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.

Correction to: Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology.

Following publication of the original article [1]. The authors reported that there is a mistake in Fig. 1: the number of patients in the control group its 449 patients, instead of 455.

Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology.

BACKGROUND: Rapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. Stool is an alternative sample type that is easy to collect from children, and studies investigating the use of stool for molecular detection of Mycobacterium tuberculosis (Mtb) have led to promising results. Our objective was to evaluate stool as an alternative specimen to sputum for Mtb detection in children. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform. METHODS: We tested stool samples from 259 children aged 0-14 years old, in Lima, Peru who presented with TB symptoms. Following extraction with TruTip, we detected the presence of Mtb DNA by IS6110 real-time PCR. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N = 22); and (2) children with clinically-diagnosed unconfirmed TB (N = 84). We calculated specificity among children in whom TB was ruled out (N = 153). Among children who were diagnosed with TB, we examined factors associated with a positive stool test. RESULTS: Assay sensitivity was 59% (95% confidence interval [CI]: 39-80%) and 1.2% (95% CI: 0.0-6.5%) in children with culture-confirmed and clinically-diagnosed unconfirmed TB, respectively, and specificity was 97% (95% CI: 93-99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB (100% sensitivity; 95% CI: 59-100%), and in 6/15 of children with smear-negative, culture-confirmed TB (40% sensitivity; 95% CI: 16-68%). Older age, smear positivity, culture positivity, ability to produce sputum and cavitary disease were associated with a positive stool result. CONCLUSION: Testing of stool samples with the TruTip workstation and IS6110 amplification yielded sensitivity and specificity estimates comparable to other tests such as Xpert. Future work should include detection of resistance using the TruTip closed amplification system and assay optimization to improve sensitivity in children with low bacillary loads.

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.

[Ln16] complexes (Ln = GdIII, DyIII): molecular analogues of natural minerals such as hydrotalcite.

Two new hexadecanuclear lanthanide complexes employing the 1,5-bis(salicylidene)carbohydrazide (H2bsc) ligand are reported herein. These polynuclear aggregates crystallize in the R3[combining macron] space group, which is unprecedented in a family of lanthanide complexes with such high nuclearity. Magnetic susceptibility measurements reveal weak intramolecular interactions between the magnetic centres, and in the case of compound 2 (DyIII), single-molecule magnet properties are observed.

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.

A bench-top automated workstation for nucleic acid isolation from clinical sample types.

Systems that automate extraction of nucleic acid from cells or viruses in complex clinical matrices have tremendous value even in the absence of an integrated downstream detector. We describe our bench-top automated workstation that integrates our previously-reported extraction method - TruTip - with our newly-developed mechanical lysis method. This is the first report of this method for homogenizing viscous and heterogeneous samples and lysing difficult-to-disrupt cells using "MagVor": a rotating magnet that rotates a miniature stir disk amidst glass beads confined inside of a disposable tube. Using this system, we demonstrate automated nucleic acid extraction from methicillin-resistant Staphylococcus aureus (MRSA) in nasopharyngeal aspirate (NPA), influenza A in nasopharyngeal swabs (NPS), human genomic DNA from whole blood, and Mycobacterium tuberculosis in NPA. The automated workstation yields nucleic acid with comparable extraction efficiency to manual protocols, which include commercially-available Qiagen spin column kits, across each of these sample types. This work expands the scope of applications beyond previous reports of TruTip to include difficult-to-disrupt cell types and automates the process, including a method for removal of organics, inside a compact bench-top workstation.

Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test.

There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.

Cycloheptatrienyl trianion: an elusive bridge in the search of exchange coupled dinuclear organolanthanide single-molecule magnets.

The preparation of η-cyclopentadienyl (η5-C5R5), η-arene (η6-C6R6), and η-cyclooctatetraenyl (η8-C8R8) bridging motifs are common in organometallic chemistry; however, the synthetic preparation of η-cycloheptatrienyl (η7-C7R7) bridging motifs has remained a synthetic challenge in 4f chemistry. To this end, we have developed a synthetic route towards a series of rare dinuclear organolanthanide inverse sandwich complexes containing the elusive η7-C7H7 bridge. Herein, we present the structures and magnetic properties of the lanthanide inverse sandwich complexes [KLn2(C7H7)(N(SiMe3)2)4] (Ln = GdIII (1), DyIII (2), ErIII (3)) and [K(THF)2Er2(C7H7)(N(SiMe3)2)4] (4). These compounds are the first single-molecule magnets (SMMs) to feature this type of bridging motif. Furthermore, η7-C7H7 was found to efficiently promote ferromagnetic exchange interactions between metal ions. Variable temperature dc magnetic susceptibility measurements and subsequent simulations give significant exchange constants of J = +1.384, +1.798, and +3.149 cm-1 and dipolar constants of J = -0.603, -0.601, and -0.475 cm-1 for compounds 2-4, respectively. Frequency dependent ac susceptibility measurements under an applied static field resulted in the observation of dual relaxation processes, and brought forth a greater understanding of the intermolecularly driven process at high frequency. In particular, this type of analysis of compound 3 under 800 Oe elicited an energy barrier of Ueff = 58 K. Ab initio calculations were performed in order to understand the nature of magnetic coupling and the origin of slow relaxation of magnetisation. Through these studies, the effect of the amido ancillary ligands on the magnetic axiality of the lanthanide ions was found to be competitive with the crystal field of the η7-C7H7 π-electron cloud. Our findings suggest that the tunability of the dipolar and exchange components of the magnetic interactions lie within the dihedral angle imposed by the amido ligands, thus offering potential for the development of new exchange coupled lanthanide systems.

A propeller-shaped µ4-carbonate hexanuclear dysprosium complex with a high energetic barrier to magnetisation relaxation.

A Dy6 complex composed of two Dy3 triangular units, [Dy6(µ3-OH)(CO3)3(bsc)3(MeOH)14(H2O)](Cl)5·(H2O)·(MeOH)2 (1), was isolated and found to exhibit slow relaxation of the magnetisation under zero applied dc field, resulting in a high energetic barrier to relaxation.

Supramolecular Assembly of Molecular Rare-Earth-3,5-Dichlorobenzoic Acid-2,2':6',2″-Terpyridine Materials: Structural Systematics, Luminescence Properties, and Magnetic Behavior.

The syntheses and crystal structures of 16 new rare-earth (RE = La(3+)-Y(3+))-3,5-dichlorobenzoic acid-terpyridine molecular materials characterized via single-crystal and powder X-ray diffraction are reported. These 16 complexes consist of four unique structure types ranging from molecular dimers (La(3+) and Ce(3+)) to tetramers (Pr(3+)-Y(3+)) as one moves across the RE(3+) series. This structural evolution is accompanied by subsequent changes in modes of supramolecular assembly (halogen bonding, halogen-π, halogen-halogen, and π-π interactions). Solid-state visible and near-infrared lifetime measurements were performed on complexes 6 (Sm(3+)), 7 (Eu(3+)), 9 (Tb(3+)), 10 (Dy(3+)), 11 (Ho(3+)), 12 (Er(3+)), and 14 (Yb(3+)), and characteristic emission was observed for all complexes except 11. Lifetime data for 11, 12, and 14 suggest sensitization by the terpy antenna does occur in near-infrared systems, although not as efficiently as in the visible region. Additionally, direct current magnetic susceptibility measurements were taken for complexes 10 (Dy(3+)) and 12 (Er(3+)) and showed dominant ferromagnetic behavior.

Impact of the coordination environment on the magnetic properties of single-molecule magnets based on homo- and hetero-dinuclear terbium(iii) heteroleptic tris(crownphthalocyaninate).

A series of Tb(III) triple-decker heteroleptic crownphthalocyaninate complexes consisting of a homodinuclear compound [(15C5)4Pc]Tb[(15C5)4Pc]Tb(Pc) (), and two novel heterodinuclear compounds [(15C5)4Pc]Tb[(15C5)4Pc]Y(Pc), () and [(15C5)4Pc]Y[(15C5)4Pc]Tb(Pc) (), have been synthesized. All compounds were characterised using UV-Vis spectroscopy, HR-ESI-MS, MALDI-TOF-MS, and (1)H NMR spectroscopy, followed by exploration into the effects of lanthanide coupling and ligand field symmetry on the magnetic properties of these complexes using SQUID magnetometry. Magnetic measurements on the homonuclear Tb(III) complex () displayed non-negligible ferromagnetic coupling between magnetic ions, eliciting a high zero-field energetic barrier to the magnetic relaxation of Ueff = 229.9(0) K, while the heteronuclear Tb(III)/Y(III) complexes displayed single-ion field-induced slow relaxation of the magnetization; yielding energetic barriers of Ueff = 129.8(0) K for , and 169.1(8) K for .

Intercalation of Coordinatively Unsaturated Fe(III) Ion within Interpenetrated Metal-Organic Framework MOF-5.

Coordinatively unsaturated Fe(III) metal sites were successfully incorporated into the iconic MOF-5 framework. This new structure, Fe(III) -iMOF-5, is the first example of an interpenetrated MOF linked through intercalated metal ions. Structural characterization was performed with single-crystal and powder XRD, followed by extensive analysis by spectroscopic methods and solid-state NMR, which reveals the paramagnetic ion through its interaction with the framework. EPR and Mössbauer spectroscopy confirmed that the intercalated ions were indeed Fe(III) , whereas DFT calculations were employed to ascertain the unique pentacoordinate architecture around the Fe(III) ion. Interestingly, this is also the first crystallographic evidence of pentacoordinate Zn(II) within the MOF-5 SBU. This new MOF structure displays the potential for metal-site addition as a framework connector, thus creating further opportunity for the innovative development of new MOF materials.