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.

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.

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.

Yvonne Linger

Akonni Biosystems, Inc.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray