

Immunohistochemical distinction of ABC and GCB in extranodal DLBCL is not reflected in mutation patterns

Gene expression profiling (GEP) separated diffuse large B-cell lymphoma (DLBCL) in two different entities, i.e. activated B cell-like (ABC) and germinal center B cell-like (GCB) lymphomas with ABC lymphomas demonstrating a less favorable outcome. NF-kB pathway activating mutations in MYD88, CD79A/B and CARD11 are predominantly found in ABC type lymphomas. Targeted therapies affecting NF-kB pathways have shown therapeutic potential in this subtype. Immunohistochemistry algorithms have been developed as a tool for distinguishing these entities in routine clinical diagnostics. To test whether this immunohistochemistry classifier would detect the biological differences between the entities 147 DLBCLs were subtyped into ABC and GCB using the Visco-Young algorithm. Mutation analysis demonstrated mutations in MYD88 or CD79 A/B in 21% (10/47) of non-GCB type but only in 3% (1/31) of GCB lymphomas (p = 0.012) in nodal lymphomas. In primary extra nodal lymphomas, however, 17.5% (4/23) of GCB type and 37.5% (15/40) of non-GCB lymphomas carried mutations in MYD88 and CD79 A/B. While the Visco-Young algorithm was sufficient to detect biological differences (i.e. mutation patterns) in nodal DLBCL it did not distinguish GCB and non-GCB type lymphomas of primary extranodal sites. Here, the morphological sites of the lymphomas seem to be more important for their molecular pattern than their immunohistochemical status.

The Role of Brain Connectome Imaging in the Estimation of Depressive Relapse Risk.

About two-thirds of all patients with major depressive disorder (MDD) suffer from depressive relapse, the mechanisms of which are still poorly understood. In recent years, analyses of the brain's connectome have increasingly been employed to identify potential biomarkers of depressive relapse. The term "connectome" refers to the map of all structural or functional connections in the brain. Itcan be investigated by structural or functional magnetic resonance imaging followed by graph theory-based analysis to characterize network topology on the global and regional level. This review is based on a selective literature search in PubMed representing the current state of research, as well as on an already published study which was awarded the Promotionspreis of the Deutsche Röntgengesellschaft. Numerous studies point to altered network topology, e. g., of default-mode network and striatum, as being crucial for the pathophysiology of MDD. Our group was able to show that striatal centrality (or hubness) is associated with the number of depressive episodes, which is one of the best predictors for depressive relapse. These data suggest aberrant striatal network topology as a potential biomarker for depressive relapse risk. The translation of these promising findings into clinical routine diagnostics is promoted by several methodological advantages, while some unresolved issues still hinder this process. · About two-thirds of all patients with MDD suffer from depressive relapse.. · The mechanisms of depressive relapse are still poorly understood.. · Imaging the brain's connectome can contribute to better understanding of depressive relapse.. · The term "connectome" comprises all structural and functional connections of the brain.. · Altered striatal network topology could be associated with depressive relapse risk.. · Brandl F, Meng C, Zimmer C et al. The Role of Brain Connectome Imaging in the Estimation of Depressive Relapse Risk. Fortschr Röntgenstr 2018; 190: 1036 - 1043.

ETIOLOGY AND CLINICO-MORPHOLOGICAL PRESENTATION OF MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS IN RUSSIAN POPULATION

