A performance evaluation of MTBDRplus version 2 for the diagnosis of multidrug-resistant tuberculosis.

OBJECTIVE: To evaluate the performance of a recently updated rapid molecular diagnostic test, GenoType® MTBDRplus version 2, designed to detect drug resistance in both acid-fast bacilli (AFB) smear-negative and -positive specimens. DESIGN: Sputum samples from 1128 patients at risk for multidrug-resistant tuberculosis (MDR-TB) were tested using MTBDRplus v2 and compared with reference standard MGIT™ 960™ drug susceptibility testing. The relationship of participant human immunodeficiency virus (HIV) status, diabetic status, previous treatment, and smear gradation to the likelihood of obtaining an interpretable result was assessed using logistic regression. RESULTS: The sensitivity and specificity of MTBDRplus v2 for detecting MDR-TB, when compared to a reference standard, were respectively 96.0% (95%CI 93.5-97.6) and 99.2% (95%CI 97.0-99.9) in AFB smear-positive specimens and 82.8% (95%CI 63.5-93.5) and 98.3% (95%CI 89.9-99.9) in AFB smear-negative specimens. A dose-response relationship was observed between the proportion of interpretable test results and AFB smear bacterial load after adjusting for age, sex, body mass index, HIV status, previous treatment and diabetic status. CONCLUSION: While MTBDRplus v2 performs well among both AFB smear-positive and -negative specimens, smear gradation appears to influence both the probability of obtaining an interpretable result and test sensitivity, indicating a significant association between bacillary load and test performance.

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape.

Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of seven Mycobacterium tuberculosis gene regions (katG, inhA, ahpC, rpoB, gyrA, rrs, and eis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INH(r)) specimens was the katG 315ACC mutation (87%). However, in the Eastern Cape, INH(r) specimens had a lower frequency of katG mutations (44%) and higher frequencies of inhA (47%) and ahpC (10%) promoter mutations. The most common mutation among rifampin-resistant (RIF(r)) specimens was the rpoB 531TTG mutation (80%). The mutation was common in RIF(r) specimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was the gyrA 94GGC mutation (44%). The rrs 1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KAN(r)) specimens, respectively. The eis promoter mutation -12T was found in 26% of KAN(r) and 4% of KAN-susceptible (KAN(s)) specimens. Inclusion of the ahpC and eis promoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered at ClinicalTrials.gov under registration number NCT02170441.).

Redefining MTBDRplus test results: what do indeterminate results actually mean?

BACKGROUND: Although line-probe assays (LPAs) are promising, little research has been conducted to elucidate the true nature of indeterminate LPA results or assess the ability of these assays to perform on a wide range of clinical samples. OBJECTIVE: To evaluate the performance of the commercially available GenoType(®) MTBDRplus LPA against conventional BACTEC™ MGIT™ 960 culture and drug susceptibility testing (DST) among 308 pulmonary tuberculosis (PTB) and 32 extra-pulmonary TB samples. RESULTS: Invalid LPA results (defined as those with a missing Mycobacterium tuberculosis identification band) were obtained for 18 PTB samples, which were excluded from further analysis. The sensitivity and specificity of the MTBDRplus assay for multidrug-resistant TB, based upon the results obtained for the remaining 322 samples, was respectively 95.2% and 95.1%. Of 290 PTB samples, 40 (13.7%) were indeterminate on LPA (defined as the absence of both wild-type and corresponding mutation bands) for isoniazid (INH) and/or rifampicin (RMP), and were further evaluated by pyrosequencing (PSQ). Contrary to standard LPA interpretation, INH and RMP susceptibility were confirmed by both DST and PSQ in respectively 7.5% (3/40) and 27.5% (11/40) of indeterminate samples. CONCLUSION: PSQ was found to be a valuable and rapid technique to resolve discrepancies in LPA test results that were not interpretable.

Performance of a pyrosequencing platform in diagnosing drug-resistant extra-pulmonary tuberculosis in India.

SETTING: Pyrosequencing diagnostic assays have shown great utility in identifying and characterizing pulmonary drug-resistant tuberculosis (TB) infections. However, the method has yet to be evaluated for the diagnosis of drug-resistant extra-pulmonary TB (EPTB). OBJECTIVE: To evaluate the performance of a pyrosequencing platform in establishing molecular drug resistance profiles for 79 clinical EPTB specimens at a referral center for drug-resistant TB in India. DESIGN: Genotypic drug resistance profiles were established for all 79 non-pulmonary, culture-positive TB clinical specimens. Acid-fast bacilli smear microscopy, MGIT™ 960™ culture and drug susceptibility testing were performed on all specimens for reference. RESULTS: In comparison to MGIT 960, the sensitivity and specificity of pyrosequencing in detecting drug resistance among specimens was found to be respectively 100% and 100%, 67% and 98%, and 100% and 100% for isoniazid, rifampicin, and the fluoroquinolones. No EPTB specimens were phenotypically resistant to any of the injectables, but the specificity of the assay was determined to be 100%, 98%, and 98% for amikacin, kanamycin, and capreomycin. CONCLUSIONS: Pyrosequencing is a rapid, appropriate technology for the diagnosis of isoniazid-, fluoroquinolone-, and potentially injectable drug-resistant EPTB clinical specimens, and should be considered as an alternative to conventional growth-based diagnostic methods for EPTB when resistance to these drugs is suspected.

Determination of MICs of levofloxacin for Mycobacterium tuberculosis with gyrA mutations.

The purpose of the present study was to correlate gyrA mutations found in Mycobacterium tuberculosis isolates using the GenoType(®) MTBDRsl assay with minimum inhibitory concentrations of the fluoroquinolone levofloxacin (LVX). Of 123 archived clinical M. tuberculosis isolates evaluated, 93 isolates had an Ala90Val, Ser91Pro, Asp94Ala, Asn/Tyr, Gly or His mutation and 30 were wild-type. Phenotypically, gyrA mutations Ala90Val, Ser91Pro or Asp94Ala showed a low level of resistance to LVX, while Asp94Asn/Tyr, Asp94Gly or Asp94His mutations had high-level resistance.

Solvation of nucleosides in aqueous mixtures of organic solvents: relevance to DNA open basepairs.

Toward the goal of understanding how open basepairs in DNA interact with their heterogeneous environment, we have studied the steady-state intrinsic fluorescence properties of the purine and pyrimidine deoxynucleosides in organic solvents in the presence of small amounts of water. The organic solvents used in the present study were: n-butanol, acetonitrile, methanol, n-propanol, isopropanol, and isobutanol. For n-butanol and acetonitrile, which have a high degree of amphiphilicity and weak hydrogen bonding ability, respectively, the fluorescence spectral properties of the purines are found to depend on the sequence of steps in which the aqueous mixtures were formed. By contrast, no such dependence was observed in the mixtures with any of the other solvents used in the present study. Moreover, no such dependence was observed for the pyrimidines. These findings suggest that the final solvation network around the purines is dependent on the nature of the environment to which they were initially exposed. This would tend to present an impediment to the closing of AT or GC basepairs in DNA that become open as a result of structural fluctuations, DNA bending, or protein-DNA interactions.

Harmonic analysis of DNA dynamics in a viscous medium.

The harmonic dynamics of normal modes of double-stranded DNA in a viscous fluid are investigated. The model DNA consists of two backbone-supported DNA strands coiling around a common helix axis with base stacking, sugar puckering, interstrand hydrogen bonding, and intrastrand sugar-base interactions assigned values based on published data. Assuming that the DNA bases are shielded from direct bombardment by the solvent, analytical solutions are obtained. The dissipation and fluctuation of the normal modes of the bases moving along the spirals display the effect of the medium indirectly through interactions with the backbone. The dynamics of the backbone are found to be overdamped with the characteristic damping times extending to the picosecond region for disturbance in position and to the sub-picosecond region for disturbance in velocity. In addition to the dynamic mode of a rigid rod, the motions of the bases are coupled to the motions of the backbone with comparable amplitudes for disturbance in position. For disturbance in velocity, however, the bases are effectively at rest, not being able to follow the motions of the backbone. The angular frequencies of the underdamped vibrational modes, identified as the ringing modes of the bases with the backbone effectively at rest, are insensitive to the viscosity and lie in the low frequency region of the Raman spectrum. These findings indicate that the backbone of DNA plays a significant role in modulating the dynamics of double-stranded DNA in an overdamping environment. This modulation of the dynamics of the motions of the bases in DNA by environmental impediments to molecular motion is briefly discussed in connection with protein- and drug- DNA interactions as well as gene regulation.

Excited-state properties of thymidine and their relevance to its heterogeneous emission in double-stranded DNA.

Published results on synthetic polynucleotides point to T as the major emitting fluorophore in DNA. We have reported also that the bases of the nonalternating polynucleotide poly(dA).poly(dT), in which T was selectively excited, undergo large-amplitude motions on the picosecond-nanosecond time scales (S. Georghiou et al., Biophys. J. 70, 1909-1922, 1996). In that study, the fluorescence decay profile of the T bases of this polynucleotide was found to contain a number of components; these may be considered to be the result of the motions of the bases that give rise to a distribution of stacked geometries of varying rigidity as well as dispersion and polar interactions. Here, we report the results of a study that we have undertaken in order to test this hypothesis. To this effect, we have studied the photophysical properties of thymidine (1) in aqueous buffer and in a number of organic solvents and (2) in aqueous sucrose solutions of viscosity extending to 149 cP. The results suggest that the fluorescence quantum yield decreases with an increase in the polarizability of the solvent, whereas it increases with an increase in the solvent polarity (on the basis of the empirical parameter of solvent polarity ETN) or viscosity. These findings suggest the following for the photophysical properties of the T bases in DNA: (1) Base stacking results in two antagonistic effects, namely it causes a reduction in fluorescence as a result of dispersion interactions and an enhancement as a result of a reduction in the motions of the bases and (2) exposure of the bases to the aqueous environment results in fluorescence enhancement.

Environmental control of the deformability of the DNA double helix.

The deformability of DNA is of crucial importance in a number of processes and interactions, such as its enzymatic recognition, its packaging into chromosomes, its interactions with drugs, and the formation of photodimers in it. Here we have studied this property by following the formation of excited-state molecular complexes (excimers) between adjacent bases of poly(dA-dT).poly(dA-dT) at 20 degrees C. The force that drives the helix distortion appears to stem mainly from charge-resonance interaction. The results indicate that the deformability of the helix on the nanosecond time scale is considerable at normal solvent viscosities, whereas it is greatly reduced by frictional forces at high viscosities (attained through sucrose addition) at which the excimer has a much less favorable geometry: the difference in the interaction energies between 1 cP and 58 cP is about 6 kcal/mol, a value that is similar to the base stacking energies for the undistorted helix. This behavior parallels the modulation by the solvent viscosity of the thermally driven motions of the bases of poly(dA).poly(dT), which we recently reported on the basis of time-resolved intrinsic fluorescence anisotropy measurements (S. Georghiou et al., Biophys, J., 70, 1909-1922, 1996). It is inferred that environmental impediments to molecular motion can modulate the conformation and dynamics of DNA. Such modulation might play a role in gene regulation: particular base configurations, which can be enzymatically recognized, may be attained as dictated by the prevailing viscosity conditions and/or geometric constraints. By contrast, up to 3 M NaCl or 0.1 M MgCl2 do not significantly reduce the deformability of the helix. The considerable plasticity of this polynucleotide is probably linked to the significant flexibility of the TA step that may account for the wide-spread use of the TATA sequence in transcription, site-specific recombination and the initiation of DNA replication.

Large-amplitude picosecond anisotropy decay of the intrinsic fluorescence of double-stranded DNA.

