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For reprint orders, comDevelopment of rapid,automated diagnostics forinfectious disease: advancesand challengesExpert Rev. Med. Devices 6(6), 641-651 (2009)Jennifer Ince andAian McNallyffAuthor for correspondenceSchool of Science andTechnology, Nottingham TrentUniversity, Clifton Lane,Nottingham NG11 8NS, UKTel.: 44 115 8483324alan.mcnally@ntu. ac. ukThe last 2 years has seen an exponential rise in the amount of research funding made availablefor the development of rapid diagnostic devices for infectious agents of medical importance.This review reports on several such projects. These highlight the development of fully automateddevices for rapid diagnostics, ranging from fully automated real-time PCR-based detectionmethods to fully automated PCR- and array-based machines for the detection and typing ofinfluenza. This review will also highlight the importance of refocusing work on classicalimmunoassay techniques, showing how biosensor-based immunoassays can greatly enhanceexisting assays and at a much reduced cost to molecular-based methods.KEYWORDS: automated infectious disease molecular diagnostics PCR point of careaureus (MRSA) and Clostridium difficile infections, and fatal outbreaks in hospitals. AccordingInfectious disease represents the greatest risk to official figures on the Health Protectionto global human health. This can range from Agency website [ioi], there were 3210 cases ofclassical infectious diseases, such as T B , chol- M R S A bacteremia reported in the U K in 2008era, dysentery and typhoid, annual epidemics, with 1652 deaths, and an astonishing 81,412such as norovirus, influenza and seasonal colds, reported cases of C. difficile infection, withemerging infectious diseases, such as avian influ- 8324 deaths (fatalityfiguresare from Office forenza and hemorrhagic fevers, through to global National Statistics data, where the organism waspandemics, such as H I V and the current newly named on death certificates [101]).Despite the obvious burden to human healthemerged H I N l v outbreak (commonly referredto as swine flu). In the U K alone, infectious and economic wealth presented by infectiousdiseases account for 10% of all deaths recorded diseases, the approach taken to diagnosis andannually and are responsible for a third of all effective treatment of these is firmly entrenchedgeneral practitioner consultations. The projected in classical microbiology of the early- and midtotal cost for treatment of infectious diseases in 20th Century. Diagnosis for almost all infectiousthe U K is approximately 6 billion per annum. diseases caused by bacteria is still based on clasIn addition, public perception and awareness sical microscopic- and culture-based methodof infectious diseases has increased in recent ology. By adopting a culture-based program ofyears owing to increased coverage in the media infectious disease diagnosis, laboratories canof high-profile outbreaks of infectious diseases. perform tests on hundreds, if not, thousands ofParticular attention has been paid to cases of the samples per day using very cheap culture mediahuman H5N1 highly pathogenic avian influenza and techniques able to be employed by semiinfection and to the introduction of the virus skilled workers. It also provides clinical microinto U K wild bird and poultry flocks. Public biologists with isolated bacterial cultures, whichperception has also increased dramatically with can be further characterized if necessary, such asregards to healthcare-associated infections, in antibiotic-resistance profiles or epidemiologicalparticular methicillin-resistant Staphylococcus testing in the event of an outbreak situation.Importance of rapid diagnosis ofinfectious diseaseWWW.experl-reviews.com10.1586/ERD.09.46 2009 Expert Reviews LtdISSN 1743-4440641
g i M-.Ince & McNallyClassical diagnostics for infectious diseasesthat are inoculated and incubated. The incubator contains aTo alleviate the burden of infectious diseases, diagnostic tests light-emitting diode, and a scanner detects color changes in thehave been developed to identify pathogenic agents. This speeds inoculation vials as a result of C 0 2 production by bacterial resup the appropriate treatment of patients and allows specific piration, producing an audible alarm to alert the microbiologisttargeted drugs to be used, slowing the spread of infection to to the presence of bacteria in what should be a sterile sample.others [i]. The types of methods used include microscopy, culture, antigen detection and immunoserology [2]. The technique Drawbackscommonly used in clinical settings is culture on selective and All diagnostic techniques have their shortcomings. A summarychromogenic agars. In the last decade, many chromogenic agars of the main challenges faced by traditional infectious diseasehave been developed to aid the rapid identification of bacte- diagnostic methods is presented in Box l . Culture-based diagrial pathogens in several disease states, for example urinary nostic methods suffer particularly from the time taken to obtaintract infections (UTIs) and bacteremia [3-5]. Agars have been a definitive result. Culture requires time to allow the bacteriadeveloped for specific pathogens, such as acrylonitrile-buta- of interest to grow, which is generally 24-72 h. Only after thisdiene-styrene medium for Salmonella. [&), Candida diagnostic time can the result be seen and this represents a huge delay toagar for Candida [7] and S. aureus ID agar or CHROMagar the treatment of patients. Ledeboer et al. investigated makingM R S A for M R S A [s-io]. Media used for potentially mixed the detection of Enterococcus spp. as rapid as possible using agar,pathogenic samples, such as those from U T I cases, include but the fastest they were able to make their culture-based assayUriSelect3, Rainbow Agar U T I medium and chromogenic was 24 h [4]. If an organism has fastidious growing requireU T I medium [3].ments, or is just slow growing, the diagnostic time is lengthenedMicroscopy and culture are also complemented in most clini- further. Culture methods also only account for those organismscal diagnostic laboratories, with techniques such as antigen that can be grown in the laboratory — those that cannot godetection and serology (FIGURE I). These methods could be con- unrecognized by these techniques.sidered the forerunners of rapid diagnostics, as they principallyApart from being time consuming, culture techniques areremove the need for time-consuming, culture-based methods laboratory bound owing to the amount of equipment andto direct detection of the infectious agent, or a response against reagents required to run them. This means that samples mustthat infectious agent. Both methods are antibody-based and are be shipped to a laboratory before they can be analyzed. If colprincipally run on the basis of ELISA or immunofluorescent lected in a hospital, this is not such a problem, as samples canantigen testing. While both techniques speed up detection and be analyzed in-house in hospital laboratories. However, this isdiagnosis, there are numerous inherent problems with these not the case with samples collected outside of hospitals. Duringtechniques, as highlighted in BOX I. Despite this, commercial transport, samples may deteriorate or change from the state inantigen detection and serology assays represent a huge mar- which they were first collected owing to changes in temperatureket and are commonly used for the diagnosis of HIV, influ- and aerobic status - such changes can have profound effects onenza, chlamydia and numerous other virus infections. Their the bacterial populations in the samples; thus, if a microbiologiuse for diagnosis of viral infections has been particularly well cal test is intended, it is important to store the samples carefullyemployed by clinical laboratories, with the ELVIS technology during transport. Disregarding changes in the sample itself,a notable success for herpes simplex virus detection, which uses transport time lengthens the diagnostic procedure. Culture alsogenetically modified cells that express the bacterial enzyme requires a considerable amount of manual labor time preparing(3-galactosidase in the presence of the virus, which is easily media, inoculating and attending to incubating cultures.detected histochemically [n]. The use of rapid diagnostics isperfect for such cases as culture and isolation of viruses, but it Impact of molecular biology on diagnosticsis extremely time consuming and cumbersome, relying on cul- More advanced methods are now being used to supplementtured eukaryotic cells or embryonated eggs, with culture often classical diagnostics utilizing nucleic acid probes specific fortaking as long as 7—14 days. Similarly, obligate intracellular pathogens of interest, most notably in the diagnosis of bloodorganisms, such as Chlamydia, are much simpler to diagnose culture samples using peptide nucleic acid FISH. This utilizesusing antigen-detection tests, as opposed to intracellular cul- a fluorescent peptide nucleic acid that hybridizes to ribosomalture of samples. In addition, many of these tests are now fully R N A of septicemia pathogens and gives a positive identificaautomated in clinical laboratories through the use of systems tion for sepsis diagnosis [12]. The technique targets the 16ssuch as the Axsym machine (Abbot Diagnostics).R N A region of bacterial chromosomes, which are unique toAs the employment o f serological and antigen-detection each species and act as a fingerprint for bacteria. By designingtests has increased for infectious organisms that are difficult a fluorescent probe that is complementary to this sequence, it isto culture, the need for rapidity in diagnosing bacterial infec- possible to detect bacteria at the molecular level by simply lysingtions has also gathered pace. Many clinical laboratories now cells in a sample in situ and adding the probe, then visualizinguse automated systems such as the B A C T E C system for forfluorescence.This is very quick but also has drawbacks withbacterial blood cultures and Mycobacterium detection (Becton regards to specificity, for example distinguishing M R S A fromDickinson). This works using barcode-labeled culture tubes methicillin-sensitive S. aureus (MSSA).642Expert Rev. Med. Devices 6(6), (2009)
