Diagnostic Accuracy of the HemoCue Hb 301, STAT-Site MHgb and URIT-12 Point-of-Care Hemoglobin Meters in a Central Laboratory and a Community Based Clinic in Durban,South Africa

In South Africa, various point-of-care hemoglobin meters are used. However, the regulatory framework for approval, implementation and oversight of use of point-of-care hemoglobin meters is suboptimal. We assessed the diagnostic accuracy of the HemoCue Hb 301, STAT-Site M^Hgb and URIT-12 point-of-care hemoglobin meters, compared to a central laboratory based reference assay, in a central laboratory and a community based clinic in Durban, South Africa. Differences in performance of the point-of-care assays, compared to the reference assay, were more pronounced in the community based clinic. Results were reasonable for the HemoCue Hb 301, but poor for the STAT-Site M^Hgb and the URIT-12. Poor test performance of point-of-care hemoglobin meters, and inadequate evaluations and oversight in South Africa, leads to suboptimal clinical care and clinical research, and increased costs. There is a need for proper evaluation and quality assurance of point-of-care tests, the results of which should be made widely available to key stakeholders.     

DeNovoCNN: a deep learning approach to de novo variant calling in next generation sequencing data

De novo mutations (DNMs) are an important cause of genetic disorders. The accurate identification of DNMs from sequencing data is therefore fundamental to rare disease research and diagnostics. Unfortunately, identifying reliable DNMs remains a major challenge due to sequence errors, uneven coverage, and mapping artifacts. Here, we developed a deep convolutional neural network (CNN) DNM caller (DeNovoCNN), that encodes the alignment of sequence reads for a trio as 160164 resolution images. DeNovoCNN was trained on DNMs of 5616 whole exome sequencing (WES) trios achieving total 96.74% recall and 96.55% precision on the test dataset. We find that DeNovoCNN has increased recall/sensitivity and precision compared to existing DNM calling approaches (GATK, DeNovoGear, DeepTrio, Samtools) based on the Genome in a Bottle reference dataset and independent WES and WGS trios. Validations of DNMs based on Sanger and PacBio HiFi sequencing confirm that DeNovoCNN outperforms existing methods. Most importantly, our results suggest that DeNovoCNN is likely robust against different exome sequencing and analyses approaches, thereby allowing the application on other datasets. DeNovoCNN is freely available as a Docker container and can be run on existing alignment (BAM/CRAM) and variant calling (VCF) files from WES and WGS without a need for variant recalling.     

Relevance of laboratory testing for the diagnosis of primary immunodeficiencies: a review of case-based examples of selected immunodeficiencies

The field of primary immunodeficiencies (PIDs) is one of several in the area of clinical immunology that has not been static, but rather has shown exponential growth due to enhanced physician, scientist and patient education and awareness, leading to identification of new diseases, new molecular diagnoses of existing clinical phenotypes, broadening of the spectrum of clinical and phenotypic presentations associated with a single or related gene defects, increased bioinformatics resources, and utilization of advanced diagnostic technology and methodology for disease diagnosis and management resulting in improved outcomes and survival. There are currently over 200 PIDs with at least 170 associated genetic defects identified, with several of these being reported in recent years. The enormous clinical and immunological heterogeneity in the PIDs makes diagnosis challenging, but there is no doubt that early and accurate diagnosis facilitates prompt intervention leading to decreased morbidity and mortality. Diagnosis of PIDs often requires correlation of data obtained from clinical and radiological findings with laboratory immunological analyses and genetic testing. The field of laboratory diagnostic immunology is also rapidly burgeoning, both in terms of novel technologies and applications, and knowledge of human immunology. Over the years, the classification of PIDs has been primarily based on the immunological defect(s) ("immunophenotype") with the relatively recent addition of genotype, though there are clinical classifications as well. There can be substantial overlap in terms of the broad immunophenotype and clinical features between PIDs, and therefore, it is relevant to refine, at a cellular and molecular level, unique immunological defects that allow for a specific and accurate diagnosis. The diagnostic testing armamentarium for PID includes flow cytometry - phenotyping and functional, cellular and molecular assays, protein analysis, and mutation identification by gene sequencing. The complexity and diversity of the laboratory diagnosis of PIDs necessitates many of the above-mentioned tests being performed in highly specialized reference laboratories. Despite these restrictions, there remains an urgent need for improved standardization and optimization of phenotypic and functional flow cytometry and protein-specific assays. A key component in the interpretation of immunological assays is the comparison of patient data to that obtained in a statistically-robust manner from age and gender-matched healthy donors. This review highlights a few of the laboratory assays available for the diagnostic work-up of broad categories of PIDs, based on immunophenotyping, followed by examples of disease-specific testing.     

Requirements for reference (calibration) laboratories in laboratory medicine

In addition to reference measurement procedures and reference materials, reference or calibration laboratories play an integral role in the implementation of measurement traceability in routine laboratories. They provide results of measurements using higher-order methods, e.g. isotope dilution mass spectrometry and may assign values to materials to be used for external quality assessment programs and to secondary reference materials. The requirements for listing of laboratories that provide reference measurement services include a statement of the metrological level or principle of measurement, accreditation as a calibration laboratory according to ISO 15195 and the participation in a proficiency testing system (regular inter-laboratory comparisons) for reference laboratories. Ring trials are currently conducted for thirty well-defined measurands and the results are made available to all laboratories. Through the use of reference laboratory services that are listed by the Joint Committee for Traceability in Laboratory Medicine there is the opportunity to further promote traceability and standardisation of laboratory measurements.     

Molecular diagnostics of medically important bacterial infections

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world's biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. In developed countries, the emergence of new, rare or already-forgotten infectious diseases, such as HIV/AIDS, Lyme disease and tuberculosis, has stimulated public interest and inspired commitments to surveillance and control. Recently, it is reported that infectious diseases are responsible for more than 17 million deaths worldwide each year, most of which are associated with bacterial infections. Hence, the control of infectious diseases control is still an important task in the world. The ability to control such bacterial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory. Diagnostic medical bacteriology consists of two main components namely identification and typing. Molecular biology has the potential to revolutionise the way in which diagnostic tests are delivered in order to optimise care of the infected patient, whether they occur in hospital or in the community. Since the discovery of PCR in the late 1980s, there has been an enormous amount of research performed which has enabled the introduction of molecular tests to several areas of routine clinical microbiology. Molecular biology techniques continue to evolve rapidly, so it has been problematic for many laboratories to decide upon which test to introduce before that technology becomes outdated. However the vast majority of diagnostic clinical bacteriology laboratories do not currently employ any form of molecular diagnostics but the use such technology is becoming more widespread in both specialized regional laboratories as well as in national reference laboratories. Presently molecular biology offers a wide repertoire of techniques and permutations of these analytical tools, hence this article wishes to explore the application of these in the diagnostic laboratory setting.     

Comparison of clinical parameters with whole exome sequencing analysis results of autosomal recessive patients; a center experience

Whole-exome sequencing (WES) is an ideal method for the diagnosis of autosomal recessive diseases. The aim of this study was to evaluate the diagnostic power of WES in patients with autosomal recessive inheritance and to determine the relationship between genotype and phenotype. Retrospective screenings of 24 patients analysed with WES were performed and clinical and genetic data were evaluated. Any pathogenic mutation that could explain the suspected disease in 4 patients was not identified. A homozygous pathogenic mutation was detected in 18 patients. 2 patients had heterozygous mutations. According to this study results, WES is a successful technique to be used at the stage of diagnosis in patients who are accompanied by various degrees of intellectual disability matching the inheritance of the autosomal recessive.

     

Next generation sequencing reveals novel homozygous frameshift in PUS7 and splice acceptor variants in AASS gene leading to intellectual disability,developmental delay,dysmorphic feature and microcephaly

Intellectual developmental disorder with abnormal behavior, microcephaly and short stature (IDDABS), (OMIM# 618342) is an autosomal recessive condition described as developmental delay, poor or absent speech, intellectual disability, short stature, mild to progressive microcephaly, delayed psychomotor development, hyperactivity, seizure, along with mild to swear aggressive behavior. Homozygous frameshift mutation in Pseudouridine Synthase 7, Putative; (PUS7) OMIM# 616,261 NM_019042.3 and splice acceptor variants in Alpha-Aminoadipic Semialdehyde Synthase; (AASS) OMIM# 605,113 NM_005763.3 was funded. Whole exome sequencing (WES) technique was used as tool to identify the molecular diagnostic test. Different bioinformatics analysis done for WES data and we identified two novel mutations one as frameshift mutation c.606_607delGA, p.Ser282CysfsTer9 in the PUS7 gene and splice acceptor variants c.1767–1 G > A in the AASS gene has been reported. The pattern of family segregation maintained the pathogenicity of this variation associated with abnormal behavior, intellectual developmental disorder, microcephaly along with short stature IDDABS. Further, the WES data was validated in the family having other affected individuals and healthy controls (n = 100) was done using Sanger sequencing. Finally, our results further explained the role of WES in the disease diagnosis and elucidated that the mutation in PUS7 and AASS genes may lead an important role for the development of IDDABS in Saudi family.     

The present and future of whole-exome sequencing in studying and treating human reproductive disorders

The causes of recurrent spontaneous abortion (RSA) and fetal malformations are multifactorial and unclear in most cases. Environmental, maternal, and genetic factors have been shown to contribute to these defects. Whole-exome sequencing (WES) is widely used to detect genetic variations associated with human diseases and has recently been successfully applied to unveil genetic causes of unexplained recurrent spontaneous abortion (URSA) and fetal malformations. Here, we review the current discovery and diagnosis strategies to identify the underlying pathogenic mutations of URSA and fetal malformations using WES technology and propose to further develop WES, both to advance our understanding of these diseases and to eventually lead to targeted therapies for reproductive disorders.     

Analysis of Downs syndrome with molecular techniques for future diagnoses

Down syndrome (DS) is a genetic disorder appeared due to the presence of trisomy in chromosome 21 in the G-group of the acrocentric region. DS is also known as non-Mendelian inheritance, due to the lack of Mendel’s laws. The disorder in children is identified through clinical symptoms and chromosomal analysis and till now there are no biochemical and molecular analyses. Presently, whole exome sequencing (WES) has largely contributed in identifying the new disease-causing genes and represented a significant breakthrough in the field of human genetics and this technique uses high throughput sequencing technologies to determine the arrangement of DNA base pairs specifying the protein coding regions of an individual’s genome. Apart from this next generation sequencing and whole genome sequencing also contribute for identifying the disease marker. From this review, the suggestion was to perform the WES is DS children to identify the marker region.     

Network-based cancer genomic data integration for pattern discovery

Background

Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency.

Results

Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%.

Conclusions

For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

     

A new tool for prioritization of sequence variants from whole exome sequencing data

Background

Whole exome sequencing (WES) has provided a means for researchers to gain access to a highly enriched subset of the human genome in which to search for variants that are likely to be pathogenic and possibly provide important insights into disease mechanisms. In developing countries, bioinformatics capacity and expertise is severely limited and wet bench scientists are required to take on the challenging task of understanding and implementing the barrage of bioinformatics tools that are available to them.

Results

We designed a novel method for the filtration of WES data called TAPER? (Tool for Automated selection and Prioritization for Efficient Retrieval of sequence variants).

Conclusions

TAPER? implements a set of logical steps by which to prioritize candidate variants that could be associated with disease and this is aimed for implementation in biomedical laboratories with limited bioinformatics capacity. TAPER? is free, can be setup on a Windows operating system (from Windows 7 and above) and does not require any programming knowledge. In summary, we have developed a freely available tool that simplifies variant prioritization from WES data in order to facilitate discovery of disease-causing genes.

     

The role of laboratory diagnostics in emerging viral infections: the example of the Middle East respiratory syndrome epidemic

Rapidly emerging infectious disease outbreaks place a great strain on laboratories to develop and implement sensitive and specific diagnostic tests for patient management and infection control in a timely manner. Furthermore, laboratories also play a role in real-time zoonotic, environmental, and epidemiological investigations to identify the ultimate source of the epidemic, facilitating measures to eventually control the outbreak. Each assay modality has unique pros and cons; therefore, incorporation of a battery of tests using traditional culture-based, molecular and serological diagnostics into diagnostic algorithms is often required. As such, laboratories face challenges in assay development, test evaluation, and subsequent quality assurance. In this review, we describe the different testing modalities available for the ongoing Middle East respiratory syndrome (MERS) epidemic including cell culture, nucleic acid amplification, antigen detection, and antibody detection assays. Applications of such tests in both acute clinical and epidemiological investigation settings are highlighted. Using the MERS epidemic as an example, we illustrate the various challenges faced by laboratories in test development and implementation in the setting of a rapidly emerging infectious disease. Future directions in the diagnosis of MERS and other emerging infectious disease investigations are also highlighted.     

