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Journal of Clinical Microbiology, December 2004, p. 5811-5818, Vol. 42, No. 12
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.12.5811-5818.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Medical Microbiology, St. George's Hospital Medical School, London,1 Beatson Cancer Institute, Glasgow, United Kingdom2
Received 2 April 2004/ Returned for modification 22 April 2004/ Accepted 27 July 2004
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ABSTRACT |
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INTRODUCTION |
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Despite the introduction of diagnostic PCR in routine laboratories, only a fraction of the potential pathogens can be identified in patients with suspected infective CNS syndromes (for a review, see reference 8). A recent audit at St. George's Hospital has shown that only 45% of physician-diagnosed cases of meningitis or encephalitis were confirmed by a laboratory diagnosis, comparable to figures reported by others (43%) (17). The principal reasons for this failure are the large number of potential pathogens involved and the small volume of the cerebrospinal fluid (CSF) sample, which must often be divided up for several analyses, often at different laboratories. A multiplex PCR approach has overcome the diagnostic limitations of multiple PCRs for the detection of individual neuroinvasive viruses (6, 8, 14, 18). However, even multiplex PCR allows only a few potential pathogens (about three to six) to be targeted at any one time. The amplification of a low-abundance pathogen by multiplex PCR often results in spurious bands by gel electrophoresis (5) and requires validation by hybridization to specific probes immobilized on membranes or microwell plates. These hybridization formats have limited capacities.
A technique capable of overcoming these limitations and increasing the diagnostic output of the assay is hybridization on a DNA microarray. Due to its unrestricted capacity to accommodate hundreds to thousands of individual gene probes, an array allows the simultaneous detection of potentially any amplifiable pathogen present in a specimen. As a result, this technology is ideal for the extensive parallel identification and differentiation of various pathogens and their strains. The technique is rapidly evolving from a novel research technology (10) to a practical tool for the identification of bacterial species (5, 11), the detection of environmental bacteria (24), and the genotyping of influenza viruses (13) and human immunodeficiency virus (HIV) type 1 (23). However, the microarray approach has not yet been extended to the clinical diagnosis of viral infections.
We have applied the DNA microarray technology to the identification of specific pathogens in cases of meningitis and encephalitis. We focused on developing a microarray for the detection not only of the most common neurotropic viruses in the United Kingdom (herpes simplex virus [HSV], varicella-zoster virus [VZV], and enteroviruses) (18) but also for the detection of several other viruses capable of causing CNS syndromes in very young and immunocompromised individuals. The greatest difficulties in clinical management are determining whether the etiology is bacterial or viral in an individual patient. However, we decided to concentrate on viral pathogens because we already had prospectively well characterized patients and cerebrospinal fluid (CSF) samples from patients with CNS infection for which the causative viral pathogen was confirmed by standard techniques and because the majority of infective CNS syndromes result from virus infections. Once the array was constructed, viral etiologic agent could be identified from a panel of 13 different neurotropic viral agents.
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MATERIALS AND METHODS |
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CSF samples from patients with viral meningitis diagnosed by virus culture and/or PCR were collected retrospectively over the past 2 years and were stored at –70°C. The laboratory worker who processed the specimens by PCR-microarray analysis was blinded to the clinical diagnoses and the viral agent.
Nucleic acid extraction from clinical specimens. Every CSF specimen was divided in two, and each half was processed separately for DNA and RNA viruses. CSF volumes of 200 µl were used. If the volume was less (30 to 100 µl), it was adjusted to 200 µl with phosphate-buffered saline. These samples were supplemented with 10 µg of carrier tRNA (Sigma-Aldrich, Poole, United Kingdom). DNA was extracted by use of a QIAamp DNA blood kit (Qiagen, Crawley, United Kingdom) and RNA was extracted by use of the Trizol LS reagent (Invitrogen, Paisley, United Kingdom), according to the directions of each manufacturer. The DNA was eluted in 50 µl of nuclease-free water and was stored at –20°C for repeated use. The total RNA from each specimen was reverse transcribed, and the cDNA was stored at –70°C.
Primers and multiplex PCR amplification. Unique pairs of gene-specific primers were designed for each viral target on the basis of a full search of the GenBank database (www.ncbi.nlm.nih.gov) with the BLAST program for known virus sequences. To ensure a nearly equal annealing efficiency, all primers were 22-mers and had similar melting temperatures (58.1 ± 1.6°C) and G+C contents (44.6% ± 3.8%). In order to prevent cross-hybridization, the target viral sequences (range, one to five per viral genome) were selected so that they represented unique regions with little or no similarity with the sequences deposited in the GenBank database. The primer sequences and their target locations within viral genomes are listed in Table 1. Three pairs of primers were designed for each virus to increase the probability of virus detection (Table 1). For enteroviral genome detection we used only one pair of primers, E1 and E2, whose sequences flank a 201-bp product within the conserved 5' nontranslated region of the genome (4) (Table 1). In the case of CMV, three newly designed primer pairs were supplemented with two known pairs already in use; the last two primer pairs (CMV4 and CMV5 in Table 1) target the late ULIIIA region and the early-late UL21.5 region of the CMV genome, respectively, and have previously been demonstrated to have high sensitivities and specificities for the detection of CMV by PCR (3). One of three pairs of primers designed to detect both HSV-2 and HHV-6B failed to amplify the viruses when they were used with positive control material and were excluded from the study, leaving only two primer sets for the detection of these viruses.
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Precautions to prevent contamination included spatial separation of pre- and post-PCR steps, the use of filter pipette tips, and regular treatment of surfaces and pipettes with DNA-destroying agents. Once a sample was found to be virus positive by the multiplex PCR and microarray assay, the extracted stored DNA or cDNA was reamplified with virus-specific primers and the purified PCR bands were validated by direct dideoxy sequencing with a USB Sequenase (version 2.0) kit (Amersham Biosciences).
Microarray production. Microarrays were printed on polylysine-coated glass microscope slides with a contact microspotting robotic system (MicroGrid II; BioRobotics, Cambridge, United Kingdom) by using split pins. The average spot size was 150 to 200 µm. The printed slides were then processed by chemically blocking the unreacted lysine groups by a method previously described for use with CMT-GAPS (Corning Ltd., High Wycombe, United Kingdom)-coated slides (7). The probe elements on the array were made from gel-purified PCR products generated from the viral DNA or RNA template (see above) by using the same gene-specific primer pair sequences (Table 1) used for multiplex PCR of the clinical samples. PCR products were suspended in 50% dimethyl sulfoxide at a DNA concentration of 0.5 µg/µl. Printed slides were stored dry in lightproof protective boxes until use.
Array format. Each array contained identical sets of probes spotted in quadruplicate (for signal averaging) and arranged in a virus-specific subgrid format (Fig. 1). The subgrids were composed of probes spotted in a pattern of seven by five spots; these were combined into larger groups of eight to form a metagrid. Each subgrid had a pattern characteristic for each virus (Fig. 1). To enable positive virus identification, the individual subgrid must have the correct spot pattern and the location of the subgrid within the metagrid template must be correct. Each subgrid had "landing-light" spots (top left and bottom right) made with cyanine 3 (Cy3)- or Cy5-labeled nucleotides to mark the array orientation (Fig. 1). The center five spot locations were left blank. The slides were analyzed in an Affymetrix (Santa Clara, Calif.) 428 array scanner with the aid of ImaGene (BioDiscovery, Marina del Rey, Calif.) software (version 4.2). Two fluorescent images of the same array for alternatively labeled hybridization control (Cy5 detection at 670 nm) and sample (Cy3 detection at 570 nm) channels were first examined on their own and were then examined as a superimposed image of the two to produce a false two-color (red-green) image. For positive samples, the resulting signal formed one of the expected spot patterns depicted in Fig. 1.
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Virus detection. The results were considered positive for virus detection when the mean hybridization signal intensity from the quadruplicate virus-specific spotted elements (probes) on the array was three times greater than the background signal intensity in one or more of the subgrids. The background signal intensity was defined as the mean signal intensity for all the nonspecific probes on the array minus the background signal intensity for the local spot. The local background signal intensity around the nonspotted area of each spot was automatically subtracted from the spot signal intensity by image acquisition software (version 4.1; ImaGene BioDiscovery). Each experiment was performed as a cohybridization between an internal hybridization control sample (prepared from a mixture of all virus-specific PCR products spotted on the array) and the multiplex PCR-amplified test sample. Control and test samples were separately labeled with the fluorescent dye conjugate Cy5 (indicated here as computer-generated false color red) or Cy3 (indicated here as computer-generated false color green), respectively. The amount of control DNA was titrated to 10 ng so that each spot on the array gave a sufficiently intense signal for spot detection (red) while still allowing the test viral PCR products to hybridize, thus producing a green color after quantitation and computer superimposition of the signal intensity ratios from the two channels. Thus, positive hybridization signals (visualized here as green) could be determined by eye from the correct patterns of spots in both the subgrid and the metagrid locations that corresponded to the target virus.
To guard against slide-to-slide and batch-to-batch variations in signal intensities, the Cy3 and Cy5 images were scanned at the same laser power and saved as eight-bit TIFF files. The mean fluorescent signal intensity was measured for each of quadruplicate Cy3-labeled sample spots or Cy5-labeled control spots with ImaGene Biodiscovery software. Statistical analysis was performed with Axum (version 6.0) software (MathSoft, Inc.).
PCR-microarray assay sensitivity. The sensitivity of our multiplex PCR-microarray assay was determined by using CMV and echovirus as representatives of the DNA and RNA virus groups, respectively. The CMV and echovirus stocks, both of which were adapted to HEL cells, were serially diluted 10-fold and titrated on replicate HEL cell cultures. The end points were determined by observing a clearly visible cytopathic effect 5 days postinfection in echovirus-infected replicate cell cultures or by immunoenzymatic staining of CMV-infected cultures (21). The RNA or DNA was extracted from 1 ml of cell culture supernatant, which was collected from each dilution in the virus titration experiments and clarified by centrifugation at 14,000 x g for 15 min and then processed by multiplex PCR. After amplification the products from replicate PCRs for each virus dilution were pooled, purified, and divided in two. One half was run on 6% polyacrylamide minigels, and the other half was randomly labeled and hybridized to the microarray, followed by comparison of end-point detection by either method. The CMV DNA copy number in the end-point dilution was estimated by a semiquantitative PCR with primers specific for the ULIIIA region (CMV5 in Table 1) and an internal DNA mimic standard (2). For echovirus RNA quantification, the purified 201-bp PCR DNA fragment was ligated to the T7 RNA polymerase promoter by use of the Lig'nScribe kit (Ambion, Austin, Tex.), and runoff RNA transcripts were produced with an in vitro transcription kit (Epicentre Technologies, Madison, Wis.), according to the instructions of each manufacturer. The RNA product was digested with 2 U of RNase-free DNase I (Ambion) and was quantified by measuring its optical density. A stock preparation of the transcript RNA (1.25 µg/µl) was serially diluted 10-fold in RNase-free water, and each dilution was processed by RT-PCR with a mixture of RNA virus-specific primers, followed by simultaneous gel and microarray hybridization analysis.
The clinical sensitivity, specificity, and positive and negative predictive values of the multiplex PCR were determined by comparison of the multiplex PCR results with the results of the virus-specific PCR and were calculated by using conventional formulas.
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RESULTS |
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A total of 60 clinical specimens from patients with clinically suspected cases of meningitis or encephalitis were processed by the diagnostic multiplex PCR-microarray procedure. Of the 60 clinical specimens test, 41 gave patterns for a specific virus on the array, including patterns for echoviruses (23 samples), HSV-2 (4 samples), VZV (4 samples), HHV-7 (1 sample), HHV-6A (1 sample), HHV-6B (2 samples), EBV (3 samples), polyomavirus JC (1 sample), and CMV (2 samples). Three samples had false-negative results (1 sample was positive for VZV, 1 was positive for HSV-2, and 1 was positive for measles virus; Table 2), and 15 had true-negative results. The samples with true-negative results comprised 1 sample from a patient with Lyme disease, 1 sample from a patient with serologically proven primary EBV infection and Guillain-Barré syndrome, and 13 samples from patients with bacterial meningitis and other neurological conditions of an ultimately noninfective etiology. A representative example of positive detection by two-color image analysis is shown in Fig. 2A for VZV, in which the quadruplicate green spots are in the correct positions within the subgrid and in the correct three subgrids of the metagrid, as predicted from the schematic in Fig. 1. Samples with positive results with two of three probes for a separate VZV-infected CSF specimen are also shown in Fig. 2B, in which one subgrid is negative for the virus-specific probe element contained within it.
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In comparison to PCR amplification only with primers specific for a particular virus followed by microarray hybridization (referred to as virus-specific PCR in Table 2), the multiplex PCR-microarray assay failed to hybridize to any probe on the array for only two samples. These were positive, however, with one of three probes (VZV) or one of two probes (HSV-2) after virus-specific PCR. For two other samples (one containing polyomavirus JC and one containing HSV-2; Table 2), the multiplex PCR-microarray assay detected one less target than the virus-specific PCR did. The virus in one specimen (a throat swab specimen) found to be positive for measles virus at the Health Protection Agency reference laboratories by a single-gene nested PCR assay was not detected by the PCR-microarray assay or virus-specific PCR assay, presumably because of very low viral titers. Thus, the performance of our multiplex PCR-microarray assay was close to that of the standard diagnostic PCR tests used by most clinical microbiology laboratories.
The analytical sensitivity of our assay was estimated for a DNA virus (CMV) and an RNA virus (echovirus) by correlating the PCR end-point signals on the gel and on the microarray by using serial dilutions of viral stocks (see Materials and Methods). The sensitivity of CMV ULIIIA DNA band detection by our polyacrylamide minigel system is 2.5 ng of DNA (2); that compared well with the reported detection limit of 1 ng of randomly labeled DNA detectable by microarray hybridization (24). In terms of infectivity or viral copy numbers, the calculated sensitivity of our multiplex PCR-microarray assay for CMV was 1.6 infectious CMV units, or 46 CMV DNA copies. For the echovirus, the values were 0.01 50% tissue culture infective doses or 28 RNA copies (125 ag), which are comparable to the sensitivities attained by nonnested PCR techniques for these two viruses reported by others (1, 15, 19).
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DISCUSSION |
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The phenomenon of PCR selection is best illustrated in our study by several examples. In the first, three of five VZV-infected samples were positive with two of three viral probes, namely, probe 1 and probe 2 (VZV1 and VZV2, respectively, in Table 1; Fig. 2B); one sample was positive for probes 1 and 3 (regardless of whether the mixture of all viral primers or only pooled VZV-specific PCR primers was used); and the fifth sample gave no signal on the array after multiplex PCR amplification (the results are summarized in Table 2). However, when this VZV array-negative sample was reamplified with only three pairs of VZV-specific primers, the positive signal on the array was due exclusively to target 3 (VZV3 in Table 1). Another example of PCR selection was seen with five HSV-2-infected specimens, all of which were positive by assays with one of two probes (Table 2). However, three specimens were positive for probe 2 (HSV-2 element 2 in Table 1; Fig. 2C) and two specimens were positive in assays with probe 1 (the results are summarized in Table 2). Similar effects were observed for CMV, as shown in Fig. 3E with four of five CMV-specific probe elements showing strong positive signal intensities of 51,477, 12,376, 11,767, and 12,691, respectively. A weak signal was seen with CMV element 5, with a signal intensity of 1,410, but the signal was still above the threefold cutoff value of 995. The variability in viral targets detected among specimens suggests that, apart from the amplification superiority of some primer pairs, some specimen-related factor(s) might affect the PCR. Indeed, two CMV-infected specimens of different origins showed only a 10-fold difference in viral loads but gave remarkably different scores for the viral targets detected by the multiplex PCR-microarray assay (Table 2)
Although PCR suppression-selection was occurring, its impact on the diagnostic utility of the multiplex PCR-microarray assay was minimized through the inclusion of multiple probes on the array for each virus such that a positive result with any one probe was sufficient for a correct identification. For both retrospective and prospective specimens, the virus detected by the multiplex PCR-microarray assay was verified by virus-specific PCR in the Department of Medical Microbiology, St. George's Hospital, and was independently confirmed by commercial PCR screening assays (18).
The majority of meningitis cases in this study (56%) were caused by enteroviruses. This was not unexpected, as their prevalence over other viral causes of meningitis is well established (for a review, see reference 20). Also, our specimens were collected over a 2-year period, inclusive of seasonal countrywide outbreaks of enterovirus infection. For 11 of 23 enterovirus-positive specimens we observed a cross-hybridization pattern between the echovirus and coxsackie B virus targets due to their known sequence similarity (see Fig. 2D and Fig. 3D, which show a weak signal intensity of 1,786 with the single coxsackie B virus-specific probe and a signal intensity of 22,659 with the echovirus-specific probe). Indeed, when the coxsackie B1 virus sequence was used to query other enterovirus sequences with the BLASTN program for sequence homology comparison, the sequence variability among the top 50 hits was 7 to 10%; these included strains of coxsackie B virus, echovirus, and other enteroviruses. Given that PCR product-based DNA microarrays cannot distinguish between DNA targets sharing more than 80% sequence identity (9, 24), the cross-hybridization between coxsackie B virus and echovirus targets was not unexpected. However, the echovirus target cross-reacted with the closely related coxsackie B virus-specific probe for only half of the samples, with a mean intensity of signal of 65% (range, 28 to 89%). This might be due to a combination of factors, such as viral load, amplification efficiency, and the degree of sequence diversity, that affect the cross-hybridization patterns of enteroviruses on the array.
The impacts of various factors on microarray scanner-generated digital images have been reviewed at length by Li et al. (13). They concluded that judging images of closely related influenza viruses just by eye can be misleading. Indeed, we observed differences in spot sizes on our arrays due to batch-to-batch or beginning-to-end printing variations. To ascertain whether the signal intensities vary from slide to slide or batch to batch, we analyzed the most prevalent echovirus target, which was detected by using 4 slides from batch 1, 15 slides from batch 2, and 3 slides from batch 3. On the basis of the average intensity of four echovirus spots per slide, the mean variations in echovirus signal intensities were 10, 7.7, and 4.6% for slide batches 1, 2, and 3, respectively. Therefore, good intra- and interbatch array reproducibilities, coupled with the lack of cross-hybridization between unrelated viruses, make the visual signal readout from the computer-assisted two-color superimposition image very clear. Of practical consideration is the ability of the multiplex PCR-microarray assay to provide a consistently robust qualitative detection of at least one target per viral genome. We found no difficulties in distinguishing by eye true-positive from true-negative results (Fig. 2). Although we showed that there was a 100% concordance for positive virus detection results and that the theoretical limits of detection were as low as 48 genome copies for CMV and 28 genome copies for echovirus, it should not be assumed that negative results indicate the absence of virus. Issues of assay sensitivity, low viral genome copy number, and sample-specific inhibition affect the interpretation of negativity. The sensitivities for the detection of virus in clinical samples are likely to be lower than those calculated here for tissue culture samples.
There is a need for multicomponent panel-type diagnostic assays that allow the differentiation and identification of the pathogens responsible for disease syndromes with many potential causes, such as CNS or respiratory infections. The capacity of microarrays to accommodate high probe element densities and the potential use of several multiplex pools of primer pairs permit the development of pan-pathogen-specific probe arrays that will encompass other, primarily bacterial, causes of CNS infection, in addition to viruses. One limitation of the present array is that strain-specific sequence variations cannot be detected, and this would be of particular relevance to RNA viruses. The primers used in this study were not designed to target strain-specific nucleotide polymorphisms. Such limitations could be overcome by the use of strain-specific primer sets that represent known polymorphisms or by choosing nonvariant target sequences for primer design. Such modifications to PCR-microarray assays are tractable and would provide great diagnostic and clinical utility for certain viral infections. Single-format pan-pathogen-specific microarrays will contribute significantly to the speed and accuracy of laboratory diagnosis. This will have a beneficial impact on clinical care and hospital economics. This study has demonstrated the simplicity, accessibility, and applicability of such an approach in the context of CNS infections and is the first time that the diagnostic power of a microarray linked to multiplex PCR amplification of multiple pathogens has been demonstrated in a clinical laboratory.
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ACKNOWLEDGMENTS |
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This work was supported by the NHS R&D Support Fund from St. George's Healthcare Trust. R.A.S. and J.H. were supported by a grant from The Wellcome Trust Functional Genomics Resources Initiative for a microbial pathogen microarray facility in the Department of Medical Microbiology, St. George's Hospital.
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FOOTNOTES |
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REFERENCES |
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