Development of rapid, automated diagnostics for infectious diseaseSample takenSample received bydiagnostic laboratory initial microscopy performedif clinically importantSample such as blood orcerebrospinal fluidCulture medium continuallyprepared in readiness forsample arrival -Serology/antigen detection if viralor intracellular pathogen suspectedSample applied to culture mediumand incubated for the required time(24-72 \Figure 1. Processes involved in classical diagnosis of infectious disease, from receipt of sample through to confirmationof result.The ability to amplify nucleic acids has opened the door toidentifying pathogens by detecting the presence of their D N A insamples without the need to culture first to increase their numbers [2,13]. D N A detection can be achieved much more rapidlythan culturing, thus diagnoses may be available in just a fewhours. It avoids the problem posed by fastidious or nonculturableorganisms and can be used to identify organisms more specifically than culture, since primers and probes may be designedfor specific organisms (FIGURE 2). Amplification-based nucleic acidmethods may be identified into three groups: target nucleic acidamplification, probe amplification or signal amplification [14].www.expert-reviews.comPolymerase chain reaction, strand-displacement amplification and isothermal R N A self-sustaining sequence replicationare examples of target amplification. The latter is the basis fornucleic acid sequence-based amplification and transcriptionmediated amplification. P C R consists of a number of temperature cycles that include a denaturing, annealing and sometimesan extension temperature [15]. The D N A template of choice(from a pathogenic bacterium for diagnostic purposes) is subjected to this cycle of temperature changes in the presenceof a polymerase enzyme, free nucleotides, primer nucleotidesequences and chemical buffers. This enables the polymerase643
Ince & McNallyM,i*\s-;'-;.--r- -QBox 1. Limitations of traditionaldiagnostic techniques.Microscopy Poor sensitivity Invasive False-positives obtainedCulture Time consuming Slow-growing organisms Uncultured/fastidious organisms BiohazardAntigen detection False-positives occur Long-term antigen shedding Narrow detection windowSerology Negative in immune-compromised patients Complex interpretation Poor correlation with disease High background titers Crossreactivityto create additional complimentary strands of D N A once theoriginal strands have denatured and primer fragments haveannealed. The product of the PCR, the D N A fragment selectedby the primer placement, doubles during every cycle, thusincreasing the amount of the chosen nucleic acid dramatically. The transcription-mediated amplification and nucleic acidsequence-based amplification reactions amplify R N A by theaction of three enzymes: reverse transcriptase, RNase and R N Apolymerase. First, c D N A i s synthesized by special primers, oneend of which is target specific and the other end is promoterR N A polymerase. Reverse transcriptase synthesizes RNA—D N A hybrid, and the RNase digests the R N A component ofthis. The reverse transcriptase then synthesizes dsDNA, andfinally, the R N A polymerase makes numerous R N A copies.The ligase chain reaction (LCR) in the gapped L C R format isa combination of target- and probe-amplification processes, andthe QB replicase amplification is an example of probe amplification [15]. In L C R , dsDNA is denatured by heating, and primersanneal to the single strands. Then, the D N A ligase joins theprimers, and the product is again released by heating. Thisproduct goes on to become the template for further ligations.Gapped L C R simply adds a D N A polymerase step to fill agap of a few bases left between the primers, before the ligasemay join them. In QB replicase amplification, the R N A probeanneals to the target and the hybrids are captured; the probeis then removed and amplified by the QB replicase. However,problems have been seen regarding the separation of hybridizedand nonhybridized probe.A l l of the aforementioned amplification reactions have beenimproved by the advent o f real-time P C R technology. Thishas the benefit over conventional amplification reactions inthat it does not require electrophoresis of products after the644amplification reaction, minimizing postmethod contamination,but rather usesfiuorescentlylabeled nucleotides (Sybr technology) or a fiuorescently labeled probe complementary to thetarget sequence, which anneals between the two primers usedin the amplification reaction (TaqMan technology; Roche).TaqMan works by addition of a fluorophore and a quenchermolecule to the probe. In the absence of complementary targetmolecules, the quencher inhibits the emission of light by thefluorophore. If bound to target D N A , the molecules are separated by distance, and fluorescence is emitted. This is detectedby a sensor that scans the reaction after each P C R cycle, hencethe real-time detection of target molecules. Real-time P C R isa much faster process, and target molecules can be detected inas little as 30 min i f abundant in a test sample [16]. The twomain benefits of this real-time scanning process are that a positive sample can be detected during the P C R assay, rather thanhaving to wait for the cycling and then a further electrophoresis step, and that as P C R and incorporation of fluorescence isa logarithmic process, one can actually quantify the amountof target in a sample by using a calibration curve composedvia positive-control samples containing known amounts of thetarget. This creates the power of quantifiable culture-baseddiagnosis using the rapidity of molecular-based tests.In addition, microrray technology could also be of use indiagnostics for infectious agents [17]. This involves glass slidesthat contain ssDNA molecules complementary to pathogenspecific sequences. By amplifying the microbial D N A or R N Apresent in a sample, it is potentially possible to identify allmicrobes present in a clinical sample on one test assay. Theamplification incorporatesfluorescentlylabeled nucleotides intothe amplified product, and hybridization is assessed using laserexcitation and fluorescence emission readings.There are an astonishing number of peer-reviewed publications on the development and implementation of PCR-baseddetection assays for infectious agents, far too many to cover ina concise review; however, a fine example of the process behindthe rationale and design of such assays is the development of aPCR assay for MRSA specifically, rather than all S. aureus strains.Methicillin resistance is conferred by a penicillin-binding proteincalled penicillin-binding protein 2a (PBP2a), which is coded forby the mecA gene [is]. A P C R assay was designed to detect thepresence of this gene [19]; however, this assay also amplified otherStaphylococcus species that were coagulase-negative in samples [20],creating a false positive for MRSA, and thus different assays weredesigned to be more specific. Reischl et al. used a duplex assay thatcombined primers for the mecA gene and primers for a S. aureusspecific marker gene, such as nuc or sa442 [20]. However, thisapproach also led to false M R S A positives by picking up MSSAand coagulase-negative methicillin-resistant Staphylococci present in the same sample. A more refined, tandem-target approachwas also used by "Warren et al., whose assay targeted a sequencefrom the mobile genetic element that holds the mecA gene,SCCmec, and a sequence from the orfX region, a conserved openreading frame in which the SCCmec can be integrated into thegenome and is uniquely found in M R S A [21]. Francois et al. usedExpert Rev. Med. Devices 6(6), (2009)
Development of rapid, automated diagnostics for infectious disease i b S S ? ; ;a triplex assay to identify between S. aureusand Staphylococcus epidermidis [22]. Theirassay included primers for mecA and femAspecific for S. aureus znifamA specificfor S epidermidis.Potential problems in developingmolecular detection methodsMolecular procedures have the riskof showing false-positive or -negativeresults [13]. False-positive results are primarily due to contamination of the reaction mixture from other samples andSample placed in automatedproducts of previous amplifications.diagnostic device - diagnosticContamination during pipetting due toprocess started (30 minaerosols is also possible, since nucleic acidamplification is very sensitive and will pickup on tiny amounts of a contaminant. Tocombat the problem of contamination,Nucleic acide used indiagnostic processurasik/V-glycosylase can be added to theommonly PCR, 1-2reaction mix [23]; however, this does notremove the necessity for good laboratorypractice when preparing molecular assays.The use of automated nucleic acid extraction and amplification techniques may alsoreduce contamination.False-negative results could be causedby a poorly designed assay that does notdetect the target sensitively enough orFigure 2. Molecular diagnostic process can, in theory, greatly simplify and speedonly detects it when the target is presentup the process of diagnosis of infectious disease.in high quantities. In such cases, the assayshould be redesigned or a more effective target should be chosen more washing steps into their D N A extraction protocol removedfor that pathogen. By their nature, molecular assays only use inhibitors to allow successful PCR, although it also meant thatsmall amounts of a sample, considerably smaller than in culture the final yield of D N A was much lower [28]. C
alan.mcnally@ntu. ac. uk Development of rapid, automated diagnostics for infectious disease: advances and challenges Expert Rev. Med. Devices 6(6), 641-651 (2009) The last 2 years has seen an exponential rise in the amount of research funding made available for the development of rapid diagno
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