THE AIM. Analysis of etiology, clinical and morphological manifestations, of membranoproliferative glomerulonephritis (MPGN).PATIENTS AND METHODS. Retrospective analysis focused on etiology, clinical presentation, light and electron microscopy, immunoglobulins (Ig) and C3 complement component (C3) deposits was done in the cohort of MPGN cases identified in the period 2000-2017. RESULTS. Two hundred and fourteen cases of MPGN were included in the study (mean age of 44 ± 16 years). Most patients had nephrotic syndrome and significant hematuria. In 58.4% of cases, eGFR was <60 mL/min/1.73 m2, and every fifth patient had CKD stages 4 or 5. The prevalence of MPGN among all biopsy-confirmed glomerulopathies was 9.3%. Idiopathic MPGN (iMPGN) was diagnosed in 30.4% of cases, while the proportion of iMPGN cases significantly reduced along the study period. Secondary MPGN (sMPGN) was identified in 69.6% of cases (autoimmune diseases – 34.1%, infectious diseases – 16.4%, monoclonal gammopathies – 9.3%, complement-mediated damage – 9.8%). Ig+C3+MPGN was mainly associated with autoimmune diseases and infections, however 26,6% of such cases remained “idiopathic”. C3-glomerulopathy or thrombotic microangiopathy were most often causes of Ig-C3+MPGN. Ig-C3-/Ig+C3-MPGN had heterogeneous etiology. CONCLUSION. MPGN is a severe variant of glomerular damage with a heterogeneous etiological structure. Targeted routine clinical and morphological diagnostics of MPGN allows identifying the cause of the disease in most cases. This approach is reliable for the adequate treatment choice and improvement of outcomes in sMPGN. Further improvement in diagnostic and classification approaches in idiopathic MPGN relies on progress in understanding of molecular etiopathogenesis of the disease.

Quantum Cascade Laser-Based Infrared Microscopy for Label-Free and Automated Cancer Classification in Tissue Sections

A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

Update on the types and usage of liquid biopsies in the clinical setting: a systematic review

BackgroundThis systematic review aimed to gather evidence from research on the current state of liquid biopsy in medical practice, specifically focusing on mutation detection and monitoring.MethodsA systematic search was performed via Medline.ResultsThe results of this investigation indicate that liquid biopsy plays a critical role in the detection and management of tumors. This technique gives healthcare providers the ability to gather critical and reliable information that may potentially shape the diagnosis, treatment, and prognosis of a variety of cancers in the near future. This study further reveals that liquid biopsy has several potential shortcomings that may limit its application and use in the healthcare setting. Nevertheless, liquid biopsy remains a valuable tool that is gradually becoming a part of routine healthcare practice in oncology departments and hospitals worldwide.ConclusionsThe evidence described herein reveals the potential relevance of liquid biopsy as an important prognostic, diagnostic, and theranostic tool. This non-invasive procedure enables healthcare practitioners to detect and monitor genomic alterations and will likely replace tumor tissue biopsy as the standard method for detecting and monitoring mutations in the future. The information obtained herein can enable physicians to make informed decisions regarding current treatment options; however, liquid biopsy has not yet been incorporated into routine clinical diagnostics for cancer patients.

Genome analysis of a MDR Streptococcus pneumoniae 23F serotype causing meningoencephalitis in a 10-months refugee infant.

Streptococcus pneumoniae is an important human pathogen causing invasive pneumococcal diseases (IPD). The re-emergence of eradicated S. pneumoniae-associated meningoencephalitis in Lebanon is a major point of concern. We aimed at conducting a comparative genome analysis of a multi-drug resistant S. pneumoniae, LAU-23F, linked to meningoencephalitis and fatality in a 10-months Syrian refugee infant in Lebanon, and 24 related publically available genome sequences. Serotype, capsular genes, MLST, SNPs, phylogenetic relatedness and repertoire of resistance genes were investigated. Genes encoding penicillin binding proteins (PBPs) were examined for mosaicity. Virulence factors were screened for SNPs as compared to reference strains. The isolate belonged to ST-277 and was of serotype 23F. It showed an intermediate resistance to ciprofloxacin, cefuroxime and penicillin and carried multiple components of different efflux pumps. Gene mosaicity was observed in pbp2x, it was also distinct from other penicillin-resistant strains; pbp1a and pbp2b appeared to be conserved between LAU-23F and the reference strain SP49. The arrangement of capsular gene loci was similar to ATCC 700669 though polymorphism was detected in the cpsABCD region, believed to be conserved among different Streptococcus species. Amplitude of virulence factors was detected showing varying degrees of conservation compared to reference strains. Observed zones of high heterogeneity were associated with phage encoded regions. The fine levels of diversity throughout the genome could account for the pronounced invasiveness of this isolate. The genomics-based methods used support the importance of implementing WGS in routine clinical diagnostics and surveillance of streptococcal diseases.

Development and evaluation of a culture-free microbiota profiling platform (MYcrobiota) for clinical diagnostics

Microbiota profiling has the potential to greatly impact on routine clinical diagnostics by detecting DNA derived from live, fastidious, and dead bacterial cells present within clinical samples. Such results could potentially be used to benefit patients by influencing antibiotic prescribing practices or to generate new classical-based diagnostic methods, e.g., culture or PCR. However, technical flaws in 16S rRNA gene next-generation sequencing (NGS) protocols, together with the requirement for access to bioinformatics, currently hinder the introduction of microbiota analysis into clinical diagnostics. Here, we report on the development and evaluation of an “end-to-end” microbiota profiling platform (MYcrobiota), which combines our previously validated micelle PCR/NGS (micPCR/NGS) methodology with an easy-to-use, dedicated bioinformatics pipeline. The newly designed bioinformatics pipeline processes micPCR/NGS data automatically and summarizes the results in interactive, but simple web reports. In order to explore the utility of MYcrobiota in clinical diagnostics, 47 clinical samples (40 “damaged skin” samples and 7 synovial fluids) were investigated using routine bacterial culture as comparator. MYcrobiota confirmed the presence of bacterial DNA in 37/37 culture-positive samples and detected bacterial taxa in 2/10 culture-negative samples. Moreover, 36/38 potentially relevant aerobic bacterial taxa and 3/3 mixtures of anaerobic bacteria were identified using culture and MYcrobiota, with the sensitivity and specificity being 95%. Interestingly, the majority of the 448 bacterial taxa identified using MYcrobiota were not identified using culture, which could potentially have an impact on clinical decision-making. Taken together, the development of MYcrobiota is a promising step towards the introduction of microbiota analysis into clinical diagnostic laboratories.

Rapid Detection of Copper in Biological Systems Using Click Chemistry.

A fast (1 min), straightforward but efficient, click chemistry-based system that enables the rapid detection of free copper (Cu) ions in either biological fluids or living cells without tedious pretreatment is provided. Cu can quickly induce the conjugation between graphene oxide (GO) and a fluorescent dye via click reaction. On the basis of the high specificity of bioorthogonal reaction and the effective quenching ability of GO, the assay studied in this paper can respond to Cu ions in less than 1 min with excellent selectivity and sensitivity, which is the fastest sensor for Cu as far as it is known. In addition, the application of this system is verified by performing assays in living cells and untreated urine samples from patients suffering from Wilson's Disease. Such a Cu detection system shows great promises in both fundamental research and routine clinical diagnostics.

Evolution of molecular genetic methods in the clinical diagnosis of hereditary endocrine tumour syndromes

The common features of hereditary endocrine tumour syndromes or multiple endocrine neoplasias (MEN) are the association of various tumours of different endocrine organs in one patient or within the same family. Different types can be distinguished from among which type 1 and type 2 are the most common. The mode of inheritance is autosomal dominant, meaning that there is a 50% chance to inherit the pathogenic alteration. The pathogenic variants of genes responsible for MEN syndromes have also been identified in sporadic endocrine tumours and many cases initially referred to as sporadic have been later categorized as familiar based on genetic analysis. The main role of the molecular genetic analysis in these syndromes is to identify the pathogenic variant, then, after appropriate genetic counseling, to perform the genetic screening of first-degree relatives. Following molecular genetic analysis, the state-of-the-art clinical follow-up of the clinically healthy mutation carriers may decrease or even prevent the morbidity and mortality. Due to technological developments in recent years, the molecular genetic analysis of hereditary tumour syndromes has also been changed. Using next generation based sequencing methods in routine clinical diagnostics, the number of pathogenic genes in endocrine tumours has also increased. The present review focuses on the genetic background of hereditary endocrine tumour syndromes and the recently used molecular biological methods will also be presented. Orv Hetil. 2018; 159(7): 285-292.

Serum Testosterone by Liquid Chromatography Tandem Mass Spectrometry for Routine Clinical Diagnostics.

In clinical diagnostics, samples containing low testosterone cannot be analyzed by random access immunoassays normally available at clinical laboratories. For these samples, sensitive and specific LC-MS/MS-based testosterone methods are required. An LC-MS/MS-based testosterone assay is described that was developed and validated for routine clinical application.

