Examples
Specific Identity of the Etiological Agent is Suspected
Multiplex Real-time PCR Real-time PCR has rapidly become the favored method of choice in clinical virology laboratories. It is routinely used in target-focused tests for many viruses, including HIV-1,[53] parvovirus,[54] BTV,[55] WNV,[56] dengue virus,[57] influenza A virus, influenza B virus and at least eight human herpes viruses. Multiplexing is utilized in some of these tests, both to enable coamplification of internal and external controls along with the target viral nucleic acid (e.g.,[55]), as well as for reasons of economy and work flow, to include several of the detectable targets in one or two reaction tubes. The level of multiplexing can be increased by various strategies. One method (utilized in[28]) entails dividing the extracted nucleic acid samples into more than one tube and using sequential real-time reactions after the positive reaction is identified by melt-curve analysis in one of the tubes. By this means it has proved possible to detect and differentiate HSV1, HSV2, VZV, Epstein–Barr virus (EBV), CMV, human herpes virus (HHV)6, HHV7 and HHV8. As mentioned earlier, some of the rarer, yet highly pathogenic, herpes viruses (e.g., herpes simiae virus) are not included in these panels. As many of these multiplex real-time PCR assays are dependent on multiple, exact-match primers directed to single target genes, they are vulnerable to sequence variation and 'drop-out of signal'. Another strategy to increase the level of multiplexing is to utilize GC-rich extensions on the tails of PCR primers, allowing discrimination of amplified targets by melt-curve analysis. Lo et al. used this approach, combined with the use of two fluorophores and the intercalating dye SYBR® Green.[57] Using this hybrid strategy, the authors were able to detect and differentiate dengue virus serotypes 1, 2, 3 and 4. However, the assay was not designed to detect JEV, which cocirculates with dengue in many locations in Asia.
Multiplex PCR-microsphere Respiratory Virus Panels The other target-focused approach that is used to identify one of a candidate panel of viruses is microspheres/microbeads. Microspheres enable capture and spatial separation of PCR products, and the products can be 'addressed' via the unique correspondence between the oligonucleotide capture sequence on the microsphere and the color of the microsphere.
There are now three commercially available respiratory virus panels that rely on targeted multiplex PCR (ResPlex II v2.0 [Qiagen], MultiCode®-PLx [EraGen Biosciences] and xTAG® [Luminex]). Balada Llasat et al. very recently evaluated these three systems.[58] The respiratory panel developed by Mahony et al. detects 20 human respiratory viruses or virus subtypes and is now commercially available as the Luminex xTAG system.[59] It relies on multiplex PCR using 14 virus-specific primers, followed by capture of the oligonucleotide 'tagged' PCR products onto universal oligonucleotoide tags on the microspheres, followed by target-specific primer extension, during which a phycoerythrin label is incorporated into the extension product. The microspheres pass through a flow cell and the signals are detected by a red laser to identify the microspheres and a green laser to measure the phycoerythrin localized to the microsphere. The list of detected viruses includes influenza A (subtypes H1, H3 and H5), influenza B, respiratory syncytial virus A and B, metapneumovirus, rhinovirus, parainfluenza 1–4, severe acute respiratory syndrome-associated coronavirus, and four other coronaviruses and adenoviruses of unspecified type. Nolte and colleagues contemporaneously developed a very similar system, utilizing modified nucleotides to label the microsphere-captured primer extension products.[60] The latter system, now commercially available as the MultiCode-PLx is designed to detect 17 respiratory viruses. All three of the commercially available multiplex PCR panels exhibited superior sensitivity compared with virus culture methods.[58] Although all of these systems cast a relatively broad net, they are target-focused, use target-specific primers and are therefore susceptible to drop out, or failure to detect variant viruses.
