综述与专论：蛋白质即时检测新技术

蛋白质是细胞各类代谢和调控等生命功能的执行者,也是致病因子、药物等对机体作用的重要靶分子。研究蛋白质表达是理解生命现象、疾病进程和药物作用的基础。临床上常规检测方法需要大型仪器支持,但随着医学事业的发展,即时检测(POCT,也称现场检测、床旁检测)成为重要的发展趋势。POCT可以改善患者和医生之间的互动方式,建立一种积极的医疗模式。除诊断、治疗疾病外,对从事应急工作的人员来说,POCT在现场和远程检测方面都有优势,因此研发既准确灵敏又简便快捷的蛋白质即时检测方法至关重要。     

蛋白质即时检测新技术

蛋白质是细胞各类代谢和调控等生命功能的执行者,也是致病因子、药物等对机体作用的重要靶分子.研究蛋白质表达是理解生命现象、疾病进程和药物作用的基础.临床上常规检测方法需要大型仪器支持,但随着医学事业的发展,即时检测(POCT,也称现场检测、床旁检测)成为重要的发展趋势.POCT可以改善患者和医生之间的互动方式,建立一种积...     

Cultural and clinical effectiveness of the 'QAAMS' point-of-care testing model for diabetes management in Australian aboriginal medical services

The national Quality Assurance for Aboriginal Medical Services (QAAMS) Program, in which point-of-care testing (POCT) for haemoglobin A(1c) (HbA(1c)) and urine albumin: creatinine ratio (ACR) is performed for diabetes management in 65 Australian Aboriginal medical services, is now embedded in the practice of diabetes care across Indigenous Australia. This paper documents the results of a detailed survey to assess levels of satisfaction with the QAAMS HbA(1c) Program among three key stakeholder groups-doctors, POCT operators and patients with diabetes. Both doctors and patients with diabetes agreed that the immediacy of POCT results contributed positively to patient care, improved the doctor-patient relationship, and made the patient more likely to be both compliant and self-motivated to improve their diabetes control. Both POCT operators and patients with diabetes reported improved satisfaction with their diabetes services after the introduction of POCT. The paper also provides evidence from two participating medical services that POCT has been an effective tool in improving the delivery of pathology services and clinical outcomes for both individuals and groups of patients with diabetes. A statistically significant reduction in HbA(1c) from 9.3% (+/- 2.0) to 8.6% (+/- 2.0) was observed in 74 diabetes patients 12 months after commencing POCT (p = 0.003, paired t-test). An improvement in the percentage of patients achieving glycaemic targets and a reduction in the percentage of patients with poor control was also observed in this group. These data provide evidence that the QAAMS POCT model delivers a culturally and clinically effective service for diabetes management in Aboriginal Australia.     

Perceptions of an ideal point-of-care test for sexually transmitted infections--a qualitative study of focus group discussions with medical providers

Background

A point-of-care test (POCT) for sexually transmitted infections (STIs), which offers immediate diagnosis resulting in patients receiving diagnosis and treatment in a single visit, has the ability to address some of the STI control needs. However, needs assessment from STI experts and end users about currently available STI POCTs and their future new development has not been evaluated since World Health Organization Sexually Transmitted Diseases Diagnostics Initiative was formed over 15 years ago. Therefore, our objective was to explore the perceptions of the ideal types of STI POCT for use in health care settings.

Methodology/Principal Findings

A qualitative study, encompassing eight focus groups, was conducted from March 2008 through April 2009. Participants included 6 STD clinic directors, 63 clinicians, and 7 public health/laboratory/epidemiology professionals in the STI field. Discussion topics included currently available POCT, perceived barriers to using POCT in clinics, priority STI for the development of new POCT, and characteristics of the ideal POCT. All discussions were recorded and transcribed verbatim. Themes raised as barriers for current POCT included complexity, long time frames of the so-called “rapid” test, multiple time-driven steps, requiring laboratory technician, difficulty in reading result, interruption of workflow, unreliability, and invasiveness. Chlamydia trachomatis was identified as the priority organism for development of a new STI POCT. Themes indicated for the ideal POCT included rapid turnaround (up to 20 minutes), ease of use, non-invasive, accurate (preferred sensitivity and specificity in the range of high 90s), Clinical Laboratory Improvement Amendments (CLIA)-waived, user-friendly (for both patients and staff), compact, durable, and sturdy.

Conclusions/Significance

Focus group discussions with STI experts and professionals highlighted chlamydia as the top priority pathogen for POCT development, and identified the qualities of new POCT for STIs. Participants endorsed ease of use, rapid turnaround and high accuracy as essential characteristics of an ideal POCT.     

Use of glycogen phosphorylase BB measurement with POCT in the diagnosis of acute coronary syndromes. A comparison with the ELISA method

Background: Glycogen Phosphorylase BB (GPBB) is considered an early and specific marker of myocardial necrosis and ischemia. A POCT kit GPBB for diagnostic use has recently been approved. Aim: an evaluation of the correspondence of qualitative POCT GBPP measurements with ELISA test results. Material and methodology: 20 individuals with non-ST elevation myocardial infarction (non-STEMI) and 20 probands without acute coronary syndrome (ACS) were tested. GPBB (POCT, ELISA) in venous plasma (lithium-heparin) was assayed in all probands. Results: individuals with non-STEMI had significantly higher GPBB ELISA values (32.3 vs. 6.1 mug/l; p < 0.01). GPBB sensitivity and specificity for non-STEMI presence 6 hours after chest pain generation were 100 %. No proband was classified in a different subgroup with POCT of GPBB (positive/negative). GPBB POCT correlate with a non- STEMI diagnosis (chi(2) 36.1; p <0.01). Conclusion: GPBB POCT measurement is comparable with ELISA test results. GPBB analysis could expand the diagnostic palette in the first hours after the onset of acute coronary syndrome.     

综述与专论: 基于核酸适配体传感器的即时检测（POCT）技术

即时检测（point-of-care testing，POCT）是一种检测成本低、检测速度快、准确度高、能自我采样获得临床诊断结果的新型诊断技术。该技术在临床诊断、病情监控与疫情防控等领域发挥了重要作用。核酸适配体是一种能够特异性识别多种靶标的分子探针，具有易合成、批间差异小、易实现信号放大等突出优势，是生物医学传感器中重要的分子识别元件。本文概述了核酸适配体探针的现有筛选方法和进展，总结了核酸适配体POCT传感器信号放大策略，着重介绍了各类核酸适配体传感器在POCT领域的应用现状，并对核酸适配体POCT传感器的发展前景进行了展望。     

分子即时检测(POCT)技术及其在新发传染病中的应用*

分子即时检测(point-of-care testing,POCT)技术具有灵敏度高、分析速度快、体积小、检测成本低廉等特点,在分子诊断领域受到广泛关注。近年来,分子POCT技术的发展与应用在应对新发、突发传染病,保护人类生命健康方面具有重大意义。介绍近五年来新兴的分子POCT技术,总结新兴分子POCT技术的最新研究进展及应用前景,分析POCT技术的优势与面临的挑战,探讨提高其检测灵敏度和选择性的技术策略。     

Rapid Syphilis Tests as Catalysts for Health Systems Strengthening: A Case Study from Peru

Objectives

Untreated maternal syphilis leads to adverse pregnancy outcomes. The use of point of care tests (POCT) offers an opportunity to improve screening coverage for syphilis and other aspects of health systems. Our objective is to present the experience of the introduction of POCT for syphilis in Peru and describe how new technology can catalyze health system strengthening.

Methods

The study was implemented from September 2009–November 2010 to assess the feasibility of the use of a POCT for syphilis for screening pregnant women in Lima, Peru. Outcomes measured included access to syphilis screening, treatment coverage, partner treatment, effect on patient flow and service efficiency, acceptability among providers and patients, and sustainability.

Results

Before the introduction of POCT, a pregnant woman needed 6 visits to the health center in 27 days before she received her syphilis result. We trained 604 health providers and implemented the POCT for syphilis as the “two for one strategy”, offering with one finger stick both syphilis and HIV testing. Implementation of the POCT resulted in testing and treatment on the first visit. Screening and treatment coverages for syphilis improved significantly compared with the previous year. Implementation of POCT has been scaled up nationally since the study ended, and coverages for screening, treatment and partner treatment have remained over 92%.

Conclusions

Implementation of POCT for syphilis proved feasible and acceptable, and led to improvement in several aspects of health services. For the process to be effective we highlight the importance of: (1) engaging the authorities; (2) dissipating tensions between providers and identifying champions; (3) training according to the needs; (4) providing monitoring, supervision, support and recognition; (5) sharing results and discussing actions together; (6) consulting and obtaining feedback from users; and (7) integrating with other services such as with rapid HIV testing.     

Paper-based point-of-care test with xeno nucleic acid probes

Bridging the unmet need of efficient point-of-care testing (POCT) in biomedical engineering research and practice with the emerging development in artificial synthetic xeno nucleic acids (XNAs), this review summarized the recent development in paper-based POCT using XNAs as sensing probes. Alongside the signal transducing mode and immobilization methods of XNA probes, a detailed evaluation of probe performance was disclosed. With these new aspects, both researchers in synthetic chemistry / biomedical engineering and physicians in clinical practice could gain new insights in designing, manufacturing and choosing suitable reagents and techniques for POCT.     

Feasibility of Performing Multiple Point of Care Testing for HIV Anti-Retroviral Treatment Initiation and Monitoring from Multiple or Single Fingersticks

Background

Point of Care testing (POCT) provides on-site, rapid, accessible results. With current South African anti-retroviral treatment guidelines, up to 4 fingersticks /patient/clinic visit could be required if utilizing POC. We determined the feasibility and accuracy of a nurse performing multiple POCT on multiple fingersticks followed by simplification of the process by performance of multiple POC on a single fingerstick.

Method and Findings

Random HIV positive adult patients presenting at a HIV treatment clinic in South Africa, for ART initiation/ monitoring, were approached to participate in the study between April-June 2012. Phase I: n=150 patients approached for multiple POCT on multiple fingersticks. Phase II: n=150 patients approached for multiple POCT on a single fingerstick. The following POC tests were performed by a dedicated nurse: PIMA (CD4), HemoCue (hemoglobin), Reflotron (alanine aminotransferase, creatinine). A venepuncture specimen was taken for predicate laboratory methodology. Normal laboratory ranges and Royal College of Pathologists Australasia (RCPA) allowable differences were used as guidelines for comparison. In 67% of participants, ≥3 tests were requested per visit. All POCT were accurate but ranged in variability. Phase I: Hemoglobin was accurate (3.2%CV) while CD4, alanine aminotransferase and creatinine showed increased variability (16.3%CV; 9.3%CV; 12.9%CV respectively). PIMA generated a misclassification of 12.4%. Phase II: Hemoglobin, alanine aminotransferase and creatinine showed good accuracy (3.2%CV, 8.7%CV, 6.4%CV respectively) with increased variability on CD4 (12.4%CV) but low clinical misclassification (4.1%). No trends were observed for the sequence in which POC was performed on a single fingerstick. Overall, PIMA CD4 generated the highest error rate (16-19%).

Conclusions

Multiple POCT for ART initiation and/or monitoring can be performed practically by a dedicated nurse on multiple fingersticks. The process is as accurate as predicate methodology and can be simplified using a single fingerstick.     

Detection of micro-RNA by a combination of nucleic acid sequence-based amplification and a novel chemiluminescent pyrophosphate assay

Micro-RNA has attracted much attention as a biomarker for disease progression and malignancy. A compact, simple, rapid, and highly sensitive method is required to perform simple genetic analyses, such as point-of-care testing (POCT), at the clinic or bedside. Nucleic acid sequence-based amplification (NASBA) is a specific amplification method for a single-stranded RNA fragment that is useful for the highly sensitive detection of miRNAs. In this work, we developed a novel miRNA analytical system for POCT by combining the NASBA and chemiluminescence methods. Because the NASBA reaction is conducted at a constant temperature (41°C) and detection by chemiluminescence reaction does not require a light source, these methods could be combined to amplify 100 ng/assay miRNA. This combined miRNA detection method could be useful for the future development of compact POCT systems.     

What Qualities Are Most Important to Making a Point of Care Test Desirable for Clinicians and Others Offering Sexually Transmitted Infection Testing?

Background

To investigate the possible effects of different levels of attributes of a point-of-care test (POCT) on sexually transmitted infection (STI) professionals'' decisions regarding an ideal POCT for STI(s).

Methods

An online survey was designed based on a large-scale in-depth focus discussion study among STI experts and professionals. The last section of the survey “build your own POCT” was designed by employing the discrete choice experiment approach. Practicing clinicians from two venues, STI-related international conference attendees and U.S. STD clinic clinicians were invited to participate in the survey. Conditional logistical regression modeling was used for data analysis.

Results

Overall, 256 subjects took the online survey with 218 (85%) completing it. Most of the participants were STD clinic clinicians who already used some POCTs in their practice. “The time frame required” was identified as a major barrier that currently made it difficult to use STI POCTs. Chlamydia trachomatis was the organism chosen as the top priority for a new POCT, followed by a test that would diagnose early seroconversion for HIV, and a syphilis POCT. Without regard to organism type selected, sensitivity of 90–99% was always the most important attribute to be considered, followed by a cost of $20. However, when the test platform was prioritized for early HIV seroconversion or syphilis, sensitivity was still ranked as most important, but specificity was rated second most important.

Conclusions

STI professionals preferred C. trachomatis as the top priority for a new POCT with sensitivity over 90%, low cost, and a very short completion time.     

Application experience of a rapid nucleic acid detection system for COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging worldwide. The COVID-19 outbreak caused severe threats to the life and health of all humans caused by SARS-CoV-2. Clinically, there is an urgent need for an in vitro diagnostic product to detect SARS-CoV-2 nucleic acid quickly. Under this background, commercial SARS-CoV-2 nucleic acid POCT products came into being. However, how to choose these products and how to use these products in a standardized way have brought new puzzles to clinical laboratories. This paper focuses on evaluating the performance of these commercial SARS-CoV-2 nucleic acid POCT products and helps the laboratory make the correct choice. At the same time, to standardize the use of this kind of product, this paper also puts forward corresponding suggestions from six elements of total quality management, namely, human, machine, material, method, environment, and measurement. In addition, this paper also puts forward some ideas on the future development direction of POCT products.     

Differential impacts of habitat heterogeneity on male and female connectivity in a spatially structured pest system

In a previous study, a model of landscape heterogeneity was developed and applied to a spatially structured wild rabbit (Oryctolagus cuniculus) population. That study showed clearly the influence of resource heterogeneity on connectivity levels. The simulation study was based on female movements and used population genetic validation data appropriate for a female study. Most models assume that males and females will exhibit similar patterns, although this has rarely been tested. In the current study we extend the analysis to consider differences between female and male connectivity in the same spatially structured pest system. Amplified fragment length polymorphism (AFLP) markers were screened on the same samples used previously for mtDNA analysis. The mtDNA data were used to validate female results, and AFLP data were used to validate combined male and female results. Connectivity patterns from the two simulations (female, and combined male and female) connectivity patterns showed no association. However, each was concordant with appropriate validation data, showing highly significant associations between pairwise population connectivity and the genetic data. A relative connectivity metric for the combined simulation was regressed against the mean of pairwise Φ_ST values, with almost 70% of the variation explained by a linear model. Demonstrating differential effects of habitat heterogeneity on male and female connectivity provides further evidence that spatial resource heterogeneity impacts on connectivity. Understanding differences in population connectivity will allow improved predictions of disease spread, local extinctions and recolonizations. Furthermore, modelling such differences in pest systems will allow management plans to be better targeted, for example by strategically introducing diseases for control purposes into populations which exhibit high male connectivity to aid spread, but low female connectivity to inhibit recolonization potential after control.     

核糖核酸调节子的构建与即时检测中的应用*

合成生物学专注于可重复利用模块和元件的工程化设计,并在生物系统中表现出良好的行为和功能。在无细胞蛋白表达系统中,核糖核酸调节子作为即时检测中的重要传感元件,通过靶标分子的诱导使其自身结构发生变化,进而调控下游基因的表达。系统介绍不同类型的核糖核酸调节子及其作用原理,包括一代核糖核酸调节子、toehold开关、功能拓展的核糖核酸调节子和核糖开关。详细阐明构建核糖核酸调节子的设计-测试-分析过程:计算机辅助设计、基因表达测试和结构功能化分析。汇总基于核糖核酸调节子的体外即时检测应用,重点总结toehold开关介导的病原菌核酸检测和核糖开关参与的小分子检测。讨论当前无细胞即时检测的特点、挑战和发展趋势,为开发新型核糖核酸调节子和即时检测工具提供思路和参考。     

From dispersal constraints to landscape connectivity: lessons from species distribution modeling

Connectivity plays a crucial role in determining the spread, viability, and persistence of populations across space. Dispersal across landscapes, or the movement of individuals or genes among resource patches, is critical for functional connectivity. Yet current connectivity modelling typically uses information on species location or habitat preference rather than movement, which unfortunately may not capture key dispersal limitations. We argue that recent developments in species distribution modelling provide insightful lessons for addressing this gap and advancing our understanding of connectivity. We suggest shifting the focus of connectivity modelling from locating where animals potentially disperse to a process‐based approach directed towards understanding and mapping factors that limit successful dispersal. To do so, we propose defining species dispersal requirements through identifying spatial, environmental and intrinsic constraints to successful dispersal, analogous to identifying environmental dimensions that define niches. We discuss the benefits of this constraint‐based framework for understanding the distribution of species, predicting species responses to climate change, and connectivity conservation practice. We illustrate how the framework can aid in identifying potential detrimental effects of human activities on connectivity and species persistence, and can spur the implementation of innovative conservation strategies. The proposed framework clarifies the validity and contextual utility of objectives and measures in existing connectivity models, and identifies gaps that may impede our understanding of connectivity and its integration into successful conservation strategies. We expect that this framework will facilitate a mechanistic approach to understanding and conserving connectivity, which will aid in effectively predicting and mitigating effects of ongoing environmental change.     

Automated pipette failure monitoring using image processing for point-of-care testing devices

Background

The accuracy and precision of liquid handling can be altered by several causes including wearing or failure of parts, and human error. The last cause is crucial since point-of-care testing (POCT) devices can be used by non-experienced users or patients themselves. Therefore it is important to improve the method of informing the users of POCT device malfunctions due to damage of parts or human error.

Methods

In this paper, image-based failure monitoring of the automated pipetting was introduced for POCT devices. An inexpensive, high-performance camera for smartphones was employed in our previous work to resolve various malfunctions such as incorrect insertion of the tip, false positioning of the tip and pump, and improper operation of the pump. The image acquired from the camera was analyzed to detect the malfunctions. In this paper, the reagent volume in the tip was estimated from the image processing to verify the pump operation. First, the color component corresponding to the reagent intrinsic color was extracted to identify the reagent area in the tip before applying the binary image processing. The extracted reagent area was projected horizontally and the support length of the projection image was calculated. As the support length was related to the reagent volume, it was referred to the volume length. The relationship between the measured volume length and the previously measured solution mass was investigated. If we can predict the mass of the solution by the volume length, we will be able to detect the pump malfunction.

Results

The cube of the volume length obtained by the proposed image processing method showed a very linear relationship with the reagent mass in the tip injected by the pumping operation (R²?=?0.996), indicating that the volume length could be utilized to estimate the reagent volume to monitor the accuracy and precision of the pumping operation.

Conclusions

An inexpensive smartphone camera was enough to detect various malfunctions of a POCT device with pumping operation. The proposed image processing could monitor the level of inaccuracy of pumping volume in limited range. The simple image processing such as a fixed threshold and projections was employed for the cost optimization and system robustness. However it delivered the promising results because the imaging condition was highly controllable in the devices.

     

Advances in paper-based sample pretreatment for point-of-care testing

In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.     

Evaluation of two devices for point-of-care testing of haemoglobin in neonatal pigs

In veterinary medicine, point-of-care testing (POCT) techniques have become popular, since they provide immediate results and only small amounts of blood are needed. However, their accuracy is controversial. Pigs are often used for research purposes and accurate measurement of haemoglobin (Hb) is important during invasive procedures. The aim of this study was to evaluate two different Hb POCT devices in neonatal pigs. A prospective study with 57 pigs of 3-6 weeks of age, weighing 4.1-6.2 kg (median 5.1 kg) was performed. Fifty-seven blood samples were analysed for Hb using a conductivity-based and a photometrical POCT device and compared with a photometrical reference method. Statistical analysis was performed with Bland-Altman analysis, Spearman correlation and Passing-Bablok regression analysis. Hb values ranged from 32 to 108 g/L (median 80 g/L) using the reference method. The bias of the photometrical method (HemoCue(?)) to the reference method was -1 g/L, with limits of agreement (LOA) of -7 to 6 g/L. The conductivity-based method (i-STAT(?)) had a bias of -15 g/L with LOA from -24 to -6 g/L. There was a significant association between protein values and the bias of i-STAT versus CellDyn (r(2) = 0.27, P < 0.05) but not with the bias of HemoCue versus CellDyn (r(2) = 0.001, P = 0.79). The lower the protein values were, the lower the Hb values were measured by the i-STAT. The conductivity-based measurement of Hb constantly underestimated Hb values, whereas the photometrical method demonstrated a better accuracy and is therefore more reliable for on-site measurement of Hb in pigs.