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Recent advances in diagnostic testing for viral infections

Recent advances in diagnostic testing for viral infections BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw010 .............................................. .................................................. .................................................. ............... Review article Recent advances in diagnostic testing for viral infections Selma Souf *Corresponding author: College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK. Email: soufselma@gmail.com Supervisor: Dr Jody Winter, College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK. .............................................. .................................................. .................................................. ............... Viral infectious diseases represent an important portion of global public health concerns with thousands of deaths annually. From serious pandemics and highly contagious infections to common influenza episodes, clinical prognosis often relies on early detection of the infectious agent. Thus, effective identification of viral pathogens is needed to help prevent transmission, set up appropriate therapy, monitor response to treatment and lead to efficient disease management and control. The aim of this review is to outline some of the recent technological advances in viral identification, including polymerase chain reaction, mass spectrometry and next-generation sequencing, and how they are applied in the diagnosis and management of viral infections. These powerful tools combine rapidity and efficiency in detecting viral pathogens and have revolutionized the field of clinical diagnostics. However, a number of drawbacks such as high cost have limited their use in many laboratories, particularly in resource-limited settings. On the contrary, the advent of microfluidic technology has attracted increasing interest from biomed- ical research groups, and could represent a challenging alternative to diagnose viral infections at lower cost. Key words: viral infections, laboratory testing, immunoassay, polymerase chain reaction, next-generation sequencing, mass spectrometry Submitted on 22 February 2016; editorial decision on 7 October 2016 .............................................. .................................................. .................................................. ............... of 2014 (WHO, 2014); more than 780 000 people die every Introduction year of Hepatitis B and up to 500 000 die of Hepatitis C- related liver diseases. The high prevalence of these diseases Global pandemics are serious threats to human life. While has raised the efforts for improving clinical diagnostics. well-established and characterized viruses such as The human immunodeficiency virus (HIV) and Hepatitis are still Effective prevention and clinical management of infec- killing millions of people, the emerging viruses are also prob- tious diseases are intimately linked to early and accurate lematic and have caused several serious outbreaks in the screening of pathogens, not only by detecting the infectious recent years. For example, the Severe Acute Respiratory particles in the organism but also by elucidating the aspects Syndrome-Coronavirus (SARS-CoV) in 2002–2003, Swine that confer resistance to therapy and immune escape pro- Influenza A (H1N1) in 2009 and Ebola Haemorrhagic fever files, including mutations and genotype disparity. outbreak in 2014 which has caused thousands of deaths in Therefore, rapid diagnosis benefits patients in allowing West Africa. timely therapy to prevent complications; and benefits public Morbidity and mortality rates are significantly high. health by collecting data for epidemiological studies, to pre- Thirty-five million people were infected with HIV in 2013, vent outbreaks and spreading of diseases. In that context, the and 350–400 millions chronic carriers of Hepatitis B virus. WHO has established many surveillance programs for disease According to the World Health Organization (WHO) report control such as the global strategy for control and assessment ............................................................................................... .................................................................. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 commercial re-use, please contact journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. of HIV drug resistance, the Global Influenza Surveillance and Specificity (also called the true negative rate) is a qualita- Response System for the control and monitoring of Influenza tive assessment, showing the capability of the test to distin- and the Global Policy on Viral Hepatitis. guish target from non-target analyte. This measure is expressed as the percentage of infection-free patients who At a smaller scale, clinical laboratories are a crucial point will have a negative result. The closer the values are to the for diagnosis of viral diseases by using a range of tools and reference, the higher the sensitivity and specificity of the test. machineries varying in cost and efficacy. On the contrary, operational parameters concern simpli- In a rapidly growing world of technology, the industry is city and ease in performing the test such as the turnaround continuously delivering up-to-date instruments but many fac- time (TAT). TAT is a key performance indicator defined as tors are limiting their implementation in healthcare settings the interval time between sample registration to result report- with low income, which unfortunately delays global benefit. ing. Sample preparation and any other pre-analytical steps This review will describe some of these advanced testing are within this interval. Assay completion in less than 60 min methods, how their specific characteristics have revolutio- is ideal so manufacturers aim to construct diagnosis instru- nized the field of laboratory diagnosis and what can be done ments allowing shorter TAT, which is particularly beneficial to overcome their limitations. for point-of-care settings (Hawkins, 2007). The WHO has established ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment- General principles of good free and Deliverable to end users) (Wu and Zaman, 2012) laboratory testing for diagnostics in resources-limited point-of care settings (Blacksell, 2012). The aim is to provide better management of Rigorous and accurate interpretation of laboratory results the disease, such as immediate delivery of the results and rapid guarantees effective clinical management of a disease and con- record of the disease status, to improve clinical decision-making. trol of its propagation (Lemon et al., 2007). However, errone- ous diagnosis could lead to financial and human loss. Traditional laboratory methods for In clinical testing of infectious diseases, it is crucial to determine precisely the presence or absence of the infectious the diagnosis of viral infections agent or its corresponding antibodies, to prove current or past exposure. Therefore, the ability to say precisely whether For long time, clinical laboratories have relied on a diverse the person is infected or not, and to determine the course of range of techniques to diagnose viral infections. the infection has a positive impact on the therapeutic strategy. In developed countries, electron microscope (EM) has for The usefulness and reliability of laboratory results depend long time been considered an efficient tool for direct detection on the performance and operational parameters of the of viruses through visualization and counting of the viral par- intended test. The performance parameters directly relate to ticles in body fluids, stools or histopathologic samples. The the results by estimating their Accuracy, Precision, Sensitivity identification is based on morphological characteristics spe- and Specificity (Lalkhen and McCluskey, 2008). While these cific to each virus family and requires a certain amount of vir- are statistical values (percentages), they have different expla- al particles (up to 106 particles/ml). However, specimen nations and involve comparison with the reference method or preparation that must be performed beforehand (Goldsmith ‘gold standard’ for the desired test (Guzman et al., 2010). and Miller, 2009) may reduce the virus concentration which makes the analysis harder. In addition, electron microscopy Accuracy describes how close the obtained results are to requires substantial technical skills and expertise; the presence those obtained with the reference method and it is expressed of unusual or look-alike structures such as cellular debris and as a percentage of correct results. Precision refers to the reli- organelles may confuse the electron microscopist with their able reproduction of one test on the same sample, and obtain- virus-like shapes (Fauquet et al., 2005). ing similar results. The combination of EM with culture-based methods has These two parameters must be regularly monitored using shown great contribution in the diagnosis of viral infections, local quality control (QC) and quality assurance (QA) pro- along with serology testing for detection of antibodies tar- cedures in order to maintain reliability of the test. In perfect geted against the virus. These conventional methods are still conditions, an ideal test would have 100% accuracy and fundamental practices in many hospital laboratories. 100% precision; however, external factors and methodo- logical differences can cause small variations. Cell culture is one of the most popular methods for isolat- ing viruses using cell lines. These latter vary according to the Sensitivity (also called the true positive rate) is the percent- targeted viruses (for example; rhesus monkey kidney cells are age of patients with confirmed infection (by the ‘gold stand- used for isolation of Influenza A virus). ard’ method) who will have positive results. It is usually measured by the lower limit of detection of the analyte produ- Evidence of virus growth is seen through the cytopathic cing a positive result. effect (CPE) exhibiting specific characteristics and alterations of ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. the cells (Robbins, Enders and Weller, 1950). The virus defini- test is time consuming, and demanding, particularly in terms tive identification is then performed using Immunofluorescence of QC. (IF) staining. Nevertheless, virus isolation using cell culture is This list of conventional techniques is not exhaustive, and not ideal in case of viruses not amenable to growth in cell lines only the most commonly used were cited. The following sec- (norovirus, hepatitis virus) or producing CPE (Papafragkou tions will discuss more recent developments in the field, which et al., 2013). replaced the conventional practices and allowed screening In addition, small volumes of the sample may not allow (qualitative test), surveillance (quantitative test) and confirm- inoculation of many cell types, and thus compromise the ation of diagnosis. results. For example, the standard inoculation of a cell culture medium with cerebrospinal fluid (CSF) sample requires a min- Recent methods in the diagnosis imum of 0.2 ml; however, much more is needed to inoculate a combination of different cell types, which could be invasive to of viral infections the patient (McIntyre, 2007, Hematiana et al., 2016). As stated before, this review will discuss only some of the main The time required for isolating viruses by cell culture is developments in diagnostic technologies that have ushered the very long (weeks), limiting the usefulness of this technique new era of clinical virology, and lay out their key advantages when rapid diagnosis is needed. Cell culture needs highly and limitations that will be summarized in Table 1. skilled and experienced personnel for accurate interpretation of the CPE and adequate facilities for handling mammalian cell lines or highly pathogenic viruses. Immunoassay-based tests In the shell vial technique, one of the rapid culture methods, Antibodies produced immediately after invasion of a foreign inoculated cells are subjected to centrifugation, incubation and substance can inform on primary infection, reinfection or a then IF staining with monoclonal antibodies, specific to the reactivation state. Therefore, measuring the level of immuno- range of viruses suspected to cause the infection, such as globulins (Ig) is a widely considered approach for the diagno- respiratory viruses, Herpes simplex virus or Varicella zoster sis of viral infections. virus. The virus-induced antigens are detected 2–4 days later. Automated immunoassay-based methods are among the Rapid culture has limited benefits as it does not target a most frequently used for testing, and are effective because of wide range of viruses and has low sensitivity. the high specificity and binding affinity between antigen and Complement fixation test (CFT) is one of the oldest meth- antibody. Therefore, the principle of the test relies in the for- ods in the history of clinical virology (Casals and Palacios, mation of an immuno-complex between antibody present in 1941). The complement reacts only with antigen–antibody the patient sample and synthetic antigen present in the reagent complex in a non-specific manner. Thus, in the presence of or vice versa, to generate a measurable signal. the complex, the complement is not free to interact with sensi- Immunoassays use labels conjugated to synthetic anti- tized sheep red blood cells (RBCs) used as an indicator, and bodies or antigens which are linked to a solid phase, and used which remain unlysed. The test is said to be ‘positive’. to capture corresponding antigens or antibodies present in CFT is supposedly easy to perform, convenient and sera samples. These labels could be radioactive isotopes, requires inexpensive material. However, it is labour intensive enzymes that cause change in colour or light-generating sub- and lacks sensitivity. In-house standardization through titra- stances. Consequently, this principle has generated several tion of the reactants and preparation of controls is crucial for methodologies for the testing. obtaining effective testing. Radio-immunoassay (RIA) is probably the initiating meth- Haemagglutination inhibition test is generally used for od (1960s); it uses radioisotopes (such as Iodine 125) to label detecting arboviruses, influenza and parainfluenza virus sub- antigen or antibody. The amount of substance to analyse is types and provides relative quantitation of the virus particles. determined by the amount of the generated radioactivity. RIA The principle relies on the capacity of haemagglutinin (HA); a is a highly sensitive method but the main drawback is the viral protein present in the envelope, to bind to erythrocytes handling and disposal of hazardous radioactive substances. (RBC) and to form a lattice pattern termed ‘agglutination’.In The enzymatic labelling alternative using alkaline phos- the assay, serial dilutions of the sample serum are added to a phatase or horseradish peroxidase as markers is, however, the fixed amount of viral HA and agglutinable RBCs. If Influenza most widely used and was long considered a reference method antibodies are present in the serum, the agglutination process (Engvall and Perlmann, 1972; Voller, Bidwell and Bartlett, is prevented. The corresponding dilution rate at which com- 1976). These enzymes induce emission of signals or change in plete haemagglutination is observed and considered. colour respectively, and allow the amount of analyte of inter- Variants of the agglutination assay are used for the diagno- est to be measured. This enzyme-linked immunoassay (EIA) sis of wider range of viral diseases other than influenza has numerous variants, including ELISA, and they differ in (Grandien et al., 1987; Sandeep et al., 2002). However, the the enzyme used and the signal detection principle. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. Table 1. Summary of the main viral diagnostic methods Diagnostic Principle Strengths Weaknesses Variants References technique Immunoassay Formation of High sensitivity Must rely on QC RIA Gupta et al. (2015), antigen– High specificity assurance EIA (FPIA, MEIA, Mixson-Hayden antibody High speed High risk of CLIA) et al. (2015) through throughput interferences recognition Quick TAT (20 min or High cost and binding less) Different types of tags, labels Automated method NAAT Amplification and High sensitivity Longer run time RT-PCR García-Arroyo et al. detection of High specificity Requires specific qPCR (2016), Reijans et al. sequences Multiplexed platforms primers for the NASBA (2008), Renois et al. from the viral Genotyping targets TMA (2010), Afshar and genome (DNA Determination of the Mollaie (2012), or RNA) viral load Mercier-Delarue New compact and et al. (2014), Wu portable formats et al. (2014) NGS Polymerization of High sensitivity High cost Pyrosequencing Capobianchi et al. DNA template High specificity Needs Fluorescently (2013), Bartolini by Identification of novel bioinformatics labelled dNTP et al. (2015), Lowe incorporation genomic sequences skills for data Detection of et al. (2016), Liu of labelled Genotyping analysis released et al. (2013), Van dNTPs, and Accurate detection of Delay in use for hydrogen ion den Hoecke et al. terminate the mutations and drug routine clinical (H+) (2015), Rothberg extension resistant mutations diagnostics et al. (2011) MS Ionization of the High sensitivity Expensive MALDI-TOF MS Lévêque et al. (2014), sample, then Versatility equipment ESI MS Mengelle et al. separation and Cost-effectiveness Limited database Often combined (2013), Qian et al. detection of High workload library with other (2014) the particles methods: PCR- according to MS their mass-to- charge ratio (m/z) CLIA, chemiluminescent immunoassay; dNTP, deoxyribonucleotide triphosphate; EIA, enzyme immunoassay; ESI, electrospray ionization; FPIA, fluorescence polarization immunoassay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MEIA, micro-particle enzyme immunoassay; MS, mass spectrometry; NGS, next-generation sequencing; NAAT, nucleic acid amplification test; NASBA, nucleic acid sequence-based amplification; qPCR, quantitative polymerase chain reaction; RIA, radio-immunoassay; RT-PCR, real-time polymerase chain reaction; TMA, transcription-mediated amplification. The main variants of EIA are as follows: In clinical practice, serological studies of Hepatitis B rely on immunoassay as a key tool for detection of Hepatitis B virus � Fluorescence polarization immunoassay (FPIA): uses fluor- (HBV) markers. The up-to-date versions of immunoassay escent label and polarized light. methods are able to detect the lowest levels of the main � Micro-particle enzyme immunoassay (MEIA)(Tassopoulos marker HBsAg, undetectable by conventional methods, par- et al.,1997): widely used and relies on alkaline phosphatase ticularly in sera samples of asymptomatic patients. The latest enzyme and a corresponding fluorescent substrate. screening standard detects as little as 0.05 IU/ml, the equiva- � Chemiluminescent immunoassay (CLIA), which uses lent of 0.2 ng/ml viral antigen (Deguchi et al., 2004) and high chemiluminescent or light-emitting labels. Companies like sensitivity (>99%) was observed at different stages of the dis- ROCHE or Abbott are exploiting this method, and high- ease or even in patients showing seroclearance. volume laboratories are gradually replacing MEIA technol- A newly developed CLIA allowed denaturation of the ogy with CLIA for its high-speed throughput and ease of HBV particle, and used monoclonal antibodies against inner measurement. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. and outer structural epitopes in order to obtain higher assay diagnostic practices such as screening donated blood for sensitivity (Shinkai et al., 2013) and sufficient information on transfusion-transmitted viruses (CMV, HIV, HCV, …) the HBV genotypes. (Jackson, 1990). Automated immunoassay techniques for virus detection The most widely used variants of conventional amplifica- overcome some of the limitations encountered with the con- tion are real-time PCR (quantitative PCR) and reverse ventional tests, particularly the delay to response. transcription-PCR (RT-PCR). Both are nowadays becoming benchmarks in assessing the viral load, and while the first Limitations method quantifies DNA throughout the reactions in real time (Ntziora et al., 2013); the second performs RT of the mRNA Despite the popularity of immunoassay in clinical testing, (RNA messenger) and amplifies the resulting cDNA (comple- erroneous results may occur for many reasons, which confer mentary DNA). It also quantifies RNA. The combination of inconsistencies to the testing. both techniques increases sensitivity in detecting viruses, par- � Immunoassays are more prone to interferences than any ticularly influenza viruses. The WHO recently approved a other assay, which leads to false-positive or false-negative newly developed reverse transcriptase-PCR assay after the results. In most cases, interferences are due to the presence first death from MERS-CoV (Middle East Respiratory of agents with structural similarities to the reagents Syndrome-Coronavirus) infection reported in 2012 (Abd El (Miller, 2004), and fluctuate according to the concentra- Wahed et al., 2013). tion of the interfering substance and the analyte. Other amplification-based tests such as nucleic acid Endogenous antibodies (autoantibodies, hetero-antibodies sequence-based amplification (NASBA) and transcription- or human anti-animal antibodies) are commonly the main mediated amplification (TMA) are suited for detection of culprit in these interferences (Emerson and Lai, 2013). RNA viruses by amplification of the mRNA instead of conver- Their binding to capture antibodies and detection anti- sion to cDNA (Mercier-Delarue et al.,2014, Wu et al.,2014). bodies in the absence of antigen (analyte) mainly leads to false-positive result (Berth and Willaert, 2016). Among the latest enhancements in PCR systems, extrac- � Accurate QC guarantees reliable results and assumes that the tion, amplification and detection steps have been combined assay is performed well. Therefore, poor QC measures lead within one unit. As an example, BioFire Film Array technol- to low accuracy and precision. In immunoassays, QC is ogy applies two sequential amplifications and can detect a assured by regular calibration and control of the reagents, panel of respiratory viruses with high sensitivity in 1 h and using standardized solutions delivered by the manufacturers. within a single instrument (Pierce et al., 2012). � The high cost of reagents and equipment is another draw- On the contrary, microfluidic technology has also benefited back in immunoassays, particularly for resource-limited PCR-based systems; and allowed further decrease in the settings, considering that the most accurate and sensitive detection time (15 min), with high sensitivity and cost- methods are automated. effectiveness. In the 2009 epidemic, detection of Influenza A virus H1N1 greatly relied on a chip-type real-time PCR Amplification-based assays instead of the tube-based principle. The portable format of such systems is very convenient during epidemics and out- Developed by Mullis and Faloona (1987), nucleic acid ampli- breaks (Song et al., 2012). fication by polymerase chain reaction (PCR) has revolutio- Multiplexed NAAT were designed to detect multiple nized the field of molecular diagnosis. The basic PCR assay viruses or subtypes in a single run. Their detection platforms relies on extraction and purification of the nucleic acid, then can comprise up to 20 viruses using diversified panels exponential amplification of the target sequence, using a (Mahony et al., 2007), for example, concurrent detection of thermostable polymerase enzyme and specific primers. The HAV, HBV and HCV infections, as well as co-infections resulting amplicons are then identified using a fluorescence- (Park et al., 2012). based detection system, and the result is reported in inter- national units IU/ml. In Hepatitis B, PCR is considered the gold standard meth- Soon after its invention, modifications in PCR were tested od for assessing the HBV DNA level with high accuracy, and patented, with the aim of improving the assay capabil- and the assays are standardized according to the WHO ities. The term nucleic acid amplification tests (NAAT) was International Standard for Hepatitis B virus (NIBSC), such as applied to this range of new variants. the reference Roche Cobas TaqMan HBV test. The test was the first FDA approved assay, with a detection limit of NAAT are very popular in the diagnosis and management 10.2 IU/ml. The combined system with Cobas AmpliPrep has of viral infections (HBV, HCV, HIV, Influenza viruses, …) an automated sample extraction process and highly sensitive because they allow determination of the viral load. In other detection for all HBV genotypes (Pyne et al., 2012). terms, quantitation of the viral nucleic acid by amplifying the In HIV patients taking antiretroviral treatment (ART), target sequence thousands-fold. In most cases, they are now ultrasensitive assays permit quantitation of low copy numbers considered a reference, or ‘gold standard’ method for ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. of HIV subtypes (<50 copies/ml) and long terminal repeats in information on species, genotypes and the occurrence of latent reservoirs and from cell lysates omitting the nucleic mutations conferring virulence or resistance to antivirals. acid extraction step (Vandergeeten et al., 2014). Such assays Pyrosequencing is currently the variant of choice within could be valuable tools in large cohort studies or pandemic NGS systems. Based on the detection of pyrophosphate (PPi) situations. after incorporation of a nucleotide in a DNA polymerization process, it uses luciferase to catalyse light-generating pro- Limitations cesses, and the collected light is then recorded. Although The limitations of PCR are an important parameter to con- Illumina is considered the most frequently used pyrosequen- sider, despite the cost-effectiveness and reliability in the diag- cing platform, 454 FLX; a subsidiary company of Roche, was nosis of viral infections. The risk of contamination is very the first high-throughput analyser in the market and was used high while handling, especially during the sample preparation to determine human papillomavirus (HPV) types (Barzon step, in addition, real-time PCR has a longer run-time (2–5h) et al., 2011), subtypes and variants, present in cervical speci- by comparison to other techniques. mens. Other variants detect hydrogen ions that are released throughout nucleotide incorporation reaction (Rothberg et al. In the case of influenza, many PCR-based assays were 2011). designed to detect only a particular subtype of the virus responsible for important pandemics (Hall et al., 2009), and Generating high volumes of sequence data has allowed the the need for designing specific primers for the target requires compilation of viral nucleotide databases and acquisition of handling by experienced operators, capable of detecting de novo sequences to understand the genetic variability of errors, particularly that PCR is prone to false-positive results. viruses (Szpara, Parsons and Enquist, 2010). HIV is by far the most sequenced because of the global priority of AIDS as a The high mutation rate of some viruses could trigger muta- serious endemic, and because of the high mutation rate of the tion within PCR primer regions of the viral genome, which virus. would lead the virus to escape the detection by this assay. So far, only gene sequencing methods have been successful in genotyping tricky HBV, unlike conventional PCR or sero- Next-generation sequencing logical assays. Therefore, sequencing has allowed better clin- ical management of HBV infection and related complications Next-generation sequencing (NGS) is one of the greatest (Margeridon-Thermet et al., 2009). achievements of the modern era. Beyond genome sequencing from known organisms, it allowed discovery of novel viruses In data analysis, recent technical approaches have responsible for unknown human diseases (Palacios et al., included adjustment of the software reading platforms for 2008), and tracking of outbreaks and pandemics such as influ- simultaneous detection of genotypes and mutants of clinical enza (Baillie et al., 2012) to understand their emergence and importance (Germer et al.,2013), and partial sequencing of transmission profiles (Leung et al., 2014; Isakov et al., 2015). the HBV S gene with high sensitivity (98.64%), considering that portion as the site of most drug-resistance mutations The journey began in the 1970s with the works of Sanger (Wang et al.,2013). Nevertheless, implementation of NGS and Barrel, followed by Maxam and Gilbert, who first in clinical settings is increasing, particularly for detecting initiated the principle of sequencing oligonucleotides via low-abundance drug-resistance patterns such as in HIV enzymatic polymerization, using radiolabelled primers. In and HCV (Palmer et al.,2005; Verbinnen et al.,2010). their experimental protocols, they relied on using dideoxy- Nucleotide sequencing of HCV sub-genomic regions is now nucleotides to terminate the extension of the polymer, hence the method of choice for genotyping. the name: chain termination or dideoxynucleotide method (Sanger, Nicklen and Coulson, 1977). Limitations Since then, the principle has remained virtually the same, The principal requirements for NGS are initially access to a but improvements and automation have dramatically in- sequencer, and considerable skills in bioinformatics and creased the speed and accuracy in delivering maximum vol- expertise in data analysis, plus adequate handling systems for ume of data comparing to dideoxynucleotide sequencing storage of generated data. Adding to that, despite the out- (Shedure and Ji, 2008). Technically, NGS is inclusive of three standing results delivered by prototypes in trials, many are main steps: sample preparation, sequencing and data analysis. still at the research level, and not yet approved for use in rou- Systems available in the market differ mostly in their tine clinical practice (Jiangqin et al., 2015). sequencing or reading techniques. Efficient and accurate clin- ical diagnosis of viral infections using NGS is increasingly NGS is undeniably a key technology in specialized clinical aiming to provide accurate longer read-length in a shortest laboratories, but its implementation is still a challenge in time and at a lower cost. Bioinformatic platforms are key many countries, where not only their resource-limited settings components of the sequencing process. They allow interpret- cannot afford a sequence analyser, sample and library prepar- ation of the sequencing output through computational ana- ation, but the vast majority of the population cannot afford lysis (Naccache et al., 2014), and then convert it into useful the cost of the test. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. Limitations Mass spectrometry The main limitation of MS is the high cost, particularly in Mass spectrometry (MS) is nowadays a benchmark of labora- high pandemic areas, which are usually the poorest; not all tory qualitative and quantitative investigation, particularly in laboratories can afford a mass analyser for their activities. bacteriology (Sauer and Kliem, 2010). The second major drawback is within the reference library. The identification is limited by known data from well- The principle of MS relies on converting the sample into identified organisms only; therefore, rare mutations cannot be charged particles (ions) by ionization process. These ions are detected if they do not exist within the reading platform, but separated according to their mass-to-charge ratio (m/z) and there is hope that MS database libraries will rapidly expand. analysed by a detector. The result obtained is compared to a reference database (library), existing within the system and delivered as an interpretive spectrum. Advantages of the recent methods In clinical laboratories, matrix-assisted laser desorption � High sensitivity and specificity assays approaching 100%. ionization (MALDI) and electrospray (ES) are the most used � Possibility to combine different methods in one assay ionization methods because they allow processing of consid- enhances detection capability and accuracy (PCR-MS, erable amounts of analyte (Emonet et al., 2010). These microfluidic chip technology-based PCR,…). approaches have extensively been evaluated experimentally � Automation of the assays reduces the number of operators and provided excellent results, either used alone or combined and manual workload. with other molecular methods, such as PCR, in order to � Small sample volumes are needed, so the assays can still be enhance sensitivity. The combination (RT-PCR/ESI-MS) was performed in particular cases (CSF, new-borns, etc.) able to detect viral pathogens usually undetected by regular � Some assays such as MS remain efficient after several freez- testing methods, and provided rapid and detailed data (types ing–thawing of the samples (archived samples). and subtypes) within a short time (Lévêque et al., 2014). � Rapid TAT: minimum time required from sample collec- Detection of genomic variations or mutations in Influenza tion to results reporting is 30 min, and microarray tools A using MALDI-TOF has been a key tool in the management can deliver results within seconds. of outbreaks (Chen et al., 2011). MS application to the struc- � Low detection limit: 10–100 copies/ml by PCR. tural investigation of biomolecules showed the efficiency of � Multiplex reactions: detection of a wide range of patho- MALDI-TOF-MS coupled to antibody magnetic nanoparti- gens in a single run is time saving. cles in detecting influenza viruses (Chou et al., 2011; Yea � Detection of rare drug-resistance patterns. et al., 2011), through concordant results with the gold stand- � NGS generates many sequencing data per run and ard PCR-based method. This blend of two powerful machin- sequences long-reads. eries (PCR-MS) can detect drug resistance to antiviral therapy � Low reaction cost when using microfluidic chips, and use as well as the presence of multiple viruses within the same in POC settings. sample and diagnose for co-infections, when assays are � Lower risk of contamination by processing single tube multiplexed. within one unit. The increasing interest in MALDI-TOF MS in clinical vir- ology has led to research advances in the diagnosis of The latest technologies and Hepatitis B. The method was in fact not only able to detect resource-limited settings HBV at relatively low viral load levels (100 HBV DNA cop- ies/ml) (Hong et al., 2004) but it also allowed detection of up The methods described above have shown outstanding per- to 60 HBV variants and genotypes at a cost lower than $10/ formance in saving thousands of lives in developed countries. sample for all the variants with high throughput (Luan et al., However, accurate diagnosis is still a challenge in resource- 2009). The same method was applied for genotyping and suc- limited settings because of the difficulty in acquiring these cessfully detected the eight HBV genotypes accurately equipment and technical expertise. While expensive tools (Ganova-Raeva et al., 2010), and also minor HCV genotypes such as PCR-based systems or mass analysers are only avail- occurring at very low level (Kim et al., 2005). able in reference laboratories or in military facilities, point-of- Mass spectrometric-based methods are versatile, sensitive, care or near-patients clinics that serve more than 80% of the rapid and cost-effective, and do not require interpretation poor population struggle with the testing devices available. As software for data analysis. The automated machinery necessi- an example, the WHO has established guidelines for monitor- tates easy sample preparation and fewer operators. The ana- ing ART efficacy in HIV-1 infected patients in developing lysis capacity can reach up to 960 specimens/day, which countries, by suggesting only CD4+ cell count and ELISA- makes it suitable for routine diagnosis in high-volume labora- based assays as alternatives to the expensive tests (WHO, tories and large-scale studies. Tests can also be performed effi- 2006a). Ultrasensitive p24 (Patton et al., 2008) and the ciently on archived specimen. ExaVir assay (Kokkayil et al., 2014), measure the level of p24 ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. antigen and the activity of reverse transcriptase, respectively, Improvement of QC programs, QA and standardization to replace viral load monitoring. Unfortunately, their lack of of assays, kits and reagents are important to fulfil require- sensitivity has induced failure of treatment and emergence of ments for accuracy. The Centre for Disease Control and resistant strains (Vekemans, John and Colebunders, 2007). Prevention (CDC) is continuously implementing laboratory- testing guidelines particularly for HIV, before further tests Hepatitis is another expensive burden to manage in poor will be approved (CDC, 2014). countries, where accurate assessment of the epidemiological profile is hard to establish. In the case of HBV, most POC The WHO and non-profit organizations such as the facilities rely on screening for HBsAg solely using rapid diag- Foundation for Innovative New Diagnostics (FIND) aim to nosis tests. These low-cost devices do not allow determination maximize efforts to implement surveillance programs and of the course of the disease to inform whether to initiate an control of communicable diseases (Loman et al., 2012), set up antiviral treatment or not. new policies and improve diagnostic services in low resource settings. Automation, microfluidics and Conclusion future prospects The recently developed viral diagnostic methods are reshap- ing the field of clinical microbiology, and could contribute Providing high-level epidemiological monitoring of viral diseases to reducing the prevalence of serious infectious diseases. is undeniably a global public health ambition, and despite rapid However, the technical capabilities alone are insufficient if progress in the development of diagnostic methods in recent not supported by health promotion strategies to increase years, improvements are needed for better cost, size (Loman awareness about the importance of early detection and regu- et al., 2012; Frey et al.,2014)and TAT (Xu et al., 2013). lar screening of persons at high risk. After the Ebola Haemorrhagic fever in 2014, the US gov- Finally, good quality diagnosis has a cost that only devel- ernmental Department of Energy has developed an innovative oped countries can afford in routine practice so far, and this rapid and portable test to detect specifically Ebola virus is delaying the implementation of new methods in the devel- within seconds. The test aims to target other RNA and exotic oping world and the endemic areas. However, there is hope viruses such as Dengue and West Nile for effective manage- that efforts will continue towards developing new good ment of viral outbreaks. Other examples include fully NAAT quality tests affordable in low-income countries, which systems and small devices to sequence single molecule DNA would substantially strengthen disease control strategies for using nanopore technology (Eisenstein, 2012). their populations. Microfluidic technology is regarded with optimism for the management of infectious diseases by allowing timely ther- Author biography apy. It provides a key potential solution for remote areas and near-patients facilities by avoiding turnaround trips of the Selma obtained an MSc with Honours in Biomedical sciences patients between the clinic and the laboratory. Their use is from Nottingham Trent University. She works as a biomed- also beneficial where time is crucial, or when physical spaces ical scientist at Institut Pasteur, within the Immunoassay div- do not allow setting up of conventional methods. ision, and has particular interest in laboratory diagnosis Lab-on-a-chip (LOC) is a very small device that integrates issues in low-income settings and how they can be solved at laboratory processes within a few square centimetres. It uses lower cost. She also has interest in structural biology and very small volume of samples to perform immediate reactions microbiology. within the chip or in a portable device. The reactions vary from nucleic acid amplification and detection, to cell count References and immunoassays; therefore, microfluidic diagnostics com- pete with large instruments in performing laboratory tests at a Abd El Wahed, A., Patel, P., Heidenreich, D. et al. (2013) Reverse tran- lower cost, to benefit low-income settings and remote areas. scription recombinase polymerase amplification assay for the detection of Middle East respiratory syndrome coronavirus, PLoS A wide range of LOC devices were approved by the FDA in Currents Outbreaks, 5, e2e8364. the diagnosis of viral infections such as Influenza (Cao et al., 2012), HIV (Alyassin et al.,2009)and HBV(Zhi et al.,2014) Afshar, R. M. and Mollaie, H. R. (2012) Detection of HBV resistance to and further development is in progress. As examples, Daktari lamivudine in patients with chronic Hepatitis B using zip nucleic Diagnostics use affinity chromatography for CD4+T-cell count acid probes in Kerman, Southeast of Iran, Asian Pacific Journal of as an alternative to flow cytometry to monitor HIV in develop- Cancer Prevention, 13 (8), 3657–3661. ing countries (Cheng et al.,2007). The bench top analyser GeneXpert made by Cepheid has an integrated sample prepar- Alyassin, M. A., Moon, S., Keles, H. O. et al. (2009) Rapid automated cell ation and PCR system for molecular diagnosis of influenza and quantification on HIV microfluidic devices, Lab on a Chip, 9 (23). 10. other bacterial infections in a light portable format. 1039/b911882a. ............................................................................................... .................................................................. 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Recent advances in diagnostic testing for viral infections

Bioscience Horizons , Volume doi (10.1093) – Nov 30, 2016

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BioscienceHorizons Volume 9 2016 10.1093/biohorizons/hzw010 .............................................. .................................................. .................................................. ............... Review article Recent advances in diagnostic testing for viral infections Selma Souf *Corresponding author: College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK. Email: soufselma@gmail.com Supervisor: Dr Jody Winter, College of Science and Technology, Nottingham Trent University, Clifton campus NG11 8NS, Nottingham, UK. .............................................. .................................................. .................................................. ............... Viral infectious diseases represent an important portion of global public health concerns with thousands of deaths annually. From serious pandemics and highly contagious infections to common influenza episodes, clinical prognosis often relies on early detection of the infectious agent. Thus, effective identification of viral pathogens is needed to help prevent transmission, set up appropriate therapy, monitor response to treatment and lead to efficient disease management and control. The aim of this review is to outline some of the recent technological advances in viral identification, including polymerase chain reaction, mass spectrometry and next-generation sequencing, and how they are applied in the diagnosis and management of viral infections. These powerful tools combine rapidity and efficiency in detecting viral pathogens and have revolutionized the field of clinical diagnostics. However, a number of drawbacks such as high cost have limited their use in many laboratories, particularly in resource-limited settings. On the contrary, the advent of microfluidic technology has attracted increasing interest from biomed- ical research groups, and could represent a challenging alternative to diagnose viral infections at lower cost. Key words: viral infections, laboratory testing, immunoassay, polymerase chain reaction, next-generation sequencing, mass spectrometry Submitted on 22 February 2016; editorial decision on 7 October 2016 .............................................. .................................................. .................................................. ............... of 2014 (WHO, 2014); more than 780 000 people die every Introduction year of Hepatitis B and up to 500 000 die of Hepatitis C- related liver diseases. The high prevalence of these diseases Global pandemics are serious threats to human life. While has raised the efforts for improving clinical diagnostics. well-established and characterized viruses such as The human immunodeficiency virus (HIV) and Hepatitis are still Effective prevention and clinical management of infec- killing millions of people, the emerging viruses are also prob- tious diseases are intimately linked to early and accurate lematic and have caused several serious outbreaks in the screening of pathogens, not only by detecting the infectious recent years. For example, the Severe Acute Respiratory particles in the organism but also by elucidating the aspects Syndrome-Coronavirus (SARS-CoV) in 2002–2003, Swine that confer resistance to therapy and immune escape pro- Influenza A (H1N1) in 2009 and Ebola Haemorrhagic fever files, including mutations and genotype disparity. outbreak in 2014 which has caused thousands of deaths in Therefore, rapid diagnosis benefits patients in allowing West Africa. timely therapy to prevent complications; and benefits public Morbidity and mortality rates are significantly high. health by collecting data for epidemiological studies, to pre- Thirty-five million people were infected with HIV in 2013, vent outbreaks and spreading of diseases. In that context, the and 350–400 millions chronic carriers of Hepatitis B virus. WHO has established many surveillance programs for disease According to the World Health Organization (WHO) report control such as the global strategy for control and assessment ............................................................................................... .................................................................. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 commercial re-use, please contact journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. of HIV drug resistance, the Global Influenza Surveillance and Specificity (also called the true negative rate) is a qualita- Response System for the control and monitoring of Influenza tive assessment, showing the capability of the test to distin- and the Global Policy on Viral Hepatitis. guish target from non-target analyte. This measure is expressed as the percentage of infection-free patients who At a smaller scale, clinical laboratories are a crucial point will have a negative result. The closer the values are to the for diagnosis of viral diseases by using a range of tools and reference, the higher the sensitivity and specificity of the test. machineries varying in cost and efficacy. On the contrary, operational parameters concern simpli- In a rapidly growing world of technology, the industry is city and ease in performing the test such as the turnaround continuously delivering up-to-date instruments but many fac- time (TAT). TAT is a key performance indicator defined as tors are limiting their implementation in healthcare settings the interval time between sample registration to result report- with low income, which unfortunately delays global benefit. ing. Sample preparation and any other pre-analytical steps This review will describe some of these advanced testing are within this interval. Assay completion in less than 60 min methods, how their specific characteristics have revolutio- is ideal so manufacturers aim to construct diagnosis instru- nized the field of laboratory diagnosis and what can be done ments allowing shorter TAT, which is particularly beneficial to overcome their limitations. for point-of-care settings (Hawkins, 2007). The WHO has established ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment- General principles of good free and Deliverable to end users) (Wu and Zaman, 2012) laboratory testing for diagnostics in resources-limited point-of care settings (Blacksell, 2012). The aim is to provide better management of Rigorous and accurate interpretation of laboratory results the disease, such as immediate delivery of the results and rapid guarantees effective clinical management of a disease and con- record of the disease status, to improve clinical decision-making. trol of its propagation (Lemon et al., 2007). However, errone- ous diagnosis could lead to financial and human loss. Traditional laboratory methods for In clinical testing of infectious diseases, it is crucial to determine precisely the presence or absence of the infectious the diagnosis of viral infections agent or its corresponding antibodies, to prove current or past exposure. Therefore, the ability to say precisely whether For long time, clinical laboratories have relied on a diverse the person is infected or not, and to determine the course of range of techniques to diagnose viral infections. the infection has a positive impact on the therapeutic strategy. In developed countries, electron microscope (EM) has for The usefulness and reliability of laboratory results depend long time been considered an efficient tool for direct detection on the performance and operational parameters of the of viruses through visualization and counting of the viral par- intended test. The performance parameters directly relate to ticles in body fluids, stools or histopathologic samples. The the results by estimating their Accuracy, Precision, Sensitivity identification is based on morphological characteristics spe- and Specificity (Lalkhen and McCluskey, 2008). While these cific to each virus family and requires a certain amount of vir- are statistical values (percentages), they have different expla- al particles (up to 106 particles/ml). However, specimen nations and involve comparison with the reference method or preparation that must be performed beforehand (Goldsmith ‘gold standard’ for the desired test (Guzman et al., 2010). and Miller, 2009) may reduce the virus concentration which makes the analysis harder. In addition, electron microscopy Accuracy describes how close the obtained results are to requires substantial technical skills and expertise; the presence those obtained with the reference method and it is expressed of unusual or look-alike structures such as cellular debris and as a percentage of correct results. Precision refers to the reli- organelles may confuse the electron microscopist with their able reproduction of one test on the same sample, and obtain- virus-like shapes (Fauquet et al., 2005). ing similar results. The combination of EM with culture-based methods has These two parameters must be regularly monitored using shown great contribution in the diagnosis of viral infections, local quality control (QC) and quality assurance (QA) pro- along with serology testing for detection of antibodies tar- cedures in order to maintain reliability of the test. In perfect geted against the virus. These conventional methods are still conditions, an ideal test would have 100% accuracy and fundamental practices in many hospital laboratories. 100% precision; however, external factors and methodo- logical differences can cause small variations. Cell culture is one of the most popular methods for isolat- ing viruses using cell lines. These latter vary according to the Sensitivity (also called the true positive rate) is the percent- targeted viruses (for example; rhesus monkey kidney cells are age of patients with confirmed infection (by the ‘gold stand- used for isolation of Influenza A virus). ard’ method) who will have positive results. It is usually measured by the lower limit of detection of the analyte produ- Evidence of virus growth is seen through the cytopathic cing a positive result. effect (CPE) exhibiting specific characteristics and alterations of ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. the cells (Robbins, Enders and Weller, 1950). The virus defini- test is time consuming, and demanding, particularly in terms tive identification is then performed using Immunofluorescence of QC. (IF) staining. Nevertheless, virus isolation using cell culture is This list of conventional techniques is not exhaustive, and not ideal in case of viruses not amenable to growth in cell lines only the most commonly used were cited. The following sec- (norovirus, hepatitis virus) or producing CPE (Papafragkou tions will discuss more recent developments in the field, which et al., 2013). replaced the conventional practices and allowed screening In addition, small volumes of the sample may not allow (qualitative test), surveillance (quantitative test) and confirm- inoculation of many cell types, and thus compromise the ation of diagnosis. results. For example, the standard inoculation of a cell culture medium with cerebrospinal fluid (CSF) sample requires a min- Recent methods in the diagnosis imum of 0.2 ml; however, much more is needed to inoculate a combination of different cell types, which could be invasive to of viral infections the patient (McIntyre, 2007, Hematiana et al., 2016). As stated before, this review will discuss only some of the main The time required for isolating viruses by cell culture is developments in diagnostic technologies that have ushered the very long (weeks), limiting the usefulness of this technique new era of clinical virology, and lay out their key advantages when rapid diagnosis is needed. Cell culture needs highly and limitations that will be summarized in Table 1. skilled and experienced personnel for accurate interpretation of the CPE and adequate facilities for handling mammalian cell lines or highly pathogenic viruses. Immunoassay-based tests In the shell vial technique, one of the rapid culture methods, Antibodies produced immediately after invasion of a foreign inoculated cells are subjected to centrifugation, incubation and substance can inform on primary infection, reinfection or a then IF staining with monoclonal antibodies, specific to the reactivation state. Therefore, measuring the level of immuno- range of viruses suspected to cause the infection, such as globulins (Ig) is a widely considered approach for the diagno- respiratory viruses, Herpes simplex virus or Varicella zoster sis of viral infections. virus. The virus-induced antigens are detected 2–4 days later. Automated immunoassay-based methods are among the Rapid culture has limited benefits as it does not target a most frequently used for testing, and are effective because of wide range of viruses and has low sensitivity. the high specificity and binding affinity between antigen and Complement fixation test (CFT) is one of the oldest meth- antibody. Therefore, the principle of the test relies in the for- ods in the history of clinical virology (Casals and Palacios, mation of an immuno-complex between antibody present in 1941). The complement reacts only with antigen–antibody the patient sample and synthetic antigen present in the reagent complex in a non-specific manner. Thus, in the presence of or vice versa, to generate a measurable signal. the complex, the complement is not free to interact with sensi- Immunoassays use labels conjugated to synthetic anti- tized sheep red blood cells (RBCs) used as an indicator, and bodies or antigens which are linked to a solid phase, and used which remain unlysed. The test is said to be ‘positive’. to capture corresponding antigens or antibodies present in CFT is supposedly easy to perform, convenient and sera samples. These labels could be radioactive isotopes, requires inexpensive material. However, it is labour intensive enzymes that cause change in colour or light-generating sub- and lacks sensitivity. In-house standardization through titra- stances. Consequently, this principle has generated several tion of the reactants and preparation of controls is crucial for methodologies for the testing. obtaining effective testing. Radio-immunoassay (RIA) is probably the initiating meth- Haemagglutination inhibition test is generally used for od (1960s); it uses radioisotopes (such as Iodine 125) to label detecting arboviruses, influenza and parainfluenza virus sub- antigen or antibody. The amount of substance to analyse is types and provides relative quantitation of the virus particles. determined by the amount of the generated radioactivity. RIA The principle relies on the capacity of haemagglutinin (HA); a is a highly sensitive method but the main drawback is the viral protein present in the envelope, to bind to erythrocytes handling and disposal of hazardous radioactive substances. (RBC) and to form a lattice pattern termed ‘agglutination’.In The enzymatic labelling alternative using alkaline phos- the assay, serial dilutions of the sample serum are added to a phatase or horseradish peroxidase as markers is, however, the fixed amount of viral HA and agglutinable RBCs. If Influenza most widely used and was long considered a reference method antibodies are present in the serum, the agglutination process (Engvall and Perlmann, 1972; Voller, Bidwell and Bartlett, is prevented. The corresponding dilution rate at which com- 1976). These enzymes induce emission of signals or change in plete haemagglutination is observed and considered. colour respectively, and allow the amount of analyte of inter- Variants of the agglutination assay are used for the diagno- est to be measured. This enzyme-linked immunoassay (EIA) sis of wider range of viral diseases other than influenza has numerous variants, including ELISA, and they differ in (Grandien et al., 1987; Sandeep et al., 2002). However, the the enzyme used and the signal detection principle. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. Table 1. Summary of the main viral diagnostic methods Diagnostic Principle Strengths Weaknesses Variants References technique Immunoassay Formation of High sensitivity Must rely on QC RIA Gupta et al. (2015), antigen– High specificity assurance EIA (FPIA, MEIA, Mixson-Hayden antibody High speed High risk of CLIA) et al. (2015) through throughput interferences recognition Quick TAT (20 min or High cost and binding less) Different types of tags, labels Automated method NAAT Amplification and High sensitivity Longer run time RT-PCR García-Arroyo et al. detection of High specificity Requires specific qPCR (2016), Reijans et al. sequences Multiplexed platforms primers for the NASBA (2008), Renois et al. from the viral Genotyping targets TMA (2010), Afshar and genome (DNA Determination of the Mollaie (2012), or RNA) viral load Mercier-Delarue New compact and et al. (2014), Wu portable formats et al. (2014) NGS Polymerization of High sensitivity High cost Pyrosequencing Capobianchi et al. DNA template High specificity Needs Fluorescently (2013), Bartolini by Identification of novel bioinformatics labelled dNTP et al. (2015), Lowe incorporation genomic sequences skills for data Detection of et al. (2016), Liu of labelled Genotyping analysis released et al. (2013), Van dNTPs, and Accurate detection of Delay in use for hydrogen ion den Hoecke et al. terminate the mutations and drug routine clinical (H+) (2015), Rothberg extension resistant mutations diagnostics et al. (2011) MS Ionization of the High sensitivity Expensive MALDI-TOF MS Lévêque et al. (2014), sample, then Versatility equipment ESI MS Mengelle et al. separation and Cost-effectiveness Limited database Often combined (2013), Qian et al. detection of High workload library with other (2014) the particles methods: PCR- according to MS their mass-to- charge ratio (m/z) CLIA, chemiluminescent immunoassay; dNTP, deoxyribonucleotide triphosphate; EIA, enzyme immunoassay; ESI, electrospray ionization; FPIA, fluorescence polarization immunoassay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MEIA, micro-particle enzyme immunoassay; MS, mass spectrometry; NGS, next-generation sequencing; NAAT, nucleic acid amplification test; NASBA, nucleic acid sequence-based amplification; qPCR, quantitative polymerase chain reaction; RIA, radio-immunoassay; RT-PCR, real-time polymerase chain reaction; TMA, transcription-mediated amplification. The main variants of EIA are as follows: In clinical practice, serological studies of Hepatitis B rely on immunoassay as a key tool for detection of Hepatitis B virus � Fluorescence polarization immunoassay (FPIA): uses fluor- (HBV) markers. The up-to-date versions of immunoassay escent label and polarized light. methods are able to detect the lowest levels of the main � Micro-particle enzyme immunoassay (MEIA)(Tassopoulos marker HBsAg, undetectable by conventional methods, par- et al.,1997): widely used and relies on alkaline phosphatase ticularly in sera samples of asymptomatic patients. The latest enzyme and a corresponding fluorescent substrate. screening standard detects as little as 0.05 IU/ml, the equiva- � Chemiluminescent immunoassay (CLIA), which uses lent of 0.2 ng/ml viral antigen (Deguchi et al., 2004) and high chemiluminescent or light-emitting labels. Companies like sensitivity (>99%) was observed at different stages of the dis- ROCHE or Abbott are exploiting this method, and high- ease or even in patients showing seroclearance. volume laboratories are gradually replacing MEIA technol- A newly developed CLIA allowed denaturation of the ogy with CLIA for its high-speed throughput and ease of HBV particle, and used monoclonal antibodies against inner measurement. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. and outer structural epitopes in order to obtain higher assay diagnostic practices such as screening donated blood for sensitivity (Shinkai et al., 2013) and sufficient information on transfusion-transmitted viruses (CMV, HIV, HCV, …) the HBV genotypes. (Jackson, 1990). Automated immunoassay techniques for virus detection The most widely used variants of conventional amplifica- overcome some of the limitations encountered with the con- tion are real-time PCR (quantitative PCR) and reverse ventional tests, particularly the delay to response. transcription-PCR (RT-PCR). Both are nowadays becoming benchmarks in assessing the viral load, and while the first Limitations method quantifies DNA throughout the reactions in real time (Ntziora et al., 2013); the second performs RT of the mRNA Despite the popularity of immunoassay in clinical testing, (RNA messenger) and amplifies the resulting cDNA (comple- erroneous results may occur for many reasons, which confer mentary DNA). It also quantifies RNA. The combination of inconsistencies to the testing. both techniques increases sensitivity in detecting viruses, par- � Immunoassays are more prone to interferences than any ticularly influenza viruses. The WHO recently approved a other assay, which leads to false-positive or false-negative newly developed reverse transcriptase-PCR assay after the results. In most cases, interferences are due to the presence first death from MERS-CoV (Middle East Respiratory of agents with structural similarities to the reagents Syndrome-Coronavirus) infection reported in 2012 (Abd El (Miller, 2004), and fluctuate according to the concentra- Wahed et al., 2013). tion of the interfering substance and the analyte. Other amplification-based tests such as nucleic acid Endogenous antibodies (autoantibodies, hetero-antibodies sequence-based amplification (NASBA) and transcription- or human anti-animal antibodies) are commonly the main mediated amplification (TMA) are suited for detection of culprit in these interferences (Emerson and Lai, 2013). RNA viruses by amplification of the mRNA instead of conver- Their binding to capture antibodies and detection anti- sion to cDNA (Mercier-Delarue et al.,2014, Wu et al.,2014). bodies in the absence of antigen (analyte) mainly leads to false-positive result (Berth and Willaert, 2016). Among the latest enhancements in PCR systems, extrac- � Accurate QC guarantees reliable results and assumes that the tion, amplification and detection steps have been combined assay is performed well. Therefore, poor QC measures lead within one unit. As an example, BioFire Film Array technol- to low accuracy and precision. In immunoassays, QC is ogy applies two sequential amplifications and can detect a assured by regular calibration and control of the reagents, panel of respiratory viruses with high sensitivity in 1 h and using standardized solutions delivered by the manufacturers. within a single instrument (Pierce et al., 2012). � The high cost of reagents and equipment is another draw- On the contrary, microfluidic technology has also benefited back in immunoassays, particularly for resource-limited PCR-based systems; and allowed further decrease in the settings, considering that the most accurate and sensitive detection time (15 min), with high sensitivity and cost- methods are automated. effectiveness. In the 2009 epidemic, detection of Influenza A virus H1N1 greatly relied on a chip-type real-time PCR Amplification-based assays instead of the tube-based principle. The portable format of such systems is very convenient during epidemics and out- Developed by Mullis and Faloona (1987), nucleic acid ampli- breaks (Song et al., 2012). fication by polymerase chain reaction (PCR) has revolutio- Multiplexed NAAT were designed to detect multiple nized the field of molecular diagnosis. The basic PCR assay viruses or subtypes in a single run. Their detection platforms relies on extraction and purification of the nucleic acid, then can comprise up to 20 viruses using diversified panels exponential amplification of the target sequence, using a (Mahony et al., 2007), for example, concurrent detection of thermostable polymerase enzyme and specific primers. The HAV, HBV and HCV infections, as well as co-infections resulting amplicons are then identified using a fluorescence- (Park et al., 2012). based detection system, and the result is reported in inter- national units IU/ml. In Hepatitis B, PCR is considered the gold standard meth- Soon after its invention, modifications in PCR were tested od for assessing the HBV DNA level with high accuracy, and patented, with the aim of improving the assay capabil- and the assays are standardized according to the WHO ities. The term nucleic acid amplification tests (NAAT) was International Standard for Hepatitis B virus (NIBSC), such as applied to this range of new variants. the reference Roche Cobas TaqMan HBV test. The test was the first FDA approved assay, with a detection limit of NAAT are very popular in the diagnosis and management 10.2 IU/ml. The combined system with Cobas AmpliPrep has of viral infections (HBV, HCV, HIV, Influenza viruses, …) an automated sample extraction process and highly sensitive because they allow determination of the viral load. In other detection for all HBV genotypes (Pyne et al., 2012). terms, quantitation of the viral nucleic acid by amplifying the In HIV patients taking antiretroviral treatment (ART), target sequence thousands-fold. In most cases, they are now ultrasensitive assays permit quantitation of low copy numbers considered a reference, or ‘gold standard’ method for ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. of HIV subtypes (<50 copies/ml) and long terminal repeats in information on species, genotypes and the occurrence of latent reservoirs and from cell lysates omitting the nucleic mutations conferring virulence or resistance to antivirals. acid extraction step (Vandergeeten et al., 2014). Such assays Pyrosequencing is currently the variant of choice within could be valuable tools in large cohort studies or pandemic NGS systems. Based on the detection of pyrophosphate (PPi) situations. after incorporation of a nucleotide in a DNA polymerization process, it uses luciferase to catalyse light-generating pro- Limitations cesses, and the collected light is then recorded. Although The limitations of PCR are an important parameter to con- Illumina is considered the most frequently used pyrosequen- sider, despite the cost-effectiveness and reliability in the diag- cing platform, 454 FLX; a subsidiary company of Roche, was nosis of viral infections. The risk of contamination is very the first high-throughput analyser in the market and was used high while handling, especially during the sample preparation to determine human papillomavirus (HPV) types (Barzon step, in addition, real-time PCR has a longer run-time (2–5h) et al., 2011), subtypes and variants, present in cervical speci- by comparison to other techniques. mens. Other variants detect hydrogen ions that are released throughout nucleotide incorporation reaction (Rothberg et al. In the case of influenza, many PCR-based assays were 2011). designed to detect only a particular subtype of the virus responsible for important pandemics (Hall et al., 2009), and Generating high volumes of sequence data has allowed the the need for designing specific primers for the target requires compilation of viral nucleotide databases and acquisition of handling by experienced operators, capable of detecting de novo sequences to understand the genetic variability of errors, particularly that PCR is prone to false-positive results. viruses (Szpara, Parsons and Enquist, 2010). HIV is by far the most sequenced because of the global priority of AIDS as a The high mutation rate of some viruses could trigger muta- serious endemic, and because of the high mutation rate of the tion within PCR primer regions of the viral genome, which virus. would lead the virus to escape the detection by this assay. So far, only gene sequencing methods have been successful in genotyping tricky HBV, unlike conventional PCR or sero- Next-generation sequencing logical assays. Therefore, sequencing has allowed better clin- ical management of HBV infection and related complications Next-generation sequencing (NGS) is one of the greatest (Margeridon-Thermet et al., 2009). achievements of the modern era. Beyond genome sequencing from known organisms, it allowed discovery of novel viruses In data analysis, recent technical approaches have responsible for unknown human diseases (Palacios et al., included adjustment of the software reading platforms for 2008), and tracking of outbreaks and pandemics such as influ- simultaneous detection of genotypes and mutants of clinical enza (Baillie et al., 2012) to understand their emergence and importance (Germer et al.,2013), and partial sequencing of transmission profiles (Leung et al., 2014; Isakov et al., 2015). the HBV S gene with high sensitivity (98.64%), considering that portion as the site of most drug-resistance mutations The journey began in the 1970s with the works of Sanger (Wang et al.,2013). Nevertheless, implementation of NGS and Barrel, followed by Maxam and Gilbert, who first in clinical settings is increasing, particularly for detecting initiated the principle of sequencing oligonucleotides via low-abundance drug-resistance patterns such as in HIV enzymatic polymerization, using radiolabelled primers. In and HCV (Palmer et al.,2005; Verbinnen et al.,2010). their experimental protocols, they relied on using dideoxy- Nucleotide sequencing of HCV sub-genomic regions is now nucleotides to terminate the extension of the polymer, hence the method of choice for genotyping. the name: chain termination or dideoxynucleotide method (Sanger, Nicklen and Coulson, 1977). Limitations Since then, the principle has remained virtually the same, The principal requirements for NGS are initially access to a but improvements and automation have dramatically in- sequencer, and considerable skills in bioinformatics and creased the speed and accuracy in delivering maximum vol- expertise in data analysis, plus adequate handling systems for ume of data comparing to dideoxynucleotide sequencing storage of generated data. Adding to that, despite the out- (Shedure and Ji, 2008). Technically, NGS is inclusive of three standing results delivered by prototypes in trials, many are main steps: sample preparation, sequencing and data analysis. still at the research level, and not yet approved for use in rou- Systems available in the market differ mostly in their tine clinical practice (Jiangqin et al., 2015). sequencing or reading techniques. Efficient and accurate clin- ical diagnosis of viral infections using NGS is increasingly NGS is undeniably a key technology in specialized clinical aiming to provide accurate longer read-length in a shortest laboratories, but its implementation is still a challenge in time and at a lower cost. Bioinformatic platforms are key many countries, where not only their resource-limited settings components of the sequencing process. They allow interpret- cannot afford a sequence analyser, sample and library prepar- ation of the sequencing output through computational ana- ation, but the vast majority of the population cannot afford lysis (Naccache et al., 2014), and then convert it into useful the cost of the test. ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Bioscience Horizons � Volume 9 2016 Review article ............................................................................................... .................................................................. Limitations Mass spectrometry The main limitation of MS is the high cost, particularly in Mass spectrometry (MS) is nowadays a benchmark of labora- high pandemic areas, which are usually the poorest; not all tory qualitative and quantitative investigation, particularly in laboratories can afford a mass analyser for their activities. bacteriology (Sauer and Kliem, 2010). The second major drawback is within the reference library. The identification is limited by known data from well- The principle of MS relies on converting the sample into identified organisms only; therefore, rare mutations cannot be charged particles (ions) by ionization process. These ions are detected if they do not exist within the reading platform, but separated according to their mass-to-charge ratio (m/z) and there is hope that MS database libraries will rapidly expand. analysed by a detector. The result obtained is compared to a reference database (library), existing within the system and delivered as an interpretive spectrum. Advantages of the recent methods In clinical laboratories, matrix-assisted laser desorption � High sensitivity and specificity assays approaching 100%. ionization (MALDI) and electrospray (ES) are the most used � Possibility to combine different methods in one assay ionization methods because they allow processing of consid- enhances detection capability and accuracy (PCR-MS, erable amounts of analyte (Emonet et al., 2010). These microfluidic chip technology-based PCR,…). approaches have extensively been evaluated experimentally � Automation of the assays reduces the number of operators and provided excellent results, either used alone or combined and manual workload. with other molecular methods, such as PCR, in order to � Small sample volumes are needed, so the assays can still be enhance sensitivity. The combination (RT-PCR/ESI-MS) was performed in particular cases (CSF, new-borns, etc.) able to detect viral pathogens usually undetected by regular � Some assays such as MS remain efficient after several freez- testing methods, and provided rapid and detailed data (types ing–thawing of the samples (archived samples). and subtypes) within a short time (Lévêque et al., 2014). � Rapid TAT: minimum time required from sample collec- Detection of genomic variations or mutations in Influenza tion to results reporting is 30 min, and microarray tools A using MALDI-TOF has been a key tool in the management can deliver results within seconds. of outbreaks (Chen et al., 2011). MS application to the struc- � Low detection limit: 10–100 copies/ml by PCR. tural investigation of biomolecules showed the efficiency of � Multiplex reactions: detection of a wide range of patho- MALDI-TOF-MS coupled to antibody magnetic nanoparti- gens in a single run is time saving. cles in detecting influenza viruses (Chou et al., 2011; Yea � Detection of rare drug-resistance patterns. et al., 2011), through concordant results with the gold stand- � NGS generates many sequencing data per run and ard PCR-based method. This blend of two powerful machin- sequences long-reads. eries (PCR-MS) can detect drug resistance to antiviral therapy � Low reaction cost when using microfluidic chips, and use as well as the presence of multiple viruses within the same in POC settings. sample and diagnose for co-infections, when assays are � Lower risk of contamination by processing single tube multiplexed. within one unit. The increasing interest in MALDI-TOF MS in clinical vir- ology has led to research advances in the diagnosis of The latest technologies and Hepatitis B. The method was in fact not only able to detect resource-limited settings HBV at relatively low viral load levels (100 HBV DNA cop- ies/ml) (Hong et al., 2004) but it also allowed detection of up The methods described above have shown outstanding per- to 60 HBV variants and genotypes at a cost lower than $10/ formance in saving thousands of lives in developed countries. sample for all the variants with high throughput (Luan et al., However, accurate diagnosis is still a challenge in resource- 2009). The same method was applied for genotyping and suc- limited settings because of the difficulty in acquiring these cessfully detected the eight HBV genotypes accurately equipment and technical expertise. While expensive tools (Ganova-Raeva et al., 2010), and also minor HCV genotypes such as PCR-based systems or mass analysers are only avail- occurring at very low level (Kim et al., 2005). able in reference laboratories or in military facilities, point-of- Mass spectrometric-based methods are versatile, sensitive, care or near-patients clinics that serve more than 80% of the rapid and cost-effective, and do not require interpretation poor population struggle with the testing devices available. As software for data analysis. The automated machinery necessi- an example, the WHO has established guidelines for monitor- tates easy sample preparation and fewer operators. The ana- ing ART efficacy in HIV-1 infected patients in developing lysis capacity can reach up to 960 specimens/day, which countries, by suggesting only CD4+ cell count and ELISA- makes it suitable for routine diagnosis in high-volume labora- based assays as alternatives to the expensive tests (WHO, tories and large-scale studies. Tests can also be performed effi- 2006a). Ultrasensitive p24 (Patton et al., 2008) and the ciently on archived specimen. ExaVir assay (Kokkayil et al., 2014), measure the level of p24 ............................................................................................... .................................................................. Downloaded from https://academic.oup.com/biohorizons/article-abstract/doi/10.1093/biohorizons/hzw010/2622464 by Ed 'DeepDyve' Gillespie user on 03 February 2018 Review article Bioscience Horizons � Volume 9 2016 ............................................................................................... .................................................................. antigen and the activity of reverse transcriptase, respectively, Improvement of QC programs, QA and standardization to replace viral load monitoring. Unfortunately, their lack of of assays, kits and reagents are important to fulfil require- sensitivity has induced failure of treatment and emergence of ments for accuracy. The Centre for Disease Control and resistant strains (Vekemans, John and Colebunders, 2007). Prevention (CDC) is continuously implementing laboratory- testing guidelines particularly for HIV, before further tests Hepatitis is another expensive burden to manage in poor will be approved (CDC, 2014). countries, where accurate assessment of the epidemiological profile is hard to establish. In the case of HBV, most POC The WHO and non-profit organizations such as the facilities rely on screening for HBsAg solely using rapid diag- Foundation for Innovative New Diagnostics (FIND) aim to nosis tests. These low-cost devices do not allow determination maximize efforts to implement surveillance programs and of the course of the disease to inform whether to initiate an control of communicable diseases (Loman et al., 2012), set up antiviral treatment or not. new policies and improve diagnostic services in low resource settings. Automation, microfluidics and Conclusion future prospects The recently developed viral diagnostic methods are reshap- ing the field of clinical microbiology, and could contribute Providing high-level epidemiological monitoring of viral diseases to reducing the prevalence of serious infectious diseases. is undeniably a global public health ambition, and despite rapid However, the technical capabilities alone are insufficient if progress in the development of diagnostic methods in recent not supported by health promotion strategies to increase years, improvements are needed for better cost, size (Loman awareness about the importance of early detection and regu- et al., 2012; Frey et al.,2014)and TAT (Xu et al., 2013). lar screening of persons at high risk. After the Ebola Haemorrhagic fever in 2014, the US gov- Finally, good quality diagnosis has a cost that only devel- ernmental Department of Energy has developed an innovative oped countries can afford in routine practice so far, and this rapid and portable test to detect specifically Ebola virus is delaying the implementation of new methods in the devel- within seconds. The test aims to target other RNA and exotic oping world and the endemic areas. However, there is hope viruses such as Dengue and West Nile for effective manage- that efforts will continue towards developing new good ment of viral outbreaks. Other examples include fully NAAT quality tests affordable in low-income countries, which systems and small devices to sequence single molecule DNA would substantially strengthen disease control strategies for using nanopore technology (Eisenstein, 2012). their populations. Microfluidic technology is regarded with optimism for the management of infectious diseases by allowing timely ther- Author biography apy. It provides a key potential solution for remote areas and near-patients facilities by avoiding turnaround trips of the Selma obtained an MSc with Honours in Biomedical sciences patients between the clinic and the laboratory. Their use is from Nottingham Trent University. She works as a biomed- also beneficial where time is crucial, or when physical spaces ical scientist at Institut Pasteur, within the Immunoassay div- do not allow setting up of conventional methods. ision, and has particular interest in laboratory diagnosis Lab-on-a-chip (LOC) is a very small device that integrates issues in low-income settings and how they can be solved at laboratory processes within a few square centimetres. It uses lower cost. She also has interest in structural biology and very small volume of samples to perform immediate reactions microbiology. within the chip or in a portable device. The reactions vary from nucleic acid amplification and detection, to cell count References and immunoassays; therefore, microfluidic diagnostics com- pete with large instruments in performing laboratory tests at a Abd El Wahed, A., Patel, P., Heidenreich, D. et al. (2013) Reverse tran- lower cost, to benefit low-income settings and remote areas. scription recombinase polymerase amplification assay for the detection of Middle East respiratory syndrome coronavirus, PLoS A wide range of LOC devices were approved by the FDA in Currents Outbreaks, 5, e2e8364. the diagnosis of viral infections such as Influenza (Cao et al., 2012), HIV (Alyassin et al.,2009)and HBV(Zhi et al.,2014) Afshar, R. M. and Mollaie, H. R. (2012) Detection of HBV resistance to and further development is in progress. As examples, Daktari lamivudine in patients with chronic Hepatitis B using zip nucleic Diagnostics use affinity chromatography for CD4+T-cell count acid probes in Kerman, Southeast of Iran, Asian Pacific Journal of as an alternative to flow cytometry to monitor HIV in develop- Cancer Prevention, 13 (8), 3657–3661. ing countries (Cheng et al.,2007). The bench top analyser GeneXpert made by Cepheid has an integrated sample prepar- Alyassin, M. A., Moon, S., Keles, H. O. et al. (2009) Rapid automated cell ation and PCR system for molecular diagnosis of influenza and quantification on HIV microfluidic devices, Lab on a Chip, 9 (23). 10. other bacterial infections in a light portable format. 1039/b911882a. ............................................................................................... .................................................................. 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