To illustrate this point, the xTAG respiratory virus panel, in addition to detecting influenza A, identifies the subtype of seasonal influenza A as H1 or H3. However, the subtype of the novel influenza A virus of swine origin (2009 outbreak) could not be identified. This emergence of an untypeable influenza A virus, using the xTAG panel, was cleverly turned from a 'negative' to a 'positive' test by Ginochio and St George, who demonstrated that untypeable influenza A virus was, at least for the 2009 outbreak, statistically very likely to be H1N1.[61] A more robust diagnostic solution (e.g., redesign of the xTAG panel) is needed, because any future change in subtype prevalence will invalidate that conclusion.
Ginocchio et al. also compared the xTAG system with the BinaxNOW® influenza A and B test (Inverness Medical International, Cranfield, UK) and the 3M™ Rapid Detection Flu A + B test (3M, St Paul, MN, USA; both antigen-detection tests).[62] The xTAG panel demonstrated far superior sensitivity for inferred detection of novel H1N1 influenza of swine origin (i.e., for detection of influenza A virus that was untypeable using the xTAG panel). However, the xTAG respiratory virus panel should be modified to allow unambiguous subtyping of influenza A virus in order to confirm these provisional comparisons with other tests.
These examples serve to demonstrate the types of problems that arise when developing diagnostic tests designed to identify specific viruses.
Identity of Etiological Agent is Suspected at the Level of Genus, Family or Group of Agents that Cause Common Clinical Syndromes
Several flaviviruses cause severe encephalitic, hemorrhagic and/or febrile illness in humans. Typically, symptoms include sudden onset of fever, anorexia and headache. Vomiting, nausea, diarrhea, muscle aches and dizziness may also occur. Clinical symptoms are not distinctive of infection by a particular virus (see Table 1). Various arboviruses cocirculate in geographically overlapping regions, with incursions of exotic flaviviruses into unanticipated regions becoming more common (see later), posing a difficult challenge for diagnostics. The pan-genus diagnostic approach has been utilized for three decades in antibody-based assays, with pan-flavivirus monoclonal antibodies developed in the early 1980s.[63] Follow-up immunological tests were necessary for species identification but these are not always definitive (see later for the example of the failure of West Nile differentiation from USUV by immunological methods). A genus-level nucleic acid-based diagnostic, capable of detecting any member of the genus, would be a useful front-line diagnostic tool, particularly if interfaced with an array facilitating rapid species identification. Such tools are currently being developed.
To address this challenge, a range of broad-spectrum RT-PCRs have been developed (e.g., Gaunt and Gould,[64] Moureau et al.,[65] Maher-Sturgess et al.[66] and Fischer et al.[67]). Maher-Sturgess et al. developed a pan-genus, one-step RT-PCR, with an extended range of positive reactions compared with earlier publications.[66] Sequence alignments of 490 flavivirus sequences were used to select highly conserved sites in all known members of the genus. A computer program capturing the method of Zheng et al.[68] was then used to chart the sequence stability in the selected, conserved flavivirus sites over the time period since the first flavivirus was entered into the National Center for Biotechnology Information database. This program (dubbed 'Lu-Tze') identifies the least rapidly changing sequences among the initially selected group of conserved sites. A region in the NS5 gene was selected and redundant (mixed-base) primers were designed,[66] amplifying a product rich in phylogenetic information and suitable for downstream analysis using array technology (see later). This assay was effective in detecting all 66 tested flaviviruses from all three groups: mosquito-borne, tick-borne and no known vector, as well as some recently isolated variant viruses (i.e., a variant Edge Hill Virus, a member of the YFV group).[69]
Bioinformatics Innovation The RT-PCR was interfaced with a prototype array detection system that utilized a novel probe design.[65] These 'dichot' or 'binary' probes successively divide a target population of sequences into two populations:[70] those that react with the probe and those that do not (Figure 1). As each probe generates a yes/no (binary) answer, they can be described in terms of a base 2 logarithm, such that Y = 2X, where X is the number of probes and Y is the maximum number of species that can be differentiated. For example, ten probes differentiate 210 = 1024 species and 20 probes differentiate >1 million. This system generates a binary barcode. The discovery of a new nonzero barcode flags a new but related virus. For example, if one compares the binary barcodes of two viruses on a dichot array consisting of nine spots, 100100100 and 100100101, these different codes can be interpreted as revealing three regions of similarity and one region of nonsimilarity between two viruses. This type of array detection yields an immediately expandable test. Newly discovered viruses will be those generating a new barcode on the existing array. The new barcode can be entered into a pattern-matching database and a new test is rapidly available. This and other array-based, nontarget-focused detection methods are examples of adaptive diagnostic tests. Wang et al. demonstrated that nucleic acid can be recovered from arrays and used for detailed characterization of new viruses.[71]
Figure 1.