Next-generation sequencing for cancer diagnostics: a practical perspective

Next-generation sequencing (NGS) is arguably one of the most significant technological advances in the biological sciences of the last 30 years. The second generation sequencing platforms have advanced rapidly to the point that several genomes can now be sequenced simultaneously in a single instrument run in under two weeks. Targeted DNA enrichment methods allow even higher genome throughput at a reduced cost per sample. Medical research has embraced the technology and the cancer field is at the forefront of these efforts given the genetic aspects of the disease. World-wide efforts to catalogue mutations in multiple cancer types are underway and this is likely to lead to new discoveries that will be translated to new diagnostic, prognostic and therapeutic targets. NGS is now maturing to the point where it is being considered by many laboratories for routine diagnostic use. The sensitivity, speed and reduced cost per sample make it a highly attractive platform compared to other sequencing modalities. Moreover, as we identify more genetic determinants of cancer there is a greater need to adopt multi-gene assays that can quickly and reliably sequence complete genes from individual patient samples. Whilst widespread and routine use of whole genome sequencing is likely to be a few years away, there are immediate opportunities to implement NGS for clinical use. Here we review the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead.     

基于全外显子组数据的ABO血型的基因判定

全外显子组测序研究已经应用在疾病、药物等方面,是临床研究中一种辅助分子诊断方法。该方法也逐步应用在临床ABO血型的判定中,由于目前临床血型判定的主要方法是血清学,疑难样本判定等问题无法解决,因此分子水平的基因测序方法提高了血型判定的准确性,比如PCR方法和基因芯片方法。为进一步提高ABO血型精细分型的准确性,通过分析全外显子组测序数据,开发了相关的VBA程序,能够快速自动化判定ABO精细分型,并且初步判定结果与临床判定结果一致,可以作为临床血型精确判定的辅助手段。     

GeneWiz browser: An Interactive Tool for Visualizing Sequenced Chromosomes

We present an interactive web application for visualizing genomic data of prokaryotic chromosomes. The tool (GeneWiz browser) allows users to carry out various analyses such as mapping alignments of homologous genes to other genomes, mapping of short sequencing reads to a reference chromosome, and calculating DNA properties such as curvature or stacking energy along the chromosome. The GeneWiz browser produces an interactive graphic that enables zooming from a global scale down to single nucleotides, without changing the size of the plot. Its ability to disproportionally zoom provides optimal readability and increased functionality compared to other browsers. The tool allows the user to select the display of various genomic features, color setting and data ranges. Custom numerical data can be added to the plot allowing, for example, visualization of gene expression and regulation data. Further, standard atlases are pre-generated for all prokaryotic genomes available in GenBank, providing a fast overview of all available genomes, including recently deposited genome sequences. The tool is available online from http://www.cbs.dtu.dk/services/gwBrowser. Supplemental material including interactive atlases is available online at http://www.cbs.dtu.dk/services/gwBrowser/suppl/.     

Comprehensive validation of a diagnostic strategy for sequencing genes with one or multiple pseudogenes using pseudoxanthoma elasticum as a model

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue. For this reason, their presence is often considered troublesome in molecular diagnostics. In pseudoxanthoma elasticum(PXE), a disease predominantly caused by mutations in ATPbinding cassette family C member 6(ABCC6), the presence of two pseudogenes complicates the analysis of sequence data. With whole-exome sequencing(WES) becoming the standard of care in molecular diagnostics, we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6. We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities, contrary to WES. We propose a time-and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE, which is highly automatable.     

Development and Challenge of HIV/AIDS Testing Laboratory Network and Quality Assurance System in China

This paper describes the development and challenge of HIV/AIDS testing laboratory network and quality assurance system in China. At present,the HIV/AIDS testing laboratories includes three classes,the National AIDS Reference Laboratory,HIV/AIDS confirmatory laboratories and HIV/AIDS screening laboratories. All of them are accredited by the health authorities,and each class of laboratories take charge of their function strictly according to the "National Management of HIV/AIDS Detection (2006)". A complete quality assurance and quality control system for HIV/AIDS testing has been developed,which includes technical training,strict laboratory monitoring and approval,examination or proficiency testing on HIV/AIDS detection,and quality evaluation and supervision of HIV/AIDS diagnostic kits. Besides conduct the routine anti-HIV antibody test,more and more laboratories began to conduct other tests,such as CD4 T lymphocyte cell counting,HIV viral load,HIV DNA PCR,genotyping,drug resistance,and HIV-1 recent infection test. The primary challenges faced by the HIV/AIDS testing laboratory network are in the areas of laboratory management and quality control. For example,the provincial PT program is inefficient,the internal quality control is conducted perfunctorily,personnel training can not met the needs of the workplace,which need to be improved.