High Prevalence of inhA Promoter Mutations among Patients with Drug-Resistant Tuberculosis in KwaZulu-Natal,South Africa

Background

Drug-resistant tuberculosis (TB) remains extremely difficult to treat because there are often few remaining active medications and limited diagnostic options to detect resistance. Resistance to isoniazid is typically caused by mutations in either katG or the inhA promoter. inhA mutations confer low-level resistance to isoniazid and cross-resistance to ethionamide while katG mutations confer high-level isoniazid resistance and no cross-resistance. Line Probe Assays (LPAs) that detect mutations in katG and inhA are currently performed on all positive TB cultures in KwaZulu-Natal province, South Africa, but the frequency of inhA mutations in drug-resistant TB patients has not been examined.

Methods

We sought to determine the proportion of patients who could potentially benefit from high-dose isoniazid and who may be resistant to ethionamide. We reviewed 994 LPA (Hain MTBDRplus) results at the TB reference laboratory in KwaZulu-Natal to determine the frequency of mutations in either katG or the inhA promoter. We stratified these results by drug-resistance category (i.e., MDR-TB, pre-XDR-TB, and XDR-TB) as determined by phenotypic drug-susceptibility testing.

Results

Among MDR- and XDR-TB isolates, the prevalence of inhA mutations without a concurrent katG mutation was 14.8% and 10.3% respectively. The prevalence of inhA mutations with OR without a katG mutation was 30.3% and 82.8%, respectively.

Conclusion

More than 10% of patients with MDR- and XDR-TB may benefit from high-dose isoniazid. Although ethionamide is empirically included in all MDR- and XDR-TB regimens, nearly a third of MDR-TB patients and a majority of XDR-TB patients likely have resistance to ethionamide. Laboratories performing line probe assays should report specific band patterns so that clinicians may adjust treatment regimens accordingly.     

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI–CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later ‘stitched’ together, forming a 3D dataset. High-resolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities.Abbreviations: DEI, diffraction-enhanced imagingDiffraction-enhanced imaging (DEI) is a biomedical imaging technique that, compared with conventional radiography, generates very detailed images with more edge contrast but deposits a lower radiation dose to the object. DEI generates enhanced contrast both from absorption, the process involved in conventional radiography, and from of X-ray refraction, a process that harnesses photons that otherwise typically are imperceptibly diffracted.⁴ The DEI technique collects information from X-rays that are refracted as they pass through tissues that have different refractive indices as it almost completely removes diffracted X-rays. In comparison, conventional radiography produces images from X-rays that are attenuated by the tissues through which they pass, but X-rays that are refracted within those same tissues confound, rather than clarify, image contrast. The creation of contrast from the refraction of X-rays, rather than exclusively from absorption, yields images that display more detail with clearer distinction between tissue interfaces. Refraction-based imaging can reveal tiny structures that are transparent to X-ray attenuation but have sufficient variation in density to produce refraction contrast. Furthermore, refraction-based imaging decreases the required radiation dose.²¹To obviate the superimposing effects in a 2-dimensional DEI refraction image, we considered that combining CT with DEI would yield images with even greater clarity. CT allows a 3D representation of the sample, such that contrast from features at different depths are no longer superimposed on one another but can be separated and viewed as independent structures. Although this advantage is valuable in traditional absorption imaging, the additional features that provide contrast in a refraction-based image enhance the value of CT. Combining DEI technology, which is capable of imaging soft-tissue detail, with CT, which allows segregation of the contrast images at different depths, overcomes limitations of conventional X-ray imaging, namely lack of distinction of soft tissues and 2-dimensionality. As we report here, DEI combined with CT and a synchrotron-generated X-ray source yields 3D images of growing joint tissues at a resolution on the order of micrometers, which is much higher than can be generated using conventional imaging techniques.A synchrotron radiation source was required for the development of DEI because a synchrotron currently is the only source capable of providing an intensely brilliant light (millions of times brighter than sunlight and conventional X-ray sources), is highly collimated (light rays in the beam remain parallel with negligible dispersion over distance), can be made to be monochromatic (having a single wavelength), and can be tuned precisely to an array of energy ranges. The Canadian Light Source (www.lightsource.ca), which began operations in 2005, is one of only 47 synchrotron facilities worldwide and the only such facility in Canada. Although nonsynchrotron sources of X-rays for DEI–CT are conceivable,^16,18 such technology requires considerable image-acquisition time. Regardless, the quality of images generated by using synchrotron technology likely would remain the standard with which any new nonsynchrotron DEI–CT technological innovations would be compared.¹⁴Despite refinements in medical imaging, conventional radiography, CT scanning, and MRI still are insufficient to discern fine details, particularly in growing joints in which soft tissues (including cartilage) predominate and change with physiologic growth. The impetus for the current research was to develop an imaging technique that better demonstrated normal joint characteristics during growth and, in the future, could be applied to pathologic joints for experimental research and eventually clinical applications. In particular, we were motivated by a need to more effectively and reliably image growing joints affected by arthritis, a disease associated with alterations of bone and cartilage growth, tissue morphology and vascularity. Childhood arthritis research likely will benefit from having an improved imaging technique to aid in early diagnosis, monitor disease progression, and assess responses to therapies. The long-term outcomes of childhood arthritis are improved with early diagnosis and prompt and effective response to treatment interventions. Clinical and laboratory-based indicators of inflammation are not always adequate to detect and monitor subclinical intraarticular inflammation which, as with overt disease, can lead to progressive joint damage. Imaging can augment clinical and laboratory assessment of arthritis activity, but even the most sensitive currently available modalities are unable to detect all joint pathology.In juvenile arthritis, joint-imaging outcomes are difficult to evaluate because variations associated with normal growth cannot always be easily discerned from variations induced by the disease. Conventional radiography tends to detect advanced joint damage that has affected bone, but cartilage can be assessed only indirectly, and soft tissue abnormalities cannot be fully evaluated. Consequently, conventional radiography has insufficient sensitivity and specificity to be considered useful for diagnosing or monitoring children with inflammatory joint disease.^6,20 MRI, which evaluates both soft tissues and osteochondral structures, can be used to detect cartilage loss, bone erosions, and synovial hypertrophy in children and adolescents, and contrast-enhanced MRI detects active synovitis.^1,10 However, standardized approaches to acquire and interpret MRI data are not established for children in general and, in particular, for children with arthritis;^12,15 it is not always clear, for example, if observed thinning of cartilage is physiologic or pathologic. Furthermore, although MRI is more sensitive than conventional radiography, MRI too has limited precision in detecting fine structures and pathologic changes; a clinical MRI has less than 50% sensitivity in detecting cartilage damage that subsequently is seen arthroscopically.^8,13CT offers another option for joint visualization, given that it provides high-resolution, 3D images of bone from any angle. Despite its high spatial resolution, however, CT cannot match MRI''s soft-tissue contrast resolution, because CT provides negligible variability of attenuation coefficients of soft tissues so attenuation is nearly the same for cartilage, muscles, and ligaments. Furthermore, CT''s value is offset by the necessity for radiation exposure, a particular concern in the pediatric population. Therefore, for joint research and clinical applications, each of the conventional imaging techniques currently available has limitations. A safe, higher resolution imaging system that generates good contrast for all joint structures is required.Because the DEI technique initially was developed by using a synchrotron light source, we similarly used synchrotron technology in the current experiments. In contrast to conventional X-ray tubes, a synchrotron generates light by using radiofrequency waves and electromagnets to energize and accelerate electrons, thus producing brilliant, highly focused light from the entire wavelength spectrum, including X-rays. For the development and evaluation of DEI–CT imaging of joints, we chose to use healthy commercial piglet stifle joints because porcine stifle joints are anatomically similar to human knees.⁵ In addition, pigs grow quickly, reaching skeletal maturity at the distal femur and proximal tibia in 20 mo,¹⁹ thus allowing for the use of the pig as a model to study growth patterns in normal and disease states in a relatively short time period. The current study aimed to develop and test a new technology for imaging growing joints by using DEI combined with CT and a synchrotron radiation source. This report is the first to document the application of DEI–CT for imaging intact, growing joints.     

Synthesis and biological evaluation of a novel 99mTc cyclopentadienyl tricarbonyl complex ([(Cp-R)99mTc(CO)3]) for sigma-2 receptor tumor imaging

We report the design, synthesis and biological evaluation of a novel ^99mTc 4-(4-cyclohexylpiperazine-1-yl)-butan-1-one-1-cyclopentadienyltricarbonyl technetium ([^99mTc]5) as a potential SPECT tracer for imaging of σ₂ receptors in tumors. [^99mTc]5 was prepared in 25 ± 5% isolated radiochemical yield with radiochemical purity of >99% via double-ligand transfer (DLT) reaction from the ferrocene precursor 2b (4-(4-cyclohexylpiperazine-1-yl)-1-ferrocenylbutan-1-one). The corresponding Re-complex 4 and the ferrocenyl complex 2b showed relatively high affinity towards σ₂ receptors in in vitro competition binding assay (K_i values of 4 and 2b were 64.4 ± 18.5 nM and 43.6 ± 21.3 nM, respectively) and moderate to high selectivity versus σ₁ receptors (K_iσ₁/K_iσ₂ ratios were 12.5 and 95.5, respectively). The log D value of [^99mTc]5 was determined to be 2.52 ± 0.33. Biodistribution studies in mice revealed comparably high initial brain uptake of [^99mTc]5 and slow washout. Administration of haloperidol 5 min prior to injection of [^99mTc]5 significantly reduced the radiotracer uptake in brain, heart, lung, and spleen by 40–50% at 2 h p.i.. Moreover, [^99mTc]5 showed high uptake in C6 glioma cell lines (8.6%) after incubation for 1 h. Blocking with haloperidol to compete with [^99mTc]5 significantly reduced the cell uptake. Preliminary blocking study in C6-brain-tumor bearing rats showed that [^99mTc]5 binds to σ receptors in the brain-tumor specifically. These results are encouraging for further exploration of ^99mTc-labeled probes for σ₂ receptor tumor imaging in vivo.     

Facies and provenance of basin-margin deposits in the Los Palacios Basin (Capdevila Formation,Cuba)

The Paleogene tectono-sedimentary evolution of the Cuban island is dominated by block tectonics with rotations and strike-slip motion along the northern boundary of the Caribbean plate. The Capdevila Formation, which is the oldest basin fill of the Los Palacios strike-slip basin, preserves this final stage of the Cuban orogeny in western Cuba during the Early Eocene. Petrographic investigations of Capdevila Formation sedimentary records were performed at two localities near to Pinar del Rio (PR) and near to San Diégo de los Baños (SDB) close to the basin-bounding Pinar Fault zone. The studied marine hybrid arenites are interpreted as deposits of tectonically active regions causing the mixture of terrigenous and marine detritus and a differentiated development of the source areas and the basin margin. Bioclastic components (foraminifera and red algae) originate from shallow-marine carbonate platforms, being exemplarily formed at the SDB site and incompletely developed at the PR site, which is shown on the basis of foraminiferal assemblages. Among the terrigenous siliciclastic constituents, lithogenic fragments, in particular mafic to intermediate volcanic rock detritus prevail, accompanied by frequent plagioclase grains. Higher quartz portions as well as metamorphic rock clasts, which lack at the SDB site, muscovite flakes and plutonic rock fragments, characterize the lithogenic spectrum of the PR arenites. Framework composition of point-counted arenites reveal magmatic arc provenance for both successions indicating the presence of Cretaceous volcanic arc rocks on top of the northerly situated Guaniguanico Mountains, from where lithogenic detritus derived. Those findings support the idea that the North American Passive margin was overthrown by the Cretaceous volcanic arc rocks during the Cuban orogeny and affirm earlier published assumptions.     

Determinants of between‐year burrow re‐occupation in a colony of the European bee‐eater Merops apiaster

Re‐occupation of existing nesting burrows in the European bee‐eater Merops apiaster has only rarely – and if so mostly anecdotically – been documented in the literature record, although such behavior would substantially save time and energy. In this study, we quantify burrow re‐occupation in a German colony over a period of eleven years and identify ecological variables determining reuse probability. Of 179 recorded broods, 54% took place in a reused burrow and the overall probability that one of 75 individually recognized burrows would be reused in a given subsequent year was estimated as 26.4%. This indicates that between‐year burrow reuse is a common behavior in the study colony which contrasts with findings from studies in other colonies. Furthermore, burrow re‐occupation probability declined highly significantly with increasing age of the breeding wall. Statistical separation of within‐ and between‐burrow effects of the age of the breeding wall revealed that a decline in re‐occupation probability with individual burrow age was responsible for this and not a selective disappearance of burrows with high re‐occupation probability over time. Limited duty cycles of individual burrows may be caused by accumulating detritus or decreasing stability with increasing burrow age. Alternatively, burrow fidelity may presuppose pair fidelity which may also explain the observed restricted burrow reuse duty cycles. A consequent next step would be to extend our within‐colony approach to other colonies and compare the ecological circumstances under which bee‐eaters reuse breeding burrows.     

