VaxArray immunoassay for the multiplexed quantification of poliovirus D-antigen.

Next generation poliovirus vaccines are critical to reaching global poliovirus eradication goals. Recent efforts have focused on creating inactivated vaccines using attenuated Sabin strains that maintain patient safety benefits and immunogenicity of conventional inactivated vaccines while increasing manufacturing safety and lowering production costs, and on developing novel oral vaccines using modified Sabin strains that provide critical mucosal immunity but are further attenuated to minimize risk of reversion to neurovirulence. In addition, there is a push to improve the analytical tools for poliovirus vaccine characterization. Conventional and Sabin inactivated poliovirus vaccines typically rely on standard plate-based ELISA as in vitro D-antigen potency assays in combination with WHO international standards as calibrants. While widely utilized, the current D-antigen ELISA assays have a long time to result (up to 72 h), can suffer from lab-to-lab inconsistency due to non-standardized protocols and reagents, and are inherently singleplex. For D-antigen quantitation, we have developed the VaxArray Polio Assay Kit, a multiplexed, microarray-based immunoassay that uses poliovirus-specific human monoclonal antibodies currently under consideration as standardized reagents for characterizing inactivated Sabin and Salk vaccines. The VaxArray assay can simultaneously quantify all 3 poliovirus serotypes with a time to result of less than 3 h. Here we demonstrate that the assay has limits of quantification suitable for both bioprocess samples and final vaccines, excellent reproducibility and precision, and improved accuracy over an analogous plate-based ELISA. The assay is suitable for adjuvanted combination vaccines, as common vaccine additives and crude matrices do not interfere with quantification, and is intended as a high throughput, standardized quantitation tool to aid inactivated poliovirus vaccine manufacturers in streamlining vaccine development and manufacturing, aiding the global polio eradication effort.

30-Minute Highly Multiplexed VaxArray Immunoassay for Pneumococcal Vaccine Antigen Characterization.

Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity. Current characterization methods are often inherently singleplex, requiring separate tests for each serotype present. In addition, identity and quantity are often determined with separate methods. We developed the VaxArray pneumococcal assay for applications in identity, quantity, and stability testing of pneumococcal polysaccharide and pneumococcal conjugate vaccines. The VaxArray pneumococcal assay has a time to result of less than 30 min and is an off-the-shelf multiplexed, microarray-based immunoassay kit that can identify and simultaneously quantify 23 pneumococcal polysaccharide serotypes common to many on-market and in-development vaccines. Here, we highlight the potential of the assay for identity testing by showing high reactivity and serotype specificity to a wide variety of native polysaccharides, CRM197-conjugated polysaccharides, and drug product. The assay also has vaccine-relevant lower limits of quantification in the low-to-mid ng/mL range and can be used for accurate quantification even in adjuvanted vaccines. Excellent correlation to the anthrone assay is demonstrated, with VaxArray resulting in significantly improved precision over this antiquated chemical method.

Rapid Identity and Quantity CQA Test for Multivalent mRNA Drug Product Formulations.

The COVID-19 pandemic highlighted mRNA as a promising platform for vaccines and therapeutics. Many of the analytical tools used to characterize the critical quality attributes of mRNA are inherently singleplex and are not necessarily optimal from a labor and cost perspective. Here, we demonstrate the feasibility of a multiplexed platform (VaxArray) for efficient identity verification and concentration determination for both monovalent and multivalent mRNA formulations. A model system comprising mRNA constructs for influenza hemagglutinin and neuraminidase was used to characterize the analytical performance metrics for a VaxArray mRNA assay. The assay presented herein had a time to result of less than 2 h, required no PCR-based amplification nor extraction of mRNA from lipid nanoparticles, and exhibited high construct specificity that enabled application to the bivalent mixture. The sensitivity for influenza hemagglutinin and neuraminidase mRNA was sub-µg/mL, which is vaccine-relevant, and the average accuracy (%recovery of a check standard) and precision were 104 ± 2% and 9 ± 2%, respectively.

Identification of Escherichia coli O157 by using a novel colorimetric detection method with DNA microarrays.

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses.

Rapid, sensitive, and precise multiplexed assays for serological analysis during candidate COVID-19 vaccine development would streamline clinical trials. The VaxArray Coronavirus (CoV) SeroAssay quantifies IgG antibody binding to 9 pandemic, potentially pandemic, and endemic human CoV spike antigens in 2 h with automated results analysis. IgG antibodies in serum bind to the CoV spike protein capture antigens printed in a microarray format and are labeled with a fluorescent anti-species IgG secondary label. The assay demonstrated excellent lower limits of quantification ranging from 0.3 to 2.0 ng/mL and linear dynamic ranges of 76 to 911-fold. Average precision of 11 % CV and accuracy (% recovery) of 92.5 % over all capture antigens were achieved over 216 replicates representing 3 days and 3 microarray lots. Clinical performance on 263 human serum samples (132 SARS-CoV-2 negatives and 131 positives based on donor-matched RT-PCR and/or date of collection) produced 98.5 % PPA and 100 % NPA.

Evaluation of a multiplexed coronavirus antigen array for detection of SARS-CoV-2 specific IgG in COVID-19 convalescent plasma.

Mitigation of the COVID-19 pandemic requires an understanding of the antibody response to SARS-CoV-2. However, throughout the development of SARS-CoV-2 IgG antibody assays during the past year, cross-reactivity to other coronaviruses remained a question. To address these issues, we evaluated IgG in COVID-19 convalescent plasma samples for reactivity against three SARS-CoV-2 antigens including full-length spike, receptor binding domain, and the proximal extracellular fusion domain, and spike antigens from other coronaviruses (SARS-CoV, MERS-CoV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63) using the VaxArray Coronavirus SeroAssay which is a multiplexed antigen assay developed by InDevR Inc. These results were compared to two commercial SARS-CoV-2 IgG ELISAs targeting either the SARS-CoV-2 nucleocapsid or spike antigens and a live virus focus reduction neutralizing antibody test (FRNT). The VaxArray platform showed high specificity for detection of SARS-CoV-2 IgG, evident from lack of reactivity to SARS-CoV-2 antigens despite significant reactivity to endemic coronavirus antigens in pre-pandemic samples. SARS-CoV-2 IgG positive samples reacted weakly to SARS-CoV spike but not to MERS-CoV. While the VaxArray platform had overall comparable results to the spike and nucleocapsid IgG ELISAs, results were more similar to the spike antigen ELISA and the platform displayed a higher sensitivity and specificity than both ELISAs. Samples with FRNT titers below 1/23 reported negative on VaxArray, while positive samples on VaxArray had significantly higher neutralizing antibody titers. These results suggest that the VaxArray Coronavirus SeroAssay performs with high sensitivity and specificity for the detection of SARS-CoV-2 IgG, and positive results on the platform indicate SARS-CoV-2 neutralizing activity.

FluChip-8G Insight: HA and NA subtyping of potentially pandemic influenza A viruses in a single assay.

BACKGROUND: Global influenza surveillance in humans and animals is a critical component of pandemic preparedness. The FluChip-8G Insight assay was developed to subtype both seasonal and potentially pandemic influenza viruses in a single assay with a same day result. FluChip-8G Insight uses whole gene segment RT-PCR-based amplification to provide robustness against genetic drift and subsequent microarray detection with artificial neural network-based data interpretation. OBJECTIVES: The objective of this study was to verify and validate the performance of the FluChip-8G Insight assay for the detection and positive identification of human and animal origin non-seasonal influenza A specimens. METHODS: We evaluated the ability of the FluChip-8G Insight technology to type and HA and NA subtype a sample set consisting of 297 results from 180 unique non-seasonal influenza A strains (49 unique subtypes). RESULTS: FluChip-8G Insight demonstrated a positive percent agreement ≥93% for 5 targeted HA and 5 targeted NA subtypes except for H9 (88%), and negative percent agreement exceeding 95% for all targeted subtypes. CONCLUSIONS: The FluChip-8G Insight neural network-based algorithm used for virus identification performed well over a data set of 297 naïve sample results, and can be easily updated to improve performance on emerging strains without changing the underlying assay chemistry.