Role of Genetic Variations in Determining Treatment Outcome in Head and Neck Cancer

Worldwide, head and neck squamous cell carcinoma (HNSCC) is responsible for >550,000 diagnoses and 380,000 deaths annually. It originates in the upper aerodigestive tract and has a multifactorial origin involving both genetic and lifestyle risk factors. The clinical management of HNSCC involves surgery, radiotherapy, and chemotherapy. Several studies point to the role of genetic variations in predicting drug efficacy and toxicity. Cancer pharmacogenomics has fast emerged as a new and promising field for the early identification of genetic markers that can predict drug response or toxicity, with the number of studies of genetic polymorphisms as prognostic factors of HNSCC treatment outcomes growing. The number of studies evaluating the association of candidate polymorphisms in drug-metabolising Phase I and II enzymes with treatment outcome far exceed the studies involving other candidate genes, such as those involved in drug metabolism, DNA repair, and cell cycle regulation. This review focusses on the relevance of genetic variations in genes, where the corresponding gene products play an important role in drug metabolism (TPMT, DPD), DNA repair (X-ray repair cross complementing 1), cell cycle (tumour protein P53), and carcinogenesis (matrix metalloproteinase 3 and 7), thereby contributing to the treatment outcome for HNSCC. This could greatly help clinicians in identifying genetic markers useful for the selection of optimal drugs, dose, and treatment duration on an individual basis, resulting in improved drug efficacy and decreased toxicity. However, further studies are needed in well characterised and larger HNSCC populations with proper validation of pharmacogenetic markers in experimental settings before application in clinical routine diagnostics.

Next generation sequencing: clinicalapplications in solid tumours.

SummaryNext generation sequencing (NGS) has unravelled the genetic alterations that underlie the pathogenesis of cancer. It is now becoming integrated into routine clinical diagnostics of malignant tumours. NGS supports diagnosis, identifies therapeutic targets, reveals resistance mechanisms and facilitates disease monitoring. It takes a central function in the implementation of cancer therapies adapted to the molecular alterations of tumours.

Rapid identification of staphylococci by Raman spectroscopy

Clinical treatment of the infections caused by various staphylococcal species differ depending on the actual cause of infection. Therefore, it is necessary to develop a fast and reliable method for identification of staphylococci. Raman spectroscopy is an optical method used in multiple scientific fields. Recent studies showed that the method has a potential for use in microbiological research, too. Our work here shows a possibility to identify staphylococci by Raman spectroscopy. We present a method that enables almost 100% successful identification of 16 of the clinically most important staphylococcal species directly from bacterial colonies grown on a Mueller-Hinton agar plate. We obtained characteristic Raman spectra of 277 staphylococcal strains belonging to 16 species from a 24-hour culture of each strain grown on the Mueller-Hinton agar plate using the Raman instrument. The results show that it is possible to distinguish among the tested species using Raman spectroscopy and therefore it has a great potential for use in routine clinical diagnostics.

Gastric cancer: immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics

Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.

Macrolide Resistance in Mycoplasma genitalium in Singapore

Background Mycoplasma genitalium was first reported as a cause of non-gonococcal urethritis in 1980. It has progressed from being an ‘emerging’ sexually transmitted infection (STI) to an accepted STI. Prevalence of infection has been reported as the Netherlands 4.5%, Sweden 6.3%, UK 1.2% and France 4%. M. genitalium has the smallest known bacterial genome and was the second bacterial genome fully sequenced. It has minimal requirements and is said to approach the minimum possible for a living cell. It is extremely fastidious; only a few strains have been cultured worldwide. Diagnosis relies on direct detection. It does not have a cell wall so it is not susceptible to antibiotics such as penicillins and cephalosporins. Therapy depends on fluoroquinolones and macrolides but resistance to macrolides has been widely reported: 13% France, 18% Sweden, 40% UK, Australia and Denmark, 100% Greenland, 30% Japan.Methods Ethics approval was granted. DNA extracts left over after routine clinical diagnostics at the Department of STI Control (DSC) Clinic, Kelantan Lane, Singapore were harvested. DNA had been extracted on a Cobas 4800 instrument (Roche) from urine and urethral swabs collected for testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). A 2-plex real-time PCR assay targeting the pdhD and mgpB genes was used to screen for M. genitalium. Samples were deemed positive if both targets were detected. If only one target was detected, the sample was retested; if reactive in either target upon retest, the sample was considered positive for M. genitalium. Positive DNA preps were then screened for macrolide resistance mutations after Sanger sequencing of the 23S rRNA gene.Results368 anonymised DNA elutes from 254 urines and 114 urethral swabs were collected between May and July 2016. One hundred eighty-four were CT/NG positive and 184 were CT/NG negative. Sixteen (4.3%) were positive for M. genitalium. Four (25%) of these 16 samples contained macrolide resistance associated mutations; A2058T (x2), A2058G (x1), and A2059G (x1).Conclusion M. genitalium was detected in 4.3% of samples. Macrolide resistance mutations were detected in 25%, similar to international rates. Some guidelines recommend testing for resistance to guide therapy and to perform a test of cure.Disclosures All authors: No reported disclosures.

Rapid Targeted Genomics in Critically Ill Newborns

Rapid diagnostic whole-genome sequencing has been explored in critically ill newborns, hoping to improve their clinical care and replace time-consuming and/or invasive diagnostic testing. A previous retrospective study in a research setting showed promising results with diagnoses in 57%, but patients were highly selected for known and likely Mendelian disorders. The aim of our prospective study was to assess the speed and yield of rapid targeted genomic diagnostics for clinical application. We included 23 critically ill children younger than 12 months in ICUs over a period of 2 years. A quick diagnosis could not be made after routine clinical evaluation and diagnostics. Targeted analysis of 3426 known disease genes was performed by using whole-genome sequencing data. We measured diagnostic yield, turnaround times, and clinical consequences. A genetic diagnosis was obtained in 7 patients (30%), with a median turnaround time of 12 days (ranging from 5 to 23 days). We identified compound heterozygous mutations in the EPG5 gene (Vici syndrome), the RMND1 gene (combined oxidative phosphorylation deficiency-11), and the EIF2B5 gene (vanishing white matter), and homozygous mutations in the KLHL41 gene (nemaline myopathy), the GFER gene (progressive mitochondrial myopathy), and the GLB1 gene (GM1-gangliosidosis). In addition, a 1p36.33p36.32 microdeletion was detected in a child with cardiomyopathy. Rapid targeted genomics combined with copy number variant detection adds important value in the neonatal and pediatric intensive care setting. It led to a fast diagnosis in 30% of critically ill children for whom the routine clinical workup was unsuccessful.

Author response: The autism “epidemic”: Ethical, legal, and social issues in a developmental spectrum disorder

We thank Machado et al. for the comments on our review,1 and for the observations of abnormal anatomic and functional connectivity using diffusion-weighted MRI (DW-MRI). Innumerable studies have compared patients with autism spectrum disorders (ASD) and nonaffected controls at rest or while performing particular tasks, most often in small selected subject samples. For example, studies linked autism with the retino-collicular magnocellular visual pathways that project via the pulvinar to the amygdala, occipital V1, and multiple other areas, including the dorsal occipito-parietal/midtemporal streams, which participate with the frontal eye fields in processing spatial attention, moving targets, and dynamic facial expression (with inputs from the amygdala and other limbic pathways).2 However, no results consistently demonstrated specific neural networks that are uniquely pathognomonic of autism in the brain despite the fact that broad networks were linked with deficient social cognition in autism.3 Complex behaviors, like ASD, do not have a single endophenotype linked to one discrete neocortical location.4 The critical need for more research in autism should be balanced by caution about generalizing interpretations of neuroimaging findings that suggest the dysregulation of specific neural pathways. At present, DW-MRI and similar techniques, such as diffusion compartment imaging, are considered beneficial for research but not for routine clinical diagnostics.

Whole-Genome Sequencing of Human Clinical Klebsiella pneumoniae Isolates Reveals Misidentification and Misunderstandings of Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae.

Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. K.variicola and K.quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K.pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K.pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K. variicola and K.quasipneumoniae. Whole-genome sequencing of 95 additional non-ESBL-producing K.pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K.pneumoniae, suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K.variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella. IMPORTANCEKlebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K.variicola and K.quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K.variicola and K. quasipneumoniae isolates are often misidentified as K.pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K.pneumoniae. The presence of KPC in K.variicola and K.quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K.variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene

Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in...