Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adults in Peru.

Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30-45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62-83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62-100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69-100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78-94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adult patients in Peru.

Diagnosis of tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic patients. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection/assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall sample sensitivity was 38% (95% Confidence Interval [CI] 30-45%). On a patient level (i.e., any of three samples positive), sensitivity was 73% (95% CI: 62-83%). Sensitivity was highest among samples from patients with smear-positive TB, 92% (95% CI: 62-100%). Specificity from a single sample from each of 10 healthy controls was 100% (95% CI: 69-100%). Adjusting our assay positivity threshold increased patient-level sensitivity to 88% (95% CI: 78-94%) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and either patient characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Detecting rifampin and isoniazid resistance in Mycobacterium tuberculosis direct from patient sputum using an automated integrated system.

While there has been progress in detection of drug resistant tuberculosis globally, WHO estimates only about half of the patients with bacteriologically confirmed tuberculosis were tested for rifampicin resistance over the past two years. To close this drug resistance diagnostic gap, an expansion of testing for rifampicin and isoniazid resistance is critically needed. The Akonni Biosystem Integrated System combines DNA extraction and a Lab-on-a-Film assembly (LFA) to perform rapid probe and PCR-based detection of resistance associated mutations to first-line anti-tuberculosis drugs. Using raw sputum samples from 25 tuberculosis patients at risk for drug resistance, we conducted a proof-of-concept study of the Integrated System with an MDR-TB assay. Performance of the Integrated System was compared to liquid Mycobacteria Growth Indicator Tube (MGIT) culture reference phenotypes using 2012 WHO endorsed critical concentrations for rifampicin and isoniazid. The overall percent agreement for rifampicin and isoniazid was 91.7% and 100% respectively, with agreement for rifampicin increasing to 95.7% after low-level resistance mutations in rpoB were excluded. The Integrated System, combining DNA extraction and LFA amplification, is a promising new tool for detection of both rifampicin and isoniazid using liquefied raw sputum.

Molecular detection of Mycobacterium tuberculosis from buccal swabs among adult in Peru.

Tuberculosis (TB) diagnosis relies on a sputum sample, which cannot be easily obtained from all symptomatic patients. Mycobacterium tuberculosis DNA can be detected from oral swabs, a noninvasive, safe alternative sample type; however, reported sensitivities have been variable and likely depend on sample collection, processing procedures and host characteristics. We analyzed three buccal swab samples from 123 adults with culture-confirmed TB in Lima, Peru. We compared the sensitivity and specificity of two sample collection devices (OmniSwab and EasiCollect FTA cards) and examined factors associated with detection. DNA was extracted with a commercially available kit and detected via real-time PCR IS6110 amplification. Overall sensitivity for buccal samples was 51% (95% Confidence Interval [CI] 42-60%). Specificity from a single sample among healthy controls was 96.7% (95% CI 83-99.9%). Positive sputum smear and cavitary disease, correlates of disease burden, were associated with detection via buccal swab. Although we observed higher sensitivities with the Omniswab samples, this appeared to be due primarily to differences in patient characteristics (e.g., cavitary disease). Overall, our findings support the potential for a buccal sample-based TB assay. Future work should focus on assay optimization and streamlining the assay workflow.

Laboratory Evaluation of a Lateral-Flow Cell for Molecular Detection of First-Line and Second-Line Antituberculosis Drug Resistance.

Despite the WHO's call for universal drug susceptibility testing for all patients being evaluated for tuberculosis (TB), a lack of rapid diagnostic tests which can fully describe TB resistance patterns is a major challenge in ensuring that all persons diagnosed with drug-resistant TB are started on an appropriate treatment regime. We evaluated the accuracy of the Akonni Biosystems XDR-TB TruArray and lateral-flow cell (XDR-LFC), a novel multiplex assay to simultaneously detect mutations across seven genes that confer resistance to both first- and second-line anti-TB drugs. The XDR-LFC includes 271 discrete three-dimensional gel elements with target-specific probes for identifying mutations in katG, inhA promoter, and ahpC promoter (isoniazid), rpoB (rifampin), gyrA (fluoroquinolones), rrs and eis promoter (kanamycin), and rrs (capreomycin and amikacin). We evaluated XDR-LFC performance with 87 phenotypically and genotypically characterized clinical Mycobacterium tuberculosis isolates. The overall assay levels of accuracy for mutation detection in specific genes were 98.6% for eis promoter and 100.0% for the genes katG, inhA promoter, ahpC promoter, rpoB, gyrA, and rrs The sensitivity and specificity against phenotypic reference were 100% and 100% for isoniazid, 98.4% and 50% for rifampin (specificity increased to 100% once the strains with documented low-level resistance mutations in rpoB were excluded), 96.2% and 100% for fluoroquinolones, 92.6% and 100% for kanamycin, 93.9% and 97.4% for capreomycin, and 80% and 100% for amikacin. The XDR-LFC solution appears to be a promising new tool for accurate detection of resistance to both first- and second-line anti-TB drugs.

Detection of Mycobacterium Tuberculosis DNA in Buccal Swab Samples from Children in Lima, Peru.

We examined Mycobacterium tuberculosis DNA detection from buccal swab samples collected from children in Lima, Peru. DNA was extracted and amplified via real-time polymerase chain reaction. Sensitivity was 21% (95% confidence interval [CI]: 7%-42%) in 24 culture-confirmed tuberculosis cases and 4.6% (95% CI: 1%-13%) in 65 clinically diagnosed unconfirmed cases. Sensitivity was highest for smear-positive tuberculosis. Specificity was 99% in the 199 controls (95% CI: 96%-100%).

A Benchtop Automated Sputum-to-Genotype System Using a Lab-on-a-Film Assembly for Detection of Multidrug-Resistant Mycobacterium tuberculosis.

Automated genotyping of drug-resistant Mycobacterium tuberculosis (MTB) directly from sputum is challenging for three primary reasons. First, the sample matrix, sputum, is highly viscous and heterogeneous, posing a challenge for sample processing. Second, acid-fast MTB bacilli are difficult to lyse. And third, there are hundreds of MTB mutations that confer drug resistance. An additional constraint is that MTB is most prevalent where test affordability is paramount. We address the challenge of sample homogenization and cell lysis using magnetic rotation of an external magnet, at high (5000) rpm, to induce the rotation of a disposable stir disc that causes chaotic mixing of glass beads ("MagVor"). Nucleic acid is purified using a pipet tip with an embedded matrix that isolates nucleic acid ("TruTip"). We address the challenge of cost and genotyping multiple mutations using 203 porous three-dimensional gel elements printed on a film substrate and enclosed in a microfluidic laminate assembly ("Lab-on-a-Film"). This Lab-on-a-Film assembly (LFA) serves as a platform for amplification, hybridization, washing, and fluorescent imaging, while maintaining a closed format to prevent amplicon contamination of the workspace. We integrated and automated MagVor homogenization, TruTip purification, and LFA amplification in a multisample, sputum-to-genotype system. Using this system, we report detection down to 43 cfu/mL of MTB bacilli from raw sputum.

Lab-on-a-Film disposable for genotyping multidrug-resistant Mycobacterium tuberculosis from sputum extracts.

We describe a Lab-on-a-Film disposable that detects multidrug-resistant tuberculosis (MDR-TB) from sputum extracts. The Lab-on-a-Film disposable consists of 203 gel elements that include DNA sequences (probes) for 37 mutations, deletions, or insertion elements across 5 genes (including an internal control). These gel elements are printed on a flexible film, which costs approximately 500 times less than microarray glass. The film with printed gel elements is then laminated to additional rollable materials (films) to form a microfluidic flow cell. We combined multiplex amplification and hybridization steps in a single microfluidic chamber, without buffer exchanges or other manipulations up to and throughout hybridization. This flow cell also incorporates post hybridization wash steps while retaining an entirely closed-amplicon system, thus minimizing the potential for sample or amplicon cross-contamination. We report analytical sensitivity of 32 cfu mL-1 across all MDR-TB markers and detection of MDR-TB positive clinical specimens using an automated TruTip workstation for extraction and the Lab-on-a-Film disposable for amplification and detection of the extracts.

Automated TruTip nucleic acid extraction and purification from raw sputum.

Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. The system efficiently homogenized primary sputa and doubled nucleic acid recovery relative to an automated protocol that did not incorporate sample homogenization. Nucleic acid recovery was at least five times higher from raw sputum as compared to that of matched sediments regardless of smear or culture grade, and the automated workstation reproducibly recovered PCR-detectable DNA to at least 80 CFU mL-1 raw sputum. M. tuberculosis DNA was recovered and detected from 122/123 (99.2%) and 124/124 (100%) primary sputum and sediment extracts, respectively. There was no detectable cross-contamination across 53 automated system runs and amplification or fluorescent inhibitors (if present) were not detectable. The open fluidic architecture of the prototype automated workstation yields purified sputum DNA that can be used for any molecular diagnostic test. The ability to transfer TruTip protocols between personalized, on-demand pipetting tools and the fully automated workstation also affords public health agencies an opportunity to standardize sputum nucleic acid sample preparation procedures, reagents, and quality control across multiple levels of the health care system.

Akonni TruTip(®) and Qiagen(®) methods for extraction of fetal circulating DNA--evaluation by real-time and digital PCR.

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed--a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.

High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots.

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

Directed evolution of novel polymerases.

DNA and RNA polymerases evolved to function in specific environments with specific substrates to propagate genetic information in all living organisms. The commercial availability of these polymerases has revolutionized the biotechnology industry, but for many applications native polymerases are limited by their stability or substrate recognition. Thus, there is great interest in the directed evolution of DNA and RNA polymerases to generate enzymes with novel, desired properties, such as thermal stability, resistance to inhibitors, and altered substrate specificity. Several screening and selection approaches have been developed, both in vivo and in vitro, and have been used to evolve polymerases with a variety of important activities. Both the techniques and the evolved polymerases are reviewed here, along with a comparison of the in vivo and in vitro approaches.

Electrochemical determination of triple helices: electrocatalytic oxidation of guanine in an intramolecular triplex.

Electrocatalytic oxidation of the oligonucleotide 5'- GAA GAG GTT TTT CCT CTT CTT TTT CTT CTC C (TS) by Ru(bpy)(3)(2+) was studied by cyclic voltammetry. This oligonucleotide forms either an intramolecular triplex, hairpin, or single strand, depending on the pH (Plum, G. E.; Breslauer, K. J. J. Mol. Biol. 1995, 248, 679-695). In the triplex form, the guanine doublet in TS is buried inside the folded structure, and as such is less susceptible to oxidation by electrogenerated Ru(bpy)(3)(3+). Digital simulations of the catalytic voltammograms gave a rate constant of 3.5 +/- 0.2 x 10(2) M(-1) s(-1) for oxidation of the triplex form, while oxidation of the duplex and single-stranded forms occurred with much faster rate constants of (3.5-9.1) x 10(4) M(-1) s(-1). Experiments using a truncated form of TS that lacked the third strand of the triplex were consistent with these measurements. The Ru(bpy)(3)(3+) complex was also generated by photolyzing Ru(bpy)(3)(2+) in the presence of Fe(CN)(6)(3-). This reaction produced strand scission following piperidine treatment, which was visualized using high-resolution gel electrophoresis. These experiments showed decreased reactivity for the triplex form, and also gave an unusual reversal of a common selectivity for the 5'-G of GG doublets generally seen in B-form DNA. This reversal was ascribed to strain caused by the location of the GG doublet adjacent to the hairpin loop.

Intramolecular electrocatalysis of 8-oxo-guanine oxidation: secondary structure control of electron transfer in osmium-labeled oligonucleotides.

A phosphoramidite containing Os(bpy)(3)(2+) (Os; bpy, 2,2'-bipyridine) with a three-carbon linker was synthesized and used to prepare oligonucleotides with the Os redox catalyst appended to the 5'-end. The electrogenerated Os(III) is capable of oxidizing 7,8-dihydro-8-oxo-guanine (8G), but 8G is not electrochemically reactive at indium tin oxide electrodes because of poor electrode kinetics for the direct reaction. The hairpin-forming oligonucleotide Os-5'-ATG TCA GAT TAG CAG GCC TGA CAT 8G was synthesized and characterized by thermal denaturation and native gel electrophoresis both in the hairpin form and when hybridized to its Watson-Crick complement. The redox potential in both forms of the appended Os(III/II) couple was 0.63 V (all potentials vs Ag/AgCl), which is identical to that for the free complex. The diffusion coefficients of the hairpin form (10.2 x 10(-)(7) cm(2)/s) and the duplex form (8.7 x 10(-)(7) cm(2)/s) were consistent with values expected from studies of noncovalently bound redox labels, which suggest that the measured diffusion coefficient should be that of the appended DNA molecule. The oligonucleotide was designed such that in the duplex form, the 8G is far from the Os(III/II) couple, but in the hairpin form, the 8G is situated close to the redox center. For the duplex form, cyclic voltammetry studies showed that mediated oxidation of the 8G nucleobase occurred only through bimolecular reaction of the electrogenerated Os(III) of one duplex with the 8G of another duplex. However, in the hairpin form, intramolecular electron transfer from 8G to Os(III) in the same molecule was apparent in both chronoamperometry and cyclic voltammetry.

Metal-ligand charge-transfer-promoted photoelectronic Bergman cyclization of copper metalloenediynes: photochemical DNA cleavage via C-4' H-atom abstraction.

Metal-to-ligand charge-transfer (MLCT) photolyses (lambda > or = 395 nm) of copper complexes of cis-1,8-bis(pyridin-3-oxy)oct-4-ene-2,6-diyne (bpod, 1), [Cu(bpod)(2)]PF(6) (2), and [Cu(bpod)(2)](NO(3))(2) (3) yield Bergman cyclization of the bound ligands. In contrast, the uncomplexed ligand 1 and Zn(bpod)(2)(CH(3)COO)(2) compound (4) are photochemically inert under the same conditions. In the case of 4, sensitized photochemical generation of the lowest energy (3)pi-pi state, which is localized on the enediyne unit, leads to production of the trans-bpod ligand bound to the Zn(II) cation by photoisomerization. Electrochemical studies show that 1, both the uncomplexed and complexed, exhibits two irreversible waves between E(p) values of -1.75 and -1.93 V (vs SCE), corresponding to reductions of the alkyne units. Irreversible, ligand-based one-electron oxidation waves are also observed at +1.94 and +2.15 V (vs SCE) for 1 and 3. Copper-centered oxidation of 2 and reduction of 3 occur at E(1/2) = +0.15 and +0.38 V, respectively. Combined with the observed Cu(I)-to-pyridine(pi) MLCT and pyridine(pi)-to-Cu(II) ligand-to-metal charge transfer (LMCT) absorption centered near approximately 315 nm, the results suggest a mechanism for photo-Bergman cyclization that is derived from energy transfer to the enediyne unit upon charge-transfer excitation. The intermediates produced upon photolysis degrade both pUC19 bacterial plasmid DNA, as well as a 25-base-pair, double-stranded oligonucleotide. Detailed analyses of the cleavage reactions reveal 5'-phosphate and 3'-phosphoglycolate termini that are derived from H-atom abstraction from the 4'-position of the deoxyribose ring rather than redox-induced base oxidation.

Digital simulation of catalytic cyclic voltammograms for oxidation of DNA by a heterobimetallic dimer: effects of DNA binding and mass transport.

The electrocatalytic oxidation of DNA by a heterodimer, [(bpy)2Ru(tpphz)Os(bpy)2]4+ (tpphz: tetrapyrido[3,2-alpha: 2',3'-c:3'',2''-h:2''',3'''-j]phenazine) (1), was studied using cyclic voltammetry with digital simulation. This dimer was chosen because the Ru(III/II) couple (E1/2 = 1.09 V vs Ag/AgCl) is capable of catalyzing guanine oxidation while the Os(III/II) couple (E1/2 = 0.63 V) provides a convenient reporter on the binding and mass transport of the complex, which can then be determined in the same voltammetric sweep as the electrocatalysis. Proper description of the electrochemical response required careful measurement of the binding constant of 1 to herring testes (HT) DNA, which was (2.0 +/- 0.1) x 10(4) M(-1) by both absorption titration and normal pulse voltammetry. Thermal denaturation experiments were consistent with a nonintercalative binding mode and gave a deltaTm of only (2.4 +/- 0.5) degrees C. The minor groove binder distamycin did not displace 1 from HT DNA, suggesting that the complex binds in the major groove. As expected, acquisition of the cyclic voltammogram of 1 in the presence of DNA produced catalytic current for the Ru(III/II) couple, while a suppression of current was observed for the Os(III/II) couple. Although the catalytic current for the Ru(III/II) couple initially appeared as a current enhancement, higher concentrations suppressed the catalytic wave as a result of the slower mass transport of the DNA-bound complex. The binding studies were used to create a model for digital simulation that reproduced the behavior of 1 with DNA and gave rate constants that were independent of DNA concentration. The apparent second-order rate constant at 25 mV/s for oxidation of guanine in HT DNA (av 1000 bp, 25% guanine) by 1 was 3 +/- 1 x 10(4) M(-1) s(-1); similar values were obtained for a 200-bp fragment (7 +/- 3 x 10(4) M(-1) s(-1)) and a 435-bp fragment (8 +/- 2 x 10(4) M(-1) s(-1)). As observed in previous studies of these reactions, biphasic kinetics in the catalytic reaction led to a dependence of the rate constant determined by simulation on the sweep rate. Increasing the sweep rate led to a systematic increase in the simulated rate constant, consistent with a fast phase of the homogeneous catalytic reaction.