Molecular detection of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2-3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.

Analytical evaluation of the microarray-based FluChip-8G Influenza A+B Assay.

BACKGROUND: Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. OBJECTIVE: To evaluate key analytical performance characteristics of the FluChip-8G Influenza A+B Assay. STUDY DESIGN: Analytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. RESULTS: The limit of detection (LOD) for the FluChip-8G influenza A+B Assay ranged from 5.8 × 102-1.5 × 105 genome copies/mL, with most samples ∼2 × 103 genome copies/mL (∼160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both "non-seasonal influenza A" and A/H1N1pdm09. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7-98.7)) regardless of operator, site, reagent lot, or testing day. CONCLUSION: The FluChip-8G Influenza A+B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.

Clinical validation of the FluChip-8G Influenza A+B Assay for influenza type and subtype identification.

BACKGROUND: The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as "non-seasonal" A viruses from human nasal specimens. OBJECTIVE: To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples. STUDY DESIGN: For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay. For non-seasonal viruses, testing was performed at a single site using contrived samples from 100 unique non-seasonal strains representing 41 subtypes. RESULTS: Sensitivity (95% CI) and specificity (95% CI) for each target group, respectively, from results of 1689 clinical specimens were: seasonal H1N1pdm2009: 96.4% (87.9-99.0), 99.3% (98.8-99.6), seasonal H3N2: 91.8% (87.7-94.7), 99.7% (99.2-99.9), Influenza B Victoria: 100% (94.0-100.0), 99.9% (99.6-100.0), and Influenza B Yamagata: 95.6% (89.2-98.3), 99.9% (99.6-100.0). The sensitivity and specificity from contrived influenza A non-seasonal viruses was determined to be 99.0% (94.6-99.8) and 100% (96.7-100.0). CONCLUSION: The FluChip-8G Influenza A+B Assay has robust sensitivity and specificity for detecting and identifying all target virus groups, including non-seasonal influenza A, with same day results.

Detection of adamantane-resistant influenza on a microarray.

BACKGROUND: Influenza A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions. OBJECTIVE: To design a cost-effective low-density microarray for use in detection of influenza resistance to the adamantanes. STUDY DESIGN: We have taken advantage of functional genomics and microarray technology to design a DNA microarray that can detect the two most common mutations in the M2 protein associated with adamantane resistance, V27A and S31N. RESULTS: In a blind study of 22 influenza isolates, the antiviral resistance-chip (AVR-Chip) had a success rate of 95% for detecting these mutations. Microarray data from a larger set of samples were further analyzed using an artificial neural network and resulted in a correct identification rate of 94% for influenza virus samples that had V27A and S31N mutations. CONCLUSIONS: The AVR-Chip provided a method for rapidly screening influenza viruses for adamantane sensitivity, and the general approach could be easily extended to detect resistance to other chemotherapeutics.

ampliPHOX colorimetric detection on a DNA microarray for influenza.

DNA microarrays have emerged as a powerful tool for pathogen detection. For instance, many examples of the ability to type and subtype influenza virus have been demonstrated. The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use, replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective. The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is ˜1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection. Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

BACKGROUND: The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10. OBJECTIVES: The work presented here represents an effort to expand and test the capabilities of the MChip to differentiate influenza A/H1N1 of various species origin. METHODS: The MChip ANN was trained to recognize fluorescence image patterns of a variety of known influenza A viruses, including examples of human H1N1, human H3N2, swine H1N1, 2009 pandemic influenza A H1N1, and a wide variety of avian, equine, canine, and swine influenza viruses. Robustness of the MChip ANN was evaluated using 296 blinded isolates. RESULTS: Training of the ANN was expanded by the addition of 71 well-characterized influenza A isolates and yielded relatively high accuracy (little misclassification) in distinguishing unique H1N1 strains: nine human A/H1N1 (88·9% correct), 35 human A/H3N2 (97·1% correct), 31 North American swine A/H1N1 (80·6% correct), 14 2009 pandemic A/H1N1 (87·7% correct), and 23 negative samples (91·3% correct). Genetic diversity among the swine H1N1 isolates may have contributed to the lower success rate for these viruses. CONCLUSIONS: The current study demonstrates the MChip has the capability to differentiate the genetic variations among influenza viruses with appropriate ANN training. Further selective enrichment of the ANN will improve its ability to rapidly and reliably characterize influenza viruses of unknown origin.

Real-time quantification of RNA polymerase activity using a "broken beacon".

A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.

Evaluation of MChip with historic subtype H1N1 influenza A viruses, including the 1918 "Spanish Flu" strain.

The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.

Identification of A/H5N1 influenza viruses using a single gene diagnostic microarray.

In previous work, a simple diagnostic DNA microarray that targeted only the matrix gene segment of influenza A (MChip) was developed and evaluated with patient samples. In this work, the analytical utility of the MChip for detection and subtyping of an emerging virus was evaluated with a diverse set of A/H5N1 influenza viruses. A total of 43 different highly pathogenic A/H5N1 viral isolates that were collected from diverse geographic locations, including Vietnam, Nigeria, Indonesia, and Kazakhstan, representing human, feline, and a variety of avian infections spanning the time period 2003-2006 were used in this study. A probabilistic artificial neural network was developed for automated microarray image interpretation through pattern recognition. The microarray assay and subsequent subtype assignment by the artificial neural network resulted in correct identification of 24 "unknown" A/H5N1 positive samples with no false positives. Analysis of a data set composed of A/H5N1, A/H3N2, and A/H1N1 positive samples and negative controls resulted in a clinical sensitivity of 97% and a clinical specificity of 100%.

Diagnostic microarray for influenza B viruses.

The importance of global influenza surveillance using simple and rapid diagnostics has been frequently highlighted. For influenza type B, the need exists for discrimination between the two currently circulating major lineages, represented by virus strains B/Victoria/2/87 and B/Yamagata/16/88, as only one of these lineages is represented in seasonal influenza vaccines. Here, the development and characterization of a low-density DNA microarray (designated BChip) designed to detect and identify the two influenza B lineages is presented. The assay involved multiplex nucleic acid amplification and microarray hybridization of viral RNA. Detection and lineage identification was achieved in less than 8 h. In a study of 62 influenza B virus samples from 19 countries, dating from 1945 to 2005, as well as 5 negative control samples, the assay exhibited 97% sensitivity and 100% specificity. Furthermore, application of a trained artificial neural network to the pattern of relative fluorescence signals resulted in correct lineage assignment for 94% of 50 applicable influenza B viruses, with no false assignments.

Comparison of the MChip to viral culture, reverse transcription-PCR, and the QuickVue influenza A+B test for rapid diagnosis of influenza.

The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A+B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.

Robust sequence selection method used to develop the FluChip diagnostic microarray for influenza virus.

DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/.

MChip: a tool for influenza surveillance.

The design and characterization of a low-density microarray for subtyping influenza A is presented. The microarray consisted of 15 distinct oligonucleotides designed to target only the matrix gene segment of influenza A. An artificial neural network was utilized to automate microarray image interpretation. The neural network was trained to recognize fluorescence image patterns for 68 known influenza viruses and subsequently used to identify 53 unknowns in a blind study that included 39 human patient samples and 14 negative control samples. The assay exhibited a clinical sensitivity of 95% and clinical specificity of 92%.