A diagnostic point-of care (POC) test would greatly improve case detection and management of pediatric tuberculosis (TB). Herein, we provide a brief overview of the challenges associated with diagnosing TB in children and recent evidence that points towards a future POC test based on transcriptomes in peripheral blood.

by Dr S. Jenum, Dr J.E. Gjøen, R. Bakken, Dr D. Sivakumaran and Prof. H.M.S. Grewal

Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), is a leading cause of childhood death and morbidity worldwide estimated to cause 1 million new cases in children <15 years of age in 2014 [1]. The gold standard for a diagnosis of pulmonary TB is growth of Mtb from respiratory specimens. However, culture-confirmed TB only occurs in about 30% of pediatric TB cases because of the paucibacillary nature of the disease and the difficulties in obtaining representative specimens [2]. For these reasons, children have been perceived less contagious and, therefore, not considered a priority for case finding within national TB programmes. This has resulted in detection rates being as low as 35% [3], with evident consequences for childhood death and morbidity. An accurate diagnostic test that provides a rapid, sensitive and specific result, enabling early initiation of treatment at the point-of-care (POC), ‘a diagnostic POC test’, could prevent this. The declaration by the World Health Organization (WHO) of pediatric TB as a neglected area of science and the stated urgent need for improved diagnostics [4], has spurred the search for TB diagnostic biomarkers in children. We provide a brief overview of the challenges associated with diagnosing TB in children and recent evidence that points towards a future POC test based on transcriptomes in peripheral blood.

Diagnosing TB in children
Diagnostic approaches and criteria for pediatric TB have recently been extensively reviewed [5]. In summary, sampling of respiratory specimens for confirmation of diagnosis by culture is strongly recommended, but requires repeated sampling of induced sputum and gastric aspirates, which are cost and labour intensive [5]. Notably, as many as 70% of children are culture negative even under optimized study conditions [2]. Therefore, in most cases, a diagnosis is based on a diagnostic algorithm including clinical signs and symptoms, radiological findings suggestive of TB and immunological evidence of previous Mtb sensitization [5]. Clinical diagnostic algorithms have not being adequately validated [6], and radiological findings are often unspecific [5]. The tuberculin skin test (TST) and the newer interferon-γ release assays used as a surrogate for Mtb infection cannot differentiate between infection and disease [7], and have reduced sensitivity in young and malnourished children [8, 9], but may be complementary by improving sensitivity in such clinical contexts [5].

Requirements for a POC test
Direct microscopy of sputum smears looking for acid-fast bacilli, is widely implemented at primary diagnostic centres for the diagnosis of pulmonary TB in adults, but the sensitivity is highly variable (20–80%) [10]. Since 2010, the use of the Xpert MTB/RIF assay on sputum samples has expanded in low- and middle-income countries [11], serving as a POC test in adults. The assay is based on direct identification of Mtb in specimens by a nucleic acid amplification test and detects in children three times the cases detected by direct microscopy and ~70% of the cases detected by liquid culture [12]. The WHO recommends that a POC triage test, meant to narrow down the population that needs further diagnostic work-up, should achieve a sensitivity of 90% and a specificity of 70%. For a diagnostic POC test, the sensitivity should be >95% and the specificity >98% [13]. A POC test based on Mtb detection will never meet these requirements in children because of the high proportion of culture-negative pulmonary TB [2], as well as the significant proportion of extrapulmonary TB [5]. Host biomarkers reflecting the ongoing pathological processes resulting from Mtb infection may hold greater promise for this purpose. Moreover, peripheral whole blood (WB) is more easily available for diagnosis than respiratory or tissue samples, particularly in children. Therefore, biomarkers based on transcriptomes in WB have gained increasing interest.

Genome-wide analysis of RNA expression
A landmark study by Berry et al., based on genome-wide analysis of RNA expression in unstimulated WB from adults, identified an 86-transcript signature able to discriminate active TB from other inflammatory and infectious diseases [14]. This transcriptomic signature consisted mainly of neutrophil-driven interferon (IFN)-inducible genes, which until then, had not been considered essential players in TB immunity. Further, a decreased abundance of B-cell transcripts in TB patients [14], challenged the paradigm of B-cells and humoral immunity being of little importance in protection against Mtb. Following this study, the upregulation of type I IFN-inducible genes and altered expression of B-cell related genes has been confirmed in adults [15–19] and adolescents [20].

To our knowledge, two previous studies in children have been published based on genome-wide transcriptional profiling of WB in analysis of RNA expression in Warao Amerindians [21], and in three African cohorts [22]. Verhagen et al. addressed the diagnostic gap in discriminating TB disease from latent TB, and identified a 5-transcript signature with a prediction error of 11% in discriminating active from latent TB. The signature classified correctly 78% of TB cases and 100% of latent TB cases in a validation cohort comprising children with non-TB pneumonia [21]. Interestingly, the 5-transcript signature, when applied on transcriptional data-sets generated by microarray analyses of WB from adults performed similarly, whereas the gene signatures identified in these adults were useless in the cohort of Warao Amerindian children [21]. This questions the validity in extrapolating results from transcriptional TB biomarker research in adults to children, highlighting the importance of studies in pediatric populations.

Applying a more real-life diagnostic setting with inclusion of children evaluated for suspected TB applying a graded diagnosis of TB in line with the consensus statement [6], Anderson et al. identified a 51-transcript signature that distinguished TB from other diseases in South African and Malawian children, subsequently validated in Kenyan children. A risk score based on the signature identified confirmed TB with a sensitivity of 82.9% and a specificity of 83.6%, and discriminated between other disease and culture-negative TB with a sensitivity ranging from 35–82% [22].

A more cost-effective method for assessing transcriptomes
Genome-wide analyses of transcriptomes are costly and resource-intensive but can be seen as a necessary first step in identifying markers with potential for subsequent refinement as POC tests. A test more suitable for hypothesis-testing or more high-throughput screening of transcriptional signatures with relevance in TB, has recently been developed [23]. The dual-colour Reverse Transcriptase-Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) can rapidly profile multiple host genes with a dynamic range and accuracy comparable to real-time qPCR and RNA sequencing at a cost of €5–7 per sample [24], most likely simplifying the translation of findings into diagnostic tools in resource-poor settings. The dcRT-MLPA method has been applied in different studies assessing TB biomarkers [19, 23–25]. Recently, incorporating the results from unbiased gene-expression profiling, the gene panels adapted for the dcRT-MLPA platform have been expanded to include type I IFN-inducible genes as well as genes covering adaptive immunity, including B-cells [25].

The dcRT-MLPA method in the context of pediatric TB
We have applied the dcRT-MLPA method to identify TB diagnostic transcriptome signatures in pediatric TB settings in India [26, 27]. We have also examined the immunological basis for discordant TST and Quantiferon® responses [28], as well as the potential interference of non-tuberculous mycobacteria on transcriptional biomarkers [29]. The younger the child, the more challenging the diagnosis of TB. We therefore explored transcriptional biomarkers in Indian children aged <3 years referred for a TB diagnostic work-up in a prospective cohort study of BCG-vaccinated neonates. We found that RAB33A alone discriminated between clinical TB and Mtb infection with an area under the curve (AUC) of 77.5%, whereas a 5-transcript signature effectively discriminated between clinical TB and controls (AUC 91.7%) [26]. Then, in a cohort of older Indian children (mean age ~9 years) with intrathoracic TB and their siblings, we assessed transcriptional biomarkers across the spectrum of TB disease. We identified 12 biomarkers consistently associated with clinical groups ‘upstream’ towards confirmed TB or ‘downstream’ towards a decreased likelihood of TB disease (Fig. 1), suggesting a correlation with Mtb-related pathology and high relevance to a future POC test. Furthermore, an 8-transcript signature separated children with TB from asymptomatic siblings (AUC 88%) [27]. Notably, these transcriptome signatures provided a superior sensitivity for confirmed TB compared with the Xpert MTB/RIF performed on gastric lavage [2]. Many of the biomarkers within these signatures have been confirmed in adults with Mtb infection or disease [19, 23, 24].

Future perspectives
We are currently exploring the performance of the extended dcRT-MLPA panels of genes which include type I IFN-signalling, myeloid cell activation, general inflammation and B-cell related genes in Indian children with intrathoracic TB. Transcriptome signatures identified in a discovery set consisting of randomly assigned TB cases and their healthy siblings will be validated in a diagnostic setting of symptomatic children aged <3 years. We are also exploring the association between transcriptional biomarkers and the extent of TB disease and the outcome of TB treatment in children. The monitoring and evaluation of TB treatment is hampered by the limited tools available to guide decision-making during the long treatment period. Based on our unpublished results, as well as from findings in adults [16, 17, 23] we believe that treatment monitoring by means other than a negative culture result at 2 months post-treatment [30] will be possible. Conclusions
Transcriptome-based tests meeting the requirements for both a screening and a diagnostic POC test would greatly improve case detection in children and subsequently reduce morbidity and mortality attributable to TB [13]. Further, a POC test capable of guiding treatment would prevent treatment failure and the subsequent emergence of resistant Mtb strains.

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The authors
Synne Jenum*¹ MD, PhD; John Espen Gjøen² MD, Rasmus Bakken²; Dhanasekaran Sivakumaran² PhD; Harleen M.S. Grewal² MD, PhD, DTMH
¹Department of Infectious Diseases, Oslo University Hospital, Pb 4950, N-0424 Oslo, Norway
²Department of Clinical Science, University of Bergen, Pb 7804, N-5021 Bergen, Norway

*Corresponding author
E-mail: synnejenum@gmail.com

Interactions of neoplastic cells with each other and the microenvironment are complex. The main factors contributing to intratumoral heterogeneity may be reflected in biomarker expression. The aim is to investigate the spatial intratumoral heterogeneity of Ki-67 immunostains in whole sections of pancreatic neuroendocrine neoplasms. The extent and range of heterogeneity has potential as a prognostic marker.

by Dr Nektarios A. Valous, Dr Frank Bergmann and Dr Niels Halama

Overview
Tumour heterogeneity means that a neoplasm comprises distinct cellular subpopulations that can vary in histology and growth rate. Phenotypic and functional heterogeneity arise among cancer cells within the neoplasm because of genetic change, environmental differences and reversible changes in cell properties [1, 2]. Genomic instability arises through various routes, leaving distinct genomic footprints and differentially affecting tumour evolution and patient outcome [3]. In addition, heterologous cell types within tumours can influence therapeutic response and shape resistance [4]. The interactions of neoplastic cells with each other and the microenvironment are complex. Clinicians assess complex cytological, histological, and morphological characteristics of tissues often in a semi-quantitative manner. In order to understand intratumoral heterogeneity, potentially subtle differences within neoplasms should be quantified.

The main factors contributing to intratumoral heterogeneity include the ischemic gradient within the neoplasm, the action of the microenvironment, mechanisms of intercellular transfer of genetic information, and differential mechanisms of modifications of genetic material/proteins [5]. This may be reflected in the expression of biomarkers and their clinical utility in the context of prognosis/stratification. A rigorous approach for assessing the spatial intratumoral heterogeneity of histological biomarker expression with accuracy and reproducibility is required, since patterns in immunohistochemical images exhibit scale-dependent changes in structure and can be challenging to identify and describe [6–8]. The aim is to determine the implications and prognostic value of observed variations, in a host of clinically relevant neoplastic properties [9].

Case study
It is recognized that proliferative heterogeneity is common in many tumours, including pancreatic neuroendocrine neoplasms [10]. These represent a rather heterogeneous group regarding histology, hormone secretion and functional activity, as well as biological behaviour. Pancreatic neuroendocrine neoplasms are classified as tumours (grade G1 and G2) and carcinomas (grade G3), based on proliferation activity. The latter is determined using a mitotic count in ten high-power fields or determination of fraction of Ki-67 positive cells within a highly proliferative area. In resected neoplasms, the proliferation-dependent tumour grade is shown to be a strong prognostic marker, whereas patient clinical management largely depends on proliferation activity. Key treatment decisions rely on the robust classification of these tumours.

Method
Earlier work has shown the advantages of using spatial statistics in histopathology [11]. A quantitative method from the fields of complex systems and image analysis that is particularly useful for characterizing complex irregular structures is lacunarity. The term lacunarity itself stems from the field of fractal geometry referring to a measure of how patterns fill space [12]. The approach has several theoretical and practical advantages for the assessment of spatial heterogeneity [13]. It is a multiscale technique, its computation is simple to implement, it exhaustively samples the image to quantify scaling changes, the analysis can be used for very sparse data, and the decay of the lacunarity index as a function of window size follows characteristic patterns for random, self-similar and structured spatial arrangements. Compared to previous approaches, the proposed methodology quantifies directly the distributional landscape of proliferative cells and not the textural content of the histological slide, thus providing a more realistic measure of heterogeneity within the sample space of the tumour region.

Workflow
Figure 1 provides an overview of the workflow for measuring the spatial intratumoral heterogeneity of proliferation in pancreatic neuroendocrine neoplasms. Immunohistochemical (IHC) staining for Ki-67 is performed on whole-slide sections taken from original tissue blocks using the avidin-biotin-peroxidase detection system on a fully automated staining facility. On IHC stains for Ki-67, the brown reaction product at the antigen site is in the cell nucleus. The slide is counterstained with hematoxylin to allow evaluation and assessment of staining localization. Glass slides are automatically imaged in bright-field mode. The resulting virtual slides are reviewed by a pathologist for determining the locality of the neoplastic region; areas are marked and large representative sections are cropped. The automated proliferative cell nuclei segmentation workflow is based on background removal, stain vector extraction/color deconvolution, and post-processing operations. Hence, for every virtual slide (large section of tumour region), a segmented image depicting proliferative nuclei is produced.

For measuring the spatial heterogeneity of proliferation using lacunarity, an automated sampling scheme is employed prior to analysis allowing better scrutiny and interpretation, thus keeping computation times manageable. The sampling method aims at capturing the spatial variability of Ki-67 positive cells in the images. Then, lacunarity is computed and subsequently visualized in a double log plot as a function of scale. These plots explicitly characterize the spatial organization of images and measure space filling capacity. From the plots, mean, median, and mode lacunarity curves are computed. These metrics capture the variability (or lack of) of the curves for the sampled sections. To ascertain that lacunarity describes the spatial organization of proliferation, the three metrics are used for partitioning the neoplasms into conceptually meaningful clusters. This is achieved by performing unsupervised learning using the k-means algorithm. First, their Mahalanobis distance is computed and then using principal component analysis vectors are decorrelated by projection into a subspace that minimizes reconstruction error in the mean squared sense. Finally, a phenomenological heterogeneity index is computed from the spatial distribution in order to provide direct numerical values.

Impact and outlook
Phenotypic heterogeneity that stems from genetic/non-genetic determinants constitutes a major source of therapeutic resistance, and is an important clinical obstacle [14]. Tissue architecture is generally not reflected in molecular assays, rendering this information underused. Heterogeneity in histological expression of biomarkers has been noted in earlier studies. However, experimental exploration has been limited by a lack of conceptual framework and tools. The critical bottleneck has become the development of computational methods to analyse, integrate, and connect data to prognostic and actionable clinical information [15].

The architectural complexity of immunohistological images has shown that single measurements are often insufficient for characterization. The selection of a region of interest as a surrogate for the complete complex structure is prey to selection bias and subsequent loss of reproducibility and precision. Especially for the neuroendocrine pancreatic tumours, the inhomogeneity of distribution depends not only on percentage content of proliferation phase but also on how the phase fills the space. An increased degree of spatial proliferative heterogeneity is observed in certain neoplasms comparing to others with similar histological grade. Whether this is a sign of different tumour biology and subsequently an association with a more benign or malignant clinical course needs to be investigated further. The approach provides an increased level of granularity for discerning different levels of spatial heterogeneity in biomarker expression, since manual inspection can be cumbersome and may not resolve finer differences. The extent and range of heterogeneity has the potential to be evaluated as a prognostic marker, e.g. for the evaluation of the clinical course utilizing the construction of survival curves. This heterogeneity is also most likely to play a role in different aspects of the clinical course. Clinical trials should incorporate such studies on heterogeneity so that the impact on therapeutic effectiveness can be understood. For practical reasons, the heterogeneity index outputted in the final step of the analysis is desirable when a single numerical value is required, e.g. in direct assessments or comparisons for the automated ranking of cases in order of increasing/decreasing heterogeneity.

In summary, the lacunarity morphometric provides information about the distribution of Ki-67 immunolabelling that corresponds to the degree of spatial organization of proliferation. This reflects a general approach with potential importance in clinical work, which is relevant to other solid tumours and a vast array of biomarkers. The additional level of understanding the distributional patterns of specific biomarkers holds promise for providing valuable information in a clinical setting. Drawing upon the richness of histopathological information and merits of computational biomedicine, the approach removes qualitative ambiguities and uncovers salient features for use in future studies of clinical relevance.

References
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The authors
Nektarios A. Valous*¹ PhD, Frank Bergmann² MD, Niels Halama³ MD, PhD
¹Applied Tumor Immunity Clinical
Cooperation Unit, National Center for Tumor Diseases, German Cancer Research Center, Heidelberg, Germany
²Institute of Pathology, Heidelberg
University Hospital, Heidelberg, Germany
³Department of Medical Oncology, National Center for Tumor Diseases,
Heidelberg University Hospital, Heidelberg, Germany

*Corresponding author
E-mail: nek.valous@nct-heidelberg.de

Point-of-care testing (POCT) enables quick test results with minimal manual interference nearer to the site of patient care, which leads to better health outcomes via rapid diagnosis, quick clinical decisions and the early start of treatment. The emerging technologies would further improve POCT by low-cost analysis with increased performance characteristics.

by Dr Sandeep Kumar Vashist and Prof. John H.T. Luong

Existing point-of-care testing (POCT) technologies
A variety of POCT technologies are being used such as POCT analysers, biosensor devices, lab-on-chips (LOC), test strips, and lateral flow assay (LFA) cartridges. Such cost-effective technologies offer rapid analysis in just a few minutes using minimal sample volumes. As well as at a patient’s bedside, POCT have been employed in the operating theatre, emergency department and critical care/maternity unit. Other deployments include nursing homes, physician’s office, prison, emergency vehicles, etc. Local pharmacies have adopted the POCT technology to provide a one-stop service for glucose, cholesterol, pregnancy, etc.

The POCT analysers are standard bench-top devices that can determine a broad range of analytes, based on spectrophotometry, reflectometry, immunoassay, turbidimetry, potentiometry/amperometry, oximetry and hematological particle counting. The target analytes are small metabolites, enzymes, drugs-of-abuse, inflammation biomarkers, heart and kidney injury biomarkers, infectious agents, humoral and cellular coagulation markers, hematological parameters, etc.

The biosensor-based devices, notably the blood glucose meters, are the most common POCT technology, which have been widely used for the detection of glucose. Different LOC platforms are being used in various POCT technologies. They are fully automated platforms that integrate all microfluidics-based bioanalytical steps such as sample treatment, separation, biomolecular detection, washing, signal detection, and data processing, storage, and transmission. The signal detection in most LOC platforms employs optical readout. The technologies and materials used for the production of LOC platforms have been fully characterized and standardized.

Test strips are another prominent POCT technology for detecting different analytes in a patient’s blood or urine sample. They are easy to use and easy to read, leading  to immediate on-the-spot analysis. The test strip comprises a solid support onto which porous matrices with dried assay reagents are integrated. The reaction starts as the biorecognition element present on the test strip detects the analyte, which leads to a visual change in colour on the test strip. The signal is read by inserting the test strip into a reader device.

LFA, one of the most widely used POCT technologies, is based on an immunochromatography format where the assay reagents are stored in dried form on various porous materials. Once the sample (urine or diluted blood) is dispensed at the designated area of the LFA cartridge, the sample flows in the lateral direction by capillary forces. It first interacts with the capture antibodies spotted at the reaction area leading to the formation of the immune complex, which is followed by binding to the detection antibodies immobilized at another area of the cartridge, thereby resulting in the formation of the sandwich immune complex. This leads to a visible colour change in the test and control lines, which facilitates rapid qualitative or semi-quantitative analysis. The POC pregnancy testing is done exclusively by LFA.

The multiplex analysis using DNA and protein microarrays is also being intensively investigated although the technology has not yet been commercialized for clinical POCT. It is envisaged that this upcoming technology would be fully automated, which would involve advanced microfluidics and the signal readout by electrochemical, chemiluminescence, fluorescence or evanescent wave techniques.

Emerging POCT technologies
Various POCT technologies have emerged during the last decade, which have tremendous potential for next-generation healthcare monitoring and management [1]. In 2011, the estimated total POCT market was US$15 billion and projected to reach US$18 billion by 2016. Of the total POCT market in 2011, 55% of it was in the US market, followed by 30% in Europe and 12% in Asia [2].

Cellphone (CP)-based devices
The most prospective emerging technology is CP-based devices. CPs have become ubiquitous with more than 7 billion global users that account for more than 95% of the world’s population. Moreover, about 70% of the CP users reside in the developing countries, where there is an imminent need for mobile healthcare (mH). The current generation of CPs are cost-effective and equipped with all the desired advanced features that facilitate personalized mH monitoring and management. The spatiotemporal tagging of the data by CP enables the real-time active response to epidemics and emergency situations. Various FDA approved and CE certified CP-based personalized healthcare devices have already been commercialized for the monitoring of basic physiological parameters such as blood glucose, blood pressure, pulse rate, blood oxygen saturation, body weight, body analysis parameters, electrocardiogram, physical activity, sleep and cardiac parameters (Fig. 1) [3]. Most of such commercial CP-based devices are developed by iHealth Labs, France. Similarly, CP-based technologies have been prepared for many POCT applications [4]. Cellmic, USA has developed a compact CP-based rapid-diagnostic-test reader for the readout of colorimetric and fluorometric LFA (Fig. 2). Of notice is the conversion of the CP into a compact and lightweight computational microscope for bright field, fluorescence, darkfield, transmission and polarized microscopy modes. Moreover, a CP-based flow cytometer based on optofluidic fluorescent imaging enabled the screening of pathogens in whole blood or water samples [5]. Another similar endeavour is the development of smartphone-based spectrophotometers for the detection of absorbance, fluorescent or chemiluminescent signals [6]. Various CP-based colorimetric readers [7], electrochemical sensing platform [8], angle-resolved surface plasmon resonance (SPR) system [9], and multiplex assays have also been developed [10].

Paper-based diagnostics
Considering simplicity and cost-effectiveness, paper-based diagnostics (PBD) are available in various formats: LFA, dipstick and microfluidic paper-based analytical devices (µPADs) [11, 12]. LFA are widely used in home pregnancy test strips to detect human chorionic gonadotropin in urine. It employs the dispensing of the sample onto the sample pad of the LFA test strip (fabricated from a nitrocellulose membrane). The sample flows laterally over a conjugate pad due to the capillary action provided by the absorbent pad, which leads to the binding of the analyte to conjugate particles (gold nanoparticles and upconversion nanoparticles). The signal detection can be visual, colorimetric, electrochemical, photoelectrochemical, chemiluminescent and electrochemiluminescent. The colorimetric PBD provide qualitative analysis by comparing the colour against a predetermined score chart. But the colour intensity can also be quantified using cameras, scanners, commercial test strip readers and hand-held colorimeters. A CP-based rapid-diagnostic-test reader is the most recent development that enables precise determination of colour intensity [13].

Paper can be patterned to fabricate two-dimensional (2D) or three-dimensional (3D) µPADs. The 3D µPADs, formed by stacking layers of the 2D paper, are ideal for multiplexing. The microfluidic paper-based electrochemical devices (µPEDs) can be fabricated by printing electrodes on paper.

The sensitivity of PBDs can be increased by employing enzymes and nanomaterials based signal enhancement strategies, which increases the costs and assay duration, and decreases the shelf-life. However, PBDs suffer from poor reproducibility, non-uniformity and variable accuracy on µPADs due to the passive capillary transport in paper substrates.

Lab-on-a-chip platforms
Various LOC platforms, such as the most widely used blood glucose testing strips, have been made for POCT. These platforms enable fully automated analysis by integrating all process steps in the operational procedure. The Piccolo Xpress™ whole blood chemistry analyser, developed by Abaxis Inc, is a prospective LOC-based POCT device that performs 14 tests on a single reagent LabDisk (8 cm diameter, barcoded).

Rapid assay formats
A prospective assay format is Optimiser™ ELISA [14] by Siloam Biosciences Inc,  which employs a novel microfluidic microtiter plate. It detects an analyte in just a few minutes using minimal reagent volumes and least number of steps. Other prospective formats are the wash-free AlphaLISA^® by Perkin Elmer, wash-free electrochemiluminescent ELISA by Meso Scale Diagnostics LLC, rapid one-step kinetics-based formats [15], CP-based easy immunoassay platforms [16], and COBAS^® Lab-in-a-Tube (LIAT) system by Roche Diagnostics.

Prolonged reagent storage strategies
The most prospective prolonged reagent storage strategies are the use of polysaccharides and saccharides (such as pullulan and trehalose), sugar alcohols, stabilizers, freeze drying, lyophilization, and reagent pouches. Use of POCT in remote areas, particularly in developing countries might be problematic due to inconsistent electrical power, lighting, and refrigeration.

Conclusions
The next decade will witness the revolutionary breakthroughs in POCT that will drastically cut down the costs and lead to considerably improved analysis with increased bioanalytical performance and capabilities. Multi-channel, high-throughput instruments are expected to expand the new concept of POCT, and there is an obvious need for the combination of POCT technology and communication technology. Non-invasive blood glucose monitoring remains the most important market for POCT, followed by coagulation, blood gas, chemistry, hematology, urinalysis, and cardiac. Also, molecular POCT technology has matured and began to move toward commercialization.

Future trends for POCT will rise due to new emerging technologies to make them well suited for low-resource or remote areas. POCT vendors increasingly offer more available types of tests with improved accuracy and minimal turnaround times. Thus, POCT will be more widely accepted as an addition to the current method of managing patients. Considering its limited test menus, clinicians still have to wait for other sophisticated laboratory tests before the action can be taken for treatment or further testing [17]. POCT evolves continuously and becomes an ‘extension’ of laboratory services, not a replacement of routine core laboratory testing.

References
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The authors
Sandeep Kumar Vashist*¹ PhD, John H.T. Luong² PhD
¹Vallo Med Health Care GmbH, Castrop-Rauxel, Germany
²Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland

*Corresponding author
E-mail: sandeep.vashist@vallomed.com

When Dr Margaret Chan, Director-General of WHO, stated that the health risks for both participants and spectators at the Rio Olympic games were “low and manageable” she was referring to possible exposure to Zika virus. But the other major health concern raised was the quality of the water, particularly for the one to two thousand athletes who competed in aquatic events.
Both the CDC and WHO provided advice on managing the risk of Zika virus infection whilst in Brazil, including use of insect repellent and wearing light clothing covering most of the body. Visitors were also urged to stay in air-conditioned accommodation so that open windows would not admit mosquitoes, and to avoid impoverished areas where lack of suitable sanitation encourages Zika vectors to breed. Abstention or the correct and consistent use of condoms was also advocated whilst in Brazil and for at least eight weeks after returning. Unfortunately we now know that the virus can persist in semen for much longer than eight weeks; two recent cases in men who contacted symptomatic Zika infection (and around 80% of cases are estimated to be asymptomatic) still had virus in their semen after 181 and 188 days respectively.
There was a rather bizarre occurrence during the games, when water in certain dedicated swimming pools turned from blue to green overnight. However the Fédération Internationale de Natation (FINA) Sports Medicine Committee confirmed that this resulted from “some of the chemicals used in the water treatment process running out”, causing the pH to be outside the normal range. FINA’s assurance that there was no risk to the health and safety of athletes was trusted; hopefully there was sufficient supporting evidence. However the potentially most serious health threat was the quality of the natural recreational water used for many aquatic events, water that is still significantly contaminated with untreated sewage. Athletes and visitors were urged to have vaccinations such as typhoid and Hepatitis A before travelling to Brazil, to avoid swallowing recreational water and to shower after being exposed to it.
But the risks to visiting athletes and participants were hopefully only transient compared with the enduring health hazards faced daily by the poorer citizens of Brazil. The best possible legacy of the Rio Olympic games would not be an increased interest in athletics but rather continued intensive Aedes mosquito control, widely disseminated information on sexual and vertical transmission of Zika virus, the sustained treatment of raw sewage and the provision of safe drinking water.