The conformational flexibility of the DNA double helix is of great interest because of its potential role in protein recognition, packaging into chromosomes, formation of photodefects, and interaction with drugs. Theory finds that DNA is very flexible; however, there is a scarcity of experimental results that examine intrinsic properties of the DNA bases for the inherent flexibility in solution. We have studied the dynamics of poly(dA).poly(dT) and (dA)20.(dT)20 in a 50 mM cacodylate, 0.1 M NaCl, pH 7 buffer by using the time-correlated picosecond fluorescence anisotropy of thymine selectively excited at 293 nm. For both nucleic acids, a large-amplitude biphasic decrease in the anisotropy is observed that has a very fast, large-amplitude component on the picosecond time scale and a slower, smaller-amplitude component on the nanosecond time scale. These modes are sensitive to sucrose concentration, and are greatly attenuated at 77% sucrose by volume. This observation suggests that motions of the bases make a significant contribution to the observed fluorescence depolarization (in the absence of sucrose). Measurements on the single-stranded systems poly(dT) and (dT)20 reveal a much smaller amplitude of the very fast depolarization mode. These observations are consistent with a mechanism that involves concerted motions in the interior of the double-stranded systems.

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length.

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.

Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution.

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.

How do mutations at phenylalanine-153 and isoleucine-155 partially suppress the effects of the aspartate-27-->serine mutation in Escherichia coli dihydrofolate reductase?

Several second-site suppressors of the D27S lesion in Escherichia coli dihydrofolate reductase (DHFR) have been identified. The activity of the primary mutant, D27S DHRF, was found to be greatly decreased at pH 7.0, consistent with aspartic acid-27 being critically involved in proton donation during catalysis. Partial suppressors of the D27S mutation have been selected by their ability to confer an increased resistance to trimethoprim upon host E. coli; the suppressors have been identified as F153S or I155N substitutions. D27S+F153S and D27S+I155N DHFRs display 2-3-fold increases in kcat over D27S DHFR values, but only the F153S mutation decreases the Km for dihydrofolate by a factor of 2. Neither double mutant approaches wild-type DHFR activity. Unexpectedly, Phe153 and Ile155 occur on the surface of the protein and are approximately 8 and 14 A distant from the active site. Ile155 is a member of a beta-bulge. A previously identified suppressing mutation, F137S, occurs nearby and is also a member of the same beta-bulge [Howell et al. (1990) Biochemistry 29, 8561-8569]. Clustering of these three second-site mutations indicates this area of the structure may be important in protein function. Conformational changes due to the presence of these suppressing mutations are likely as the F153S and I155N mutations do not affect hydride-transfer rates upon introduction in wild-type DHFR and alterations in circular dichroism spectra are associated with the double-mutant DHFRs.

Room-temperature steady-state fluorescence properties of poly(dG-dC).poly(dG-dC).

We report the steady-state fluorescence properties of the alternating polynucleotide poly(dG-dC).poly(dG-dC) in low-salt solution at room temperature for excitation at the Hg lines 265, 280 and 297 nm. Its fluorescence spectrum peaks at about 325 nm and, within the experimental error, its shape does not change significantly with the excitation wavelength. The fluorescence anisotropy is found to decrease strongly for short-wavelength excitation, a behavior which is very similar to that exhibited by free guanine. In view of the fact that the anisotropy for free cytosine is virtually constant at the aforementioned three excitation wavelengths, the results suggest that in this polynucleotide the emission stems from guanine. The values of the fluorescence quantum yield for the three excitation wavelengths are found to be very low, 0.8 x 10(-5), 0.8 x 10(-5), and 2.8 x 10(-5), respectively; these are compatible with transfer of energy from the lower-energy electronic state of guanine, before vibronic relaxation is established, to cytosine. Upon denaturation, the fluorescence spectrum becomes very broad and the fluorescence quantum yield increases; these observations support the authenticity of the emission from the nondenatured polynucleotide.

