The Impact of a Line Probe Assay Based Diagnostic Algorithm on Time to Treatment Initiation and Treatment Outcomes for Multidrug Resistant TB Patients in Arkhangelsk Region,Russia

BackgroundIn the Arkhangelsk region of Northern Russia, multidrug-resistant (MDR) tuberculosis (TB) rates in new cases are amongst the highest in the world. In 2014, MDR-TB rates reached 31.7% among new cases and 56.9% among retreatment cases. The development of new diagnostic tools allows for faster detection of both TB and MDR-TB and should lead to reduced transmission by earlier initiation of anti-TB therapy.MethodsA culture-based diagnostic algorithm used prior to LPA implementation was compared to an LPA-based algorithm that replaced BacTAlert and Löwenstein Jensen (LJ) for drug sensitivity testing. A total of 295 MDR-TB patients were included in the study, 163 diagnosed with the culture-based algorithm, 132 with the LPA-based algorithm.ResultsAmong smear positive patients, the implementation of the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 50 and 66 days compared to the culture-based algorithm (BacTAlert and LJ respectively, p<0.001). In smear negative patients, the LPA-based algorithm was associated with a median decrease in time to MDR-TB treatment initiation of 78 days when compared to the culture-based algorithm (LJ, p<0.001). However, several weeks were still needed for treatment initiation in LPA-based algorithm, 24 days in smear positive, and 62 days in smear negative patients. Overall treatment outcomes were better in LPA-based algorithm compared to culture-based algorithm (p = 0.003). Treatment success rates at 20 months of treatment were higher in patients diagnosed with the LPA-based algorithm (65.2%) as compared to those diagnosed with the culture-based algorithm (44.8%). Mortality was also lower in the LPA-based algorithm group (7.6%) compared to the culture-based algorithm group (15.9%). There was no statistically significant difference in smear and culture conversion rates between the two algorithms.ConclusionThe results of the study suggest that the introduction of LPA leads to faster time to MDR diagnosis and earlier treatment initiation as well as better treatment outcomes for patients with MDR-TB. These findings also highlight the need for further improvements within the health system to reduce both patient and diagnostic delays to truly optimize the impact of new, rapid diagnostics.     

The Distribution and Density of Water Mice (Xeromys myoides) in the Maroochy River of Southeast Queensland,Australia

The water mouse is a small and vulnerable rodent present in coastal areas of south-west Papua New Guinea, and eastern Queensland and the Northern Territory of Australia. Current knowledge regarding the distribution of the water mouse is incomplete and the loss of one local population has been documented in southeast Queensland, a region where pressures from urban and industrial development are increasing. Water mouse populations have not been studied intensively enough to enable the primary factors responsible for the local decline to be identified. We surveyed the distribution and density of the water mouse along the Maroochy River of southeast Queensland, near the southern extent of the species’ range, to gather baseline data that may prove valuable for detecting any future decline in this population’s size or health. All areas of suitable habitat were surveyed on foot or by kayak or boat over a three-year period. We found 180 water mouse nests, of which ~94% were active. Permanent camera monitoring of one nest and limited supplementary live trapping suggested that up to three individual mice occupied active nests. Water mouse density was estimated to be 0.44 per hectare of suitable habitat along the Maroochy River. Should future monitoring reveal an adverse change in the water mouse population on the Maroochy River, a concerted effort should be made to identify contributing factors and address proximate reasons for the decline.     

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33^-/- mice. Importantly, IL-33^-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

Chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals

Oxidation of biopharmaceuticals is a major product quality issue with potential impacts on activity and immunogenicity. At Eli Lilly and Company, high tryptophan oxidation was observed for two biopharmaceuticals in development produced in Chinese hamster ovary cells. A switch from historical hydrolysate‐containing media to chemically defined media with a reformulated basal powder was thought to be responsible, so mitigation efforts focused on media modification. Shake flask studies identified that increasing tryptophan, copper, and manganese and decreasing cysteine concentrations were individual approaches to lower tryptophan oxidation. When amino acid and metal changes were combined, the modified formulation had a synergistic impact that led to substantially less tryptophan oxidation for both biopharmaceuticals. Similar results were achieved in shake flasks and benchtop bioreactors, demonstrating the potential to implement these modifications at manufacturing scale. The modified formulation did not negatively impact cell growth and viability, product titer, purity, charge variants, or glycan profile. A potential mechanism of action is presented for each amino acid or metal factor based on its role in oxidation chemistry. This work served not only to mitigate the tryptophan oxidation issue in two Lilly biopharmaceuticals in development, but also to increase our knowledge and appreciation for the impact of media components on product quality. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:178–188, 2016     

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.