A theoretical barcode that might be generated using a 'dichot' or binary probe array. With the use of only four dichot probes, each generating a yes/no answer, up to 16 viruses can be differentiated. In this theoretical example, three out of four probes bind to the target virus nucleic acid, generating the barcode 1011.
Reproduced with permission from S Maher-Sturgess.
Moureau et al. also developed a one-step RT-PCR method for universal (more correctly, pan-genus) detection of flaviviruses.[65] The primers (PF1S/PF2R) were a modification of degenerate primers described earlier.[72–74] Their assay was extended to develop a real-time RT-PCR version of the test, using the SYBR green system. The real-time assay, when positive, was followed by direct sequencing using the amplification primers. This test exhibited exquisite sensitivity and, notably, was capable of detecting cell fusing agent virus in mosquito pools and Ngoye virus from crushed ticks.
The protocols of Maher-Sturgess et al.[66] and Moureau et al.[65] avoid the use of nested PCR, a feature of many earlier assays, which is prone to contamination.
Previously found only in Africa, USUV appeared in Vienna, Austria, in 2001. The association with extensive bird mortality led to a presumptive diagnosis of WNV.[26] Although serology and histochemistry was positive for WNV, samples were negative in a target-focused West Nile virus PCR.[48] Accordingly, a 'universal' RT-PCR was developed using degenerate primers based on the sequences of several mosquito-borne flaviruses.[26] The use of the new RT-PCR, targeting the NS5 region, followed by sequencing, led to the identification of USUV. Similarly, in a 2009 case of neuroinvasive infection,[51] molecular tests of cerebrospinal fluid were negative for CMV, HSV1/2, EBV, adenoviruses, parvovirus B19 and WNV. The use of a hemi-nested degenerate PCR for genus Flavivirus[75] was necessary to amplify the virus and enabled subsequent identification of USUV by direct sequencing.[50]
An expansion of arbovirus surveillance and reporting systems was implemented in North America following the appearance of WNV. ArboNET collects and reports data from humans, mosquitoes, birds, mammals and sentinel chickens. These data are integrated into a single reporting system.[76] Similar surveillance expansions have taken place in Europe after WNV and USUV outbreaks.[38] Broad-spectrum molecular tests such as those previously described should make a significant contribution to such programs.
Influenza A virus possesses molecular signatures that determine host preference, pathogenic potential and drug resistance. These signatures are located in the HA gene segment, the PB2 gene segment, the NA gene segment and the M gene segment. Although the association between signature and phenotype is well established, the signatures have not yet been utilized in front-line diagnostic tests, which currently give little more than rudimentary information about the HA and NA type. In response to this need, Castillo Alvarez et al. developed a degenerate primer, single-step, touch-down RT-PCR to detect all NA subtypes, amplifying the region of the NA gene segment containing the molecular signature (H275Y) encoding oseltamivir resistance, and the PB2 segment from all influenza subtypes containing molecular signatures for pathogenicity.[77,78] A second weakness of current front-line diagnostic tests is that most of the commercial and in-house assays target a limited range of HA and NA types. Moreover, they are prone to drop out due to sequence variation in primer and probe regions.[35] In response to this problem, a more robust, albeit target-focused, real-time RT-PCR was developed to detect H1N1 of swine origin. It was improved by using RT-PCR to amplify two gene segments, the HA and NA genes.[37] This assay detected the first case of novel H1N1 influenza in Australia.
RT-PCR Amplification Followed by Microsphere Array Detection Fischer et al. have also utilized a universal PCR for flaviruses.[67] A first phase of symmetric PCR was followed by a second round of asymmetric PCR, followed by capture of PCR products onto Luminex beads preconjugated to primers specific for 12 flaviruses and four alphaviruses. This detection assay showed better sensivity than TaqMan® PCR for Kunjin and Japanese encephalitis viruses. A disadvantage of this type of microsphere panel (a disadvantage shared with the respiratory virus panels discussed previously) is that they typically contain only one virus-specific probe per virus, rendering them susceptible to drop out in the event of sequence change.
Degenerate RT-PCR Amplification Followed by Mass Spectrometry There are more than 29 species in the genus alphavirus and many subtypes; an intractable challenge for target-focused diagnostics. Eshoo et al. developed a multilocus broad-spectrum RT-PCR utilizing degenerate oligonucleotide primers followed by electrospray ionization mass spectrometry (ESI-MS) that identifies alphaviruses by base composition.[79] A small set of mixed-base primer pairs targeted conserved sites in the alphavirus RNA genome. Base compositions from the amplicons could unambiguously assign the species or subtype of 35 of the 36 isolates of Old and New World alphaviruses. The assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. One mosquito pool from Peru contained an alphavirus with a distinctive mass spectrum signature. Subsequent sequencing confirmed that the virus was a member of a new subtype of the Mucambo virus species (subtype IIID in the Venezuelan equine encephalitis virus complex). This high-throughput assay is useful both for surveillance and discovery of uncharacterized or emerging viruses.
Identity of Etiological Agent is Completely Unknown: No Hypothesis
At this level, the metagenomics approach is adopted whereby randomized amplification is followed by undirected or semidirected sequencing. Petrosino et al. defined metagenomics as "culture-independent studies of the collective set of genomes of mixed microbial communities".[80] It is an approach that can be used for pathogen identification in mixed microbial communities in the absence of an hypothesis, or when the first two diagnostic approaches listed in the section entitled 'Technological approaches to broad spectrum virus diagnosis' have failed. Metagenomic approaches have the power to sample the spectrum of known and novel viruses present in clinical samples. The detection of novel viruses facilitates follow-up research to determine the role of these novel viruses in etiology and epidemiology.
The studies of Van den Hoogen et al.,[81] Allander et al.,[82] Gaynor et al.,[83] Finkelbeiner et al.,[84] Nanda et al.,[85] Uhlenhaut et al.[86,87] and Epstein et al.[24] all involve the use of various versions of random amplification PCR or degenerate oligonucleotide PCR, prior to cloning into plasmid vectors and sequencing[84] or direct high-throughput pyrosequencing.[24,87]
The work of Gaynor et al. is instructive on the general principles involved in the metagenomic approach and how it fits into the diagnostic armory.[83] A nasopharyngeal aspirate (NPA) was obtained from a child with pneumonia. A total of 17 target-focused PCR assays for known respiratory viruses produced negative results. Total nucleic acid from the NPA was randomly amplified using the sequential, random primed 'AB' protocol of Wang et al..[71] The primer B products were cloned and 384 clones were sequenced using standard dye terminator chemistry. The strategy resulted in 327 human sequences, six known bacterial sequences and six viral sequences, all with limited homology (34–50% predicted amino acid identity) to known polyoma viruses. This led to the discovery of a novel polyoma virus. Specific primers were then designed and prevalence studies undertaken on 1254 respiratory samples, revealing a 3% prevalence. Finkbeiner et al. used the same approach, including the random primed 'AB' amplification method, followed by cloning and sequencing to identify a spectrum of known and novel viruses from human diarrhea, including highly divergent astrovirus and, unexpectedly, sequences from nodaviruses not hitherto associated with infection in mammals.[84]
Epstein et al. utilized a random octamer amplification combined with an unbiased pyrosequencing approach as a discovery tool, to screen sera from Pteropus bats for the presence of known or novel infectious agents.[24] PCR products greater than 70 bp in size were selected by column chromatography and then ligated to linkers for unbiased pyrosequencing. By this means, a novel GB-like flavivirus was discovered.