Room-temperature fluorescence properties of the polynucleotide polydA.polydT.

The room-temperature fluorescence spectrum of the non-alternating polynucleotide polydA.polydT is found to have its maximum at about 325 nm and, when exciting in the spectral region where both adenine (A) and thymine (T) absorb, to coincide with that obtained for excitation at 293 nm where thymine is selectively excited. The fluorescence anisotropy is found to be equal to 0.18 and independent of the excitation and emission wavelengths. These observations are consistent with: (i) emission stemming from T; and (ii) transfer of electronic energy from A to T being not efficient. These inferences are also supported by the observed dependence of the fluorescence quantum yield on the excitation wavelength.

Excited-state properties of the alternating polynucleotide poly(dA-dT).poly(dA-dT).

Measurements of the steady-state fluorescence spectrum and anisotropy, r, of the alternating polynucleotide poly(dA-dT).poly(dA-dT) were carried out in order to characterize its photophysical properties at room temperature. The shape of the fluorescence spectrum depends on the excitation wavelength, namely, the relative fluorescence intensity of the short-wavelength peak decreases for excitation at short wavelengths. When monitoring the emission at short wavelengths, r is 0.18 and independent of the excitation wavelength. When monitoring the emission at long wavelengths, however, r is very low, about 0.03. These results suggest that: (i) the short-wavelength emission stems from thymine; and (ii) the long-wavelength emission stems from an excited-state complex (excimer), with the same one being formed regardless of whether thymine or adenine is excited. The corresponding fluorescence spectra have been resolved. The occurrence of transfer of electronic energy is discussed.

Kinetics of melittin binding to phospholipid small unilamellar vesicles.

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.

Singlet-singlet energy transfer along the helix of a double-stranded nucleic acid at room temperature.

An irreversible electronic energy trap has been formed in calf thymus DNA by methylating about 75% of its G bases at position N-7. This has allowed us to measure for the first time the efficiency of transfer of energy along the helix of a double-stranded nucleic acid at room temperature. It is found that about one out of every three photons absorbed by the other bases is trapped. We have also simulated the data with a stochastic model that uses the dipole-dipole interaction to calculate the efficiency of transfer. In order to approximate the experimental results, the model requires that: (i) the fluorescence quantum yield of T, C, and G in DNA be about 2 x 10(-3), which is about two orders of magnitude larger than the value of the fluorescence quantum yield reported for DNA; and (ii) the fluorescence quantum yield of A in DNA be negligibly small. Requirement (i) is consistent with energy transfer taking place before a very efficient fluorescence quenching process sets in, which could be formation of excited-state complexes (excimers) that do not fluoresce appreciably. Requirement (ii) implies a very short fluorescence lifetime for A, which is consistent with the reported absence of a significant number of photoproducts formed by A in DNA. The simulations find that, on the average, the excitation energy takes about 1.2 steps to reach the trap; that is to say, bases that are nearest and next nearest neighbors of the trap are, in effect, the only energy donors. Both intra- as well as interstrand energy transfer (the latter only for the C-trap base pair) make significant contributions. The value of the efficiency for pairwise base-base intrastrand transfer is about 60%, whereas those for base-trap intra- and interstand transfer are 90% and 80%, respectively. The corresponding values for the rate constant of transfer are 2 x 10(11), 1 x 10(12), and 4 x 10(11) s-1. Transfer is inefficient when A is the donor or the acceptor. In addition to the dipole-dipole term, the only other significant term in the expansion of the interaction potential is the dipole-quadrupole term which, however, makes only a small contribution to the overall transfer efficiency. The electron exchange interaction appears to be much less efficient than the coulombic interaction.

Fluorometric analysis of the long-wavelength absorption band of N-7 methylated GMP into the constituent bands of the two electronic states.

Steady-state measurements of fluorescence anisotropy are used to resolve the long-wavelength absorption spectrum of 7-methyl guanosine 5'-monophosphate (GMP) in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle theta between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on theta. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and approximately 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their CD spectra is discussed.