demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Demonstrating a Multi-drug Resistant <em>Mycobacterium tuberculosis</em> Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Akonni Biosystems, Inc.

A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

Diagnostic oligonucleotide microarray fingerprinting of Bacillus isolates.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

The objective of this study was to develop and validate a simple, field-portable, microarray system for monitoring microbial community structure and dynamics in groundwater and subsurface environments, using samples representing site status before acetate injection, during Fe-reduction, in the transition from Fe- to SO(4)(2-)-reduction, and into the SO(4)(2-)-reduction phase. Limits of detection for the array are approximately 10(2)-10(3) cell equivalents of DNA per reaction. Sample-to-answer results for the field deployment were obtained in 4 h. Retrospective analysis of 50 samples showed the expected progression of microbial signatures from Fe- to SO(4)(2-) -reducers with changes in acetate amendment and in situ field conditions. The microarray response for Geobacter was highly correlated with qPCR for the same target gene (R(2) = 0.84). Microarray results were in concordance with quantitative PCR data, aqueous chemistry, site lithology, and the expected microbial community response, indicating that the field-portable microarray is an accurate indicator of microbial presence and response to in situ remediation of a uranium-contaminated site.

Monitoring microbial community structure and dynamics during in situ U(VI) bioremediation with a field-portable microarray analysis system.

By modifying polymer compositions and cross-linking reagents, we have developed a simple yet effective manufacturing strategy for copolymerized three-dimensional gel element arrays. A new gel-forming monomer, 2-(hydroxyethyl) methacrylamide (HEMAA), was used. HEMAA possesses low volatility and improves the stability of copolymerized gel element arrays to on-chip thermal cycling procedures relative to previously used monomers. Probe immobilization efficiency within the new polymer was 55%, equivalent to that obtained with acrylamide (AA) and methacrylamide (MA) monomers. Nonspecific binding of single-stranded targets was equivalent for all monomers. Increasing cross-linker chain length improved hybridization kinetics and end-point signal intensities relative to N,N-methylenebisacrylamide (Bis). The new copolymer formulation was successfully applied to a model orthopox array. Because HEMAA greatly simplifies gel element array manufacture, we expect it (in combination with new cross-linkers described here) to find widespread application in microarray science.

Nonvolatile copolymer compositions for fabricating gel element microarrays.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) is important for prevention and control of MRSA infections, but the discovery of mecA dropouts and SCCmec junction sequences with homology to coagulase-negative staphylococci (CoNS) has challenged several real-time PCR tests. The objective of this study was to develop a user-friendly, gel element microarray test for MRSA detection, to estimate the analytical performance characteristics of the test on bacterial isolates, and to perform an initial evaluation of the test on nasopharyngeal swabs from patients known to have a high prevalence of S. aureus containing mecA dropouts. The assay limit of detection for the test was 250 fg (or less) of genomic DNA per amplification reaction (approximately 80 cell equivalents) and MRSA was consistently detected at a ratio of 1:12,000 in a non-target background. Of 87 bacterial isolates, the test accurately classified 86 (98.8%) overall, and correctly identified 14 mecA dropout specimens that were falsely positive in the BD GeneOhm MRSA test or BD GeneOhm StaphSR test. A retrospective analysis of 246 nasal swab samples acquired from a high-risk patient population (overall prevalence=10.8% by culture) resulted in 80.5% sensitivity (95% CI=68.4%, 92.6%) and 96.6% specificity. Of these 246 samples, 174 (71%) were positive for mecA, 86 (35%) were positive for S. aureus tufA and 46 (19%) were positive for a SCCmec junction sequence. To estimate method repeatability, 48 samples representing the full range of phenotypes, genotypes and microarray probe SNR values were tested in triplicate, with three discordant results for a concordance rate of 97.9% (141/144 tests). These data demonstrate that a very simple microarray test can identify mecA dropouts with high specificity in either cultured isolates or nasal swabs from a high-prevalence, high-risk patient population. However, the clinical sensitivity of the test will likely depend on local microbial ecology and the prevalence of mecA positive CoNS in any given patient population.

Development of a simple, low-density array to detect methicillin-resistant Staphylococcus aureus and mecA dropouts in nasal swabs.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO(3), but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Profiling in situ microbial community structure with an amplification microarray.

Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection.

Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva.

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB and rpsL, and compared the analytical performance of the test relative to phenotypic DST. The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of those mutations for which there was a sequence-specific probe on the microarray, and 100% of 96 wildtype sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first line drugs relative to phenotypic DST (true resistance) was 100% for RIF (14/14), 90.0% for INH (36/40), 70% for EMB (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113) and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug STR, overall concordance between agar proportion and the microarray was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false susceptible discrepants were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical MTB isolates, and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable.

A simplified microarray system for simultaneously detecting rifampin, isoniazid, ethambutol, and streptomycin resistance markers in Mycobacterium tuberculosis.

Julia Golova

Akonni Biosystems, Inc.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray