Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of Drug-Resistant Tuberculosis in Harare,Zimbabwe

Introduction

Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings.

Methods

We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method.

Results

One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69–95%) and specificity was 93% (95% CI, 84–98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7–15 days) than MGIT (12 days; IQR 6–16 days) or LJ (28 days; IQR 21–35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68–97%), 96% (95% CI, 79–100%), and 91% (95% CI, 72–99%), respectively; specificity was 89% (95% CI, 52–100%), 89% (95% CI, 52–100%), and 90% (95% CI, 56–100%), respectively.

Conclusion

In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods.     

Choroid Sprouting Assay: An Ex Vivo Model of Microvascular Angiogenesis

Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research.     

Oral Exposure to Phytomonas serpens Attenuates Thrombocytopenia and Leukopenia during Acute Infection with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi, the agent of Chagas disease, rapidly develop anemia and thrombocytopenia. These effects are partially promoted by the parasite trans-sialidase (TS), which is shed in the blood and depletes sialic acid from the platelets, inducing accelerated platelet clearance and causing thrombocytopenia during the acute phase of disease. Here, we demonstrate that oral immunization of C57BL/6 mice with Phytomonas serpens, a phytoflagellate parasite that shares common antigens with T. cruzi but has no TS activity, reduces parasite burden and prevents thrombocytopenia and leukopenia. Immunization also reduces platelet loss after intraperitoneal injection of TS. In addition, passive transfer of immune sera raised in mice against P. serpens prevented platelet clearance. Thus, oral exposure to P. serpens attenuates the progression of thrombocytopenia induced by TS from T. cruzi. These findings are not only important for the understanding of the pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.     

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B₄ as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions.The emerging notion that cells can communicate by packaged information represents a major shift in our understanding of cell-to-cell interaction in complex settings including inflammation (1). Packaging of mediators (irrespective of their chemical nature) in structures that can be transported through the vascular and lymphatic systems might avoid their rapid dilution and removal by biological fluids and allow the target cell or tissue to receive a biologically relevant amount of a given molecule. As an example, TNF-α produced by mast cells in the mouse paw can reach the lymph nodes unmodified, wrapped up in small structures or vesicles (2). In this respect, the last few years have witnessed augmented understanding in microparticle function.Described over 50 years ago (reviewed in (3, 4), microparticles are heterogeneous in nature with their size varying between 0.2 and 1.0 μm, and are characterized by an outer membrane composed of a phospholipid bilayer and cell surface proteins. The mechanism of microparticle production is not fully understood, though it may follow processes not dissimilar from those observed in apoptosis, involving membrane detachment from the anchoring cytoskeleton and loss of membrane symmetry, which leads to exposure of negatively charged phospholipids (5–7). Proteins found on the outer leaflet of the microparticle cell membrane are believed to reflect both the origin and activation status of the parental cell (8, 9); for instance, microparticles from neutrophils express CD66b and CD62L (10, 11). We have recently identified the selective expression of the potent anti-inflammatory and proresolving protein Annexin A1 (ANXA1) on the surface of microparticles generated from neutrophils adherent to endothelial monolayers, when compared with those prepared from quiescent neutrophils (12). Microparticle production is not restricted to one subset of cells and using cell specific antigens the relative contribution of different cell types to the total microparticle population in a particular environment can be assessed. This has allowed for the analysis of different microparticle populations (the focus being by and large platelet- and endothelial-derived microparticles) in a number of pathologies in the quest to identify robust biomarkers for disease and treatment (13–15). With regard to inflammatory diseases, examples would include plasma samples in sepsis (16), psoriatic arthritis (17), and scleroderma (18). However, the vