Rapid Diagnostic Algorithms as a Screening Tool for Tuberculosis: An Assessor Blinded Cross-Sectional Study

Background

A major obstacle to effectively treat and control tuberculosis is the absence of an accurate, rapid, and low-cost diagnostic tool. A new approach for the screening of patients for tuberculosis is the use of rapid diagnostic classification algorithms.

Methods

We tested a previously published diagnostic algorithm based on four biomarkers as a screening tool for tuberculosis in a Central European patient population using an assessor-blinded cross-sectional study design. In addition, we developed an improved diagnostic classification algorithm based on a study population at a tertiary hospital in Vienna, Austria, by supervised computational statistics.

Results

The diagnostic accuracy of the previously published diagnostic algorithm for our patient population consisting of 206 patients was 54% (CI: 47%–61%). An improved model was constructed using inflammation parameters and clinical information. A diagnostic accuracy of 86% (CI: 80%–90%) was demonstrated by 10-fold cross validation. An alternative model relying solely on clinical parameters exhibited a diagnostic accuracy of 85% (CI: 79%–89%).

Conclusion

Here we show that a rapid diagnostic algorithm based on clinical parameters is only slightly improved by inclusion of inflammation markers in our cohort. Our results also emphasize the need for validation of new diagnostic algorithms in different settings and patient populations.     

Identification of Hepatotropic Viruses from Plasma Using Deep Sequencing: A Next Generation Diagnostic Tool

We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.     

CRISPR/Cas12a系统：核酸检测的多功能工具

规律成簇的间隔短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）是原核生物的适应性免疫系统,对抗外来遗传物质（如质粒和噬菌体）的攻击。近年来,科学家们发现了一种新型基因编辑工具,一种强大的分子剪刀CRISPR/Cas12a系统,该系统在对靶标DNA进行切割的同时还具有对体系内单链DNA进行任意切割的活性,并将其转移到体外检测系统。本文对CRISPR/Cas12a系统组成、结构、Cas12a与Cas9的对比和CRISPR/Cas12a系统在核酸检测中的应用进行了综述。     

fcGENE: A Versatile Tool for Processing and Transforming SNP Datasets

Background

Modern analysis of high-dimensional SNP data requires a number of biometrical and statistical methods such as pre-processing, analysis of population structure, association analysis and genotype imputation. Software used for these purposes often rely on specific and incompatible input and output data formats. Therefore extensive data management including multiple format conversions is necessary during analyses.

Methods

In order to support fast and efficient management and bio-statistical quality control of high-dimensional SNP data, we developed the publically available software fcGENE using C++ object-oriented programming language. This software simplifies and automates the use of different existing analysis packages, especially during the workflow of genotype imputations and corresponding analyses.

Results

fcGENE transforms SNP data and imputation results into different formats required for a large variety of analysis packages such as PLINK, SNPTEST, HAPLOVIEW, EIGENSOFT, GenABEL and tools used for genotype imputation such as MaCH, IMPUTE, BEAGLE and others. Data Management tasks like merging, splitting, extracting SNP and pedigree information can be performed. fcGENE also supports a number of bio-statistical quality control processes and quality based filtering processes at SNP- and sample-wise level. The tool also generates templates of commands required to run specific software packages, especially those required for genotype imputation. We demonstrate the functionality of fcGENE by example workflows of SNP data analyses and provide a comprehensive manual of commands, options and applications.

Conclusions

We have developed a user-friendly open-source software fcGENE, which comprehensively supports SNP data management, quality control and analysis workflows. Download statistics and corresponding feedbacks indicate that software is highly recognised and extensively applied by the scientific community.     

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.     

基因芯片技术检测重要人兽共患病病毒方法的建立

为了建立能对25种重要人兽共患病病毒进行筛查及鉴定用的基因芯片技术,本实验首先设计针对每种病毒的寡核苷酸探针并进行探针特异性的生物信息学验证.然后探索病毒核酸随机扩增方法,优化杂交动力学条件,建立本芯片标准的数据处理分析方法.最后用细胞培养的病毒和模拟临床标本验证芯片的敏感性与特异性．结果表明,锚定随机PCR扩增法适合于本芯片病毒核酸的扩增;芯片杂交前用0.25% NaBH4进行封闭,最优杂交条件为51 ℃,2 h及50%甲酰胺浓度;芯片具有较好的敏感性及检测特异性．初步结果表明,本实验所建立的基因芯片技术可应用于对25种重要人兽共患病病毒进行筛查及鉴定．     

FusionPro,a Versatile Proteogenomic Tool for Identification of Novel Fusion Transcripts and Their Potential Translation Products in Cancer Cells,

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Highlights

• •FusionPro, a versatile tool for studying fusion proteoforms, has been developed.
• •Fusion peptides were identified against a customized database built by FusionPro.
• •Types and features of fusion proteoforms were efficiently predicted by FusionPro.

     

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.     

Neural Organoids,a Versatile Model for Neuroscience

Three-dimensional cultures of human neural tissue/organ-like structures in vitro can be achieved by mimicking the developmental processes occurring in vivo. Rapid progress in the field of neural organoids has fueled the hope (and hype) for improved understanding of brain development and functions, modeling of neural diseases, discovery of new drugs, and supply of surrogate sources of transplantation. In this short review, we summarize the state-of-the-art applications of this fascinating tool in various research fields and discuss the reality of the technique hoping that the current limitations will soon be overcome by the efforts of ingenious researchers.     

口蹄疫等5种动物病毒基因芯片检测技术的研究

用分子克隆方法获得口蹄疫病毒、水泡性口炎病毒、蓝舌病病毒、鹿流行性出血热病毒和赤羽病病毒各一段高度保守的基因片段,用芯片点样仪点样到包被过的玻璃片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测,可同时诊断上述5种动物传染病,此方法不但快速、准确、敏感,而且可同时进行多种病毒的检测,达到大批动物高通量检疫的目的。     

Screening of Natural Waters for Viruses Which Infect Chlorella Cells

By using a plaque assay with the unicellular green alga Chlorella sp. strain NC64A as a host, viruses were screened from natural pond waters collected in Kyoto and Higashi-Hiroshima, Japan. From some samples tested, two kinds of plaques, large ( = 6 to 10 mm) and small ( = 2 to 3 mm), were detected with various frequencies. The frequency of plaques in each of the water sources was seasonal; generally, it reached a peak value (8,000 PFU/ml) in May and gradually decreased to the limit of detection (<1) in November before increasing again in early spring. Electron microscopy revealed that the purified and negatively stained viruses were very large (125 to 200 nm) icosahedral particles. The genome isolated from these particles was always a linear double-stranded DNA of 340 to 370 kbp. Electrophoresis patterns of the DNA fragments produced by digestion with restriction enzymes differed considerably from plaque to plaque, even for plaques from the same water source. However, Southern hybridization showed strong homology among all of the virus DNAs tested, indicating relatedness of those viruses. A possible use of the Chlorella virus assay system to monitor the natural population of algal cells and water quality is discussed.     

Accuracy of McMonnies Questionnaire as a Screening Tool for Chinese Ophthalmic Outpatients

ObjectiveTo evaluate the accuracy of the McMonnies questionnaire (MQ) as a screening tool for dry eye (DE) among Chinese ophthalmic outpatients.MethodsWe recruited 27718 cases from 94 hospitals (research centers), randomly selected from 45 cities in 23 provinces from July to November in 2013. Only symptomatic outpatients were included and they were in a high risk of DE. Outpatients meeting the criteria filled out questionnaires and then underwent clinical examinations by qualified medical practitioners. We mainly evaluated sensitivity, specificity, diagnostic odds ratio (DOR), and area under the receiver-operating characteristic curve (AUC) to evaluate the accuracy of the questionnaire in the diagnosis of dry eye.ResultsOf all the subjects included in the study, sensitivity, specificity, and DOR were 0.77, 0.86 and 20.6, respectively. AUC was 0.865 with a 95% CI (0.861, 0.869). The prevalence of DE among the outpatients claiming “constantly” as the frequency of symptom was over 90%. Scratchiness was a more accurate diagnostic indication than dryness, soreness, grittiness or burning. Different cut points of McMonnies Index (MI) scores can be utilized to optimize the screening results.ConclusionsMQ can be an effective screening tool for dry eye. We can take full advantage of MI score during the screening process.     

利用siRNA高通量测序技术筛查蚊子体内携带的RNA病毒

昆虫可以利用小干扰RNA(siRNA)机制干扰体内RNA病毒的增殖,从而获得对该病毒的免疫能力。通过第二代测序技术对蚊子的小RNA进行高通量测序,再通过生物信息学方法寻找其中的RNA病毒序列,发现在我国云南地区的白纹伊蚊和致倦库蚊体内存在Marayo,Omsk hemorrhagic fever和Ilheus等RNA病毒的基因片段。至此建立了发现媒介昆虫体内携带病毒的一种新方法,可用于媒介昆虫携带RNA病毒的本底调查。     

Past Anomalies as a Diagnostic Tool for Evaluating Multiple Marine Ecological Disturbance Events

In the search for the best ecological and economic indicators of ecosystem change, a unifying solution for joining data from disparate fields appears as a general rule: Organize data into space/time/topic hierarchies that permit convergence of data resulting from shared and appropriate scaling. The scale of the data selects for compatible methodologies, leading to data integration and the discovery of new relationships. Information technology approaches include bibliographic keyword searches, data-mining, data-modeling and geographic information system design. The approach was used within the “HEED” (Health Ecological and Economic Dimensions) study, which reconstructed historic marine disturbance events within the Northwestern Atlantic, Gulf of Mexico and Caribbean Sea. The object of the study was to retrospectively derive co-occurring Multiple Marine Ecological Disturbances (MMEDs). Disturbances include indices of morbidity, mortality and disease events affecting humans, marine invertebrates, flora, and wildlife populations. Correlations between space/time occurrence, event coincidence, climate and oceanographic forcing are used to better define multiple marine ecological disturbance types. Systematic derivation of these types is part of diagnostic approach that can assist or guide marine ecological risk assessment.     

Validation of a Rapid Rabies Diagnostic Tool for Field Surveillance in Developing Countries

BackgroundOne root cause of the neglect of rabies is the lack of adequate diagnostic tests in the context of low income countries. A rapid, performance friendly and low cost method to detect rabies virus (RABV) in brain samples will contribute positively to surveillance and consequently to accurate data reporting, which is presently missing in the majority of rabies endemic countries.Conclusion/SignificanceThe RIDT shows excellent performance qualities both in regard to user friendliness and reliability of the result. In addition, the test cassettes can be used as a vehicle to ship viral RNA to reference laboratories for further laboratory confirmation of the diagnosis and for epidemiological investigations using nucleotide sequencing. The potential for satisfactory use in remote locations is therefore very high to improve the global knowledge of rabies epidemiology. However, we suggest some changes to the protocol, as well as careful further validation, before promotion and wider use.     

Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4^th to 6^th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.     

Further Results in Indices for Diagnostic Screening II

This article is in continuation of a previous one on properties of diagnostic indices (Bennett, 1976). Results are presented on biases in sample estimates of the sensitivity (ξ) and specificity (η) of a diagnostic test T for a disease, as well as their asymptotic variances. The problem of combining estimates of ξ, η from various clinical centres and obtaining appropriate confidence limits is also discussed. A numerical example is also given. (Tables 1a, b). The log-linear model for ξ, η is also discussed.     

Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research

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