Changes in Regional Blood–Brain Transfer of l-Leucine Elicited by Arginine-Vasopressin

Arginine-vasopressin (AVP), injected into the carotid artery in physiological concentration together with L-leucine, changed kinetic constants of the blood-brain barrier (BBB) transport of this neutral amino acid without changing the cerebral blood flow (CBF). The maximum velocity of transport (Vmax), the half-saturation constant (Km), the nonsaturable transport constant (KD), and CBF were estimated in nine brain regions of male Wistar rats anesthetized with ether. In cerebral hemisphere, Vmax decreased from 21 nmol . min-1 . g-1 (control) to 7.6 nmol . min-1 . g-1 (AVP). Km decreased from 0.11 to 0.029 mM. Regional differences of the kinetic constants were found in controls as well as in AVP-treated animals. In all regions, the calculated constants Vmax and Km of animals coinjected with AVP were significantly decreased when compared to controls. A direct or indirect interaction of AVP with the transport system of large neutral amino acids is suggested.     

Antigen-gelonin conjugates. Preparation and application in experimental myasthenia gravis

The antigen-specific immune suppression by gelonin-antigen conjugates was tested in two different systems: (i) the horseradish-peroxidase-stimulated T-cell proliferation in vitro and (ii) in vivo with experimental autoimmune myasthenia gravis (EAMG) in the rat. For this, the phytotoxin gelonin, a glycoprotein from Gelonium multiflorum, was purified and linked to the respective antigens. For the in-vitro assay a lymph node cell suspension from rats immunized with horseradish peroxidase was cultured in the presence of this protein and proliferation was measured by [3H]thymidine uptake. In-vitro proliferation was significantly inhibited by adding gelonin-horseradish peroxidase conjugates. The therapeutic effects of antigen-gelonin conjugates were tested in the rat model EAMG. For these experiments rats were immunized with purified nicotinic acetylcholine receptor from electric fish in order to develop EAMG. The success of the immunization was monitored by the change in physical performance tests, the change in anti-acetylcholine receptor antibody titer, and by the change in the number of ionic endplate channels using a novel electrophysiological method. The latter method permits a very accurate assay of functional damage of acetylcholine receptor at the endplate and correlates well with the clinical severity of the disease. Rats were conventionally immunized with acetylcholine receptor from electric fish. After the onset of EAMG as measured by physical performance tests and rise in antibody titer a group of the animals was injected with an acetylcholine receptor-gelonin conjugate and this treatment was repeated seven days later. The loss in functional acetylcholine receptor was significantly smaller in the therapy group than in the untreated EAMG group.(ABSTRACT TRUNCATED AT 250 WORDS)     

An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

Blood-Brain Transfer of l-Phenylalanine Declines After Peripheral but Not Central Nervous Administration of Vasopressin

To determine whether a previously reported effect of vasopressin on blood-brain transfer of leucine extends to other large neutral amino acids, we measured the regional blood-brain transfer of L-phenylalanine with the integral technique. Intravenous co-injection of L-phenylalanine and arginine vasopressin (30 nmol to 10 pmol) resulted in a decrease of the permeability-surface area (PaS) product of phenylalanine of between 11 and 39%. In addition, the peptide elicited a decrease of the cerebral blood flow of between 11 and 56% combined with a drastic decrease of the cardiac output (32-64%) and an elevation of the blood pressure to approximately 150% of control values. However, we found no changes of the cardiac output, the blood pressure, or the PaS product of phenylalanine after microdialysis (30 min, 5 microliters min-1) of arginine vasopressin (15 mumol L-1) into the dorsal hippocampus, but cerebral blood flow was decreased. The results support the hypothesis that arginine vasopressin receptors at the blood-brain barrier are involved in the regulation of large neutral amino acid transfer from blood to brain and indicate that these receptors are located at the luminal membrane of the endothelial cells.     

The relationship between codon boundaries and multiple reading-frame preferences: coding organization of bacterial insertion sequences

Theoretical considerations have shown that the five possible overlapping reading-frame configurations differ significantly in their coding flexibility and thus in their information content (Siegel and Fitch 1980; Smith and Waterman 1980). Contrary to expectation, the overlapping frame configuration allowing the greatest coding flexibility is rarely seen, whereas one of the most constraining is common. We point out here that this overlapping reading-frame paradox and an observed but unexplained preference in coding regions for a pyrimidine-purine at codon boundaries (Shepherd 1981; Jones and Kafatos 1982; Smith et al. 1983) are intimately linked. The codon boundary preference, which may be related to translation efficiency or accuracy, places constraints on the evolution of overlapping coding regions. These considerations may help identify actual coding regions in DNA sequences. We have analyzed five sequenced (enteric) bacterial insertion sequences for codon boundary incidences and reading-frame configurations and find that they are consistent with these proposed constraints.