Metagenomics is not yet widely used in clinical virology laboratories, but work in bacterial pathogen identification[88] has mapped a path for the introduction of metagenomics into routine pathogen testing in a clinical setting. One of the challenges highlighted by Luna et al. is setting the decision criteria for sending samples for pyrosequencing, rather than utilizing more routine, target-focused assays.[88] Even in those cases where metagenomics and pyrosequencing is not part of the routine work flow and is rarely used (i.e., only when all routine assays are negative), the results of the metagenomic approach could be rapidly introduced into routine clinical diagnostics, if only as an expanded version in the panel of target-focused tests.
Metagenomics Approaches: Random Amplification Followed by Microarray Detection The road to incorporating metagenomics into the clinical virology lab may be smoothed by combining random amplification strategies with densely tiled arrays or dichot arrays (see earlier). Wang et al. combined the random primed AB PCR protocol with a densely tiled array containing the most highly conserved 70-mer sequences from every fully sequenced genome in GenBank.[71] By this means, rapid identification by pattern-matching is possible, and novel viruses can be discovered by simply scraping hybridized viral sequences from the array. A novel coronavirus was discovered by this method.[71]
Although pathogen arrays have proven their utility for discovering novel viruses, there have been technical problems related to accuracy and sensitivity, resulting in resistance to their use in routine patient care. Wong et al. demonstrated that amplification efficiency by random primers is the crucial determinant of the probe hybridization signal.[89] They developed an algorithm to predict the random primed viral amplification efficiency score.[90] In order to correlate the predicted virus amplification efficiency score with the array hybridization signal, they utilized Nimblegen array synthesis technology to detect 35 RNA viruses, using 40-mer probes tiled across the full length of each virus genome (390,482 probes, including replicates of each probe and controls). The prediction algorithm was used to design an amplification efficiency score optimized primer that had the highest score for all 35 viruses represented on the array. By utilizing the optimized amplification, the microarray platform identified pathogens with 76% sensitivity and 100% specificity relative to real-time PCR.
CombiMatrix corporation have developed sequencing microarrays, including a broad-scan microarray for subtyping influenza A virus.[91] The system is technologically elegant; however, the multiplex RT-PCR amplification of influenza virus nucleic acid prior to hybridization depends on the use of target-specific reverse primers[91] and, moreover, the probes on the array are unique to each of the target subtypes. This configuration means that the system is potentially vulnerable to drop out and will need to be redesigned when new subtypes arise or a mutation arises in regions of the virus recognized by the primers or probes. Bolotin et al. evaluated the CombiMatrix influenza detection system, comparing it with the FDA-approved Luminex respiratory virus panel (see earlier), for subtyping influenza A virus.[92] Although the limit of detection for the CombiMatrix test was three orders of magnitude greater than the respiratory syncytial virus panel, the sensitivity for detecting either H1 or H3 seasonal influenza viruses was still 95% relative to the Luminex respiratory virus panel.
Nanotechnology & Biosensors The core elements of biosensors are the amplification and detection technologies (that may be built using nanotechnology). However, assay design remains fundamental if the challenges of the emerging disease environment are to be met (sensitivity, specificity, use of multiple target genes to avoid drop out and capability to detect unexpected and novel viruses).
A range of new detection technologies has emerged over the last decade. The ones that will make the most impact will be compatible with assay designs that can support a high degree of multiplexing and adaptability to the detection of new viruses. Some of the adaptability will reside in astute primer design using new bioinformatics approaches (see earlier). High degrees of multiplexing, in turn, depend on resolution of signals from simultaneous assays, either by detection at different energy frequencies or by spatially/temporally resolving signals in one, two, three or four dimensions. Azzazy et al. reviewed some of the core technologies in nanodiagnostics, comparing quantum dots, cantilevers and nanoparticles.[93] Quantum dots and nanoparticle-based assays offer the potential for large-scale multiplexing of nucleic acid-based diagnostic tests, generally via the use of capture oligonucleotides to address and separate reactions for analysis. Nucleic acid amplification on microspheres is the core technology of new-generation sequencing,[94] but is yet to be fully exploited in first-line clinical molecular diagnostics.
At the frontier is research on addressable, real-time nucleic acid tests. These have the potential to be used on microsphere platforms, but need to be interfaced with technology (akin to that used for 454 sequencing[94]) to allow continuous reading of the fluorescent (or other) signal as it develops on microspheres. In one such system under development,[95] real-time PCR reactions can be carried out on microspheres (or other substrates, such as microplates or nanowires (see later) using large sets of oligonucleotide sequence tags. With these oligonucleotide tags, one set of microspheres can be adapted to perform many different real-time assays.
Closed, self-contained and fully integrated automated platforms that combine on-board sample preparation with real-time PCR and fluorescence detection are now available, with a range of target-focused, real-time PCR-based diagnostic tests. One assay developed for this type of fully integrated platform is an influenza diagnostic tool that detects and distinguishes (with excellent sensitivity and specificity[96]) between seasonal influenza, influenza B and the novel 2009 H1N1 influenza. However, independent of the efficient hardware used to execute the test, assay design remains the key issue with this type of target-focused assay. Next season, the prevalent strain of influenza A virus may change, necessitating redesign of the assay and renegotiation of the regulatory process.
Although sophisticated assays have been designed to increase the breadth of analytes detected in real-time assays, such as the use of extensions on PCR primers and melt-curve analysis,[57] fluorescence systems are dependent on labels and remain limited by the number of optical detection channels. They are therefore limited in the number of target analytes that can be selected, necessitating redesign to accommodate the emergence of novel, recombinant, reassorted or genetic variants.
Nanowires may overcome the challenge of multiplexing because, as no label is required, they are not limited by the number of available labels. Each wire is less than 50 nm wide and may be coated or covalently linked to oligonucleotides. Nanowire arrays, where nucleic acids are amplified and hybridized to nanowires, result in direct conversion of the hybridization event to an electrical signal and direct interface with information systems. Stern and colleagues have developed nanowire complementary metal oxide semiconductor-based label-free immunodetection.[97] To avoid the problem of Debye screening, the salt concentrations in the buffers used for macromolecular sensing experiments must be chosen so that Debye distance is sufficiently long to enable sensing, but sufficiently short to screen unbound macromolecules.[97] Others are developing etched silicon-based nanowire sensors for direct measurement of nucleic acid hybridization.[98–100] Bunimovich et al. demonstrated that a single-stranded complementary oligonucleotide is able to significantly change the conductance of a group of 20-nm-diameter silicon nanowires in a 0.165 M solution, by hybridizing to a primary DNA strand that has been electrostatically adsorbed onto an amine-terminated organic monolayer on the nanowires.[100] This intimate contact of the primary strand with the amine groups of the nanowire surface brings the binding event within the Debye distance, allowing the binding event to be electronically detected. Moreover, within a 0.165 M ionic strength solution, the DNA hybridization is efficient, compared with solutions of lower ionic strength.[100]
This work will lead to multichannel devices capable of very high degrees of multiplexing. If such systems are interfaced with sets of 'universal' capture oligonucleotides, coated or covalently linked onto the nanowires, they will become highly parallel and rapidly adaptable diagnostic systems. The longer-term vision for these devices is for integration with amplification, using nanowire chips that can be dropped into a PCR tube and relay the electrical signal via radio-frequency identification device from remote locations to a central server (Figure 2).
Figure 2.
Nanowire chip in PCR tube. Electronic 'drop-in-tube' biochip. The chip could include oligonucleotide-coated nanowires at the lower end of the chip (yellow section), each of which is connected to a discrete radiofrequency identification transponder (red section) [102]. The process by which nucleic acids are coated on the nanowires, and the distance between the wire surface and the hybridization event, will be critical (see [99,100]) to allow this type of device to operate at the ionic strengths encountered in PCR reactions. Reproduced with permission from Biochip Innovations Ltd (Brisbane, Australia).
Expert Rev Mol Diagn. 2011;11(4):409-423. © 2011 Expert Reviews Ltd.
Cite this: Expecting the Unexpected - Medscape - May 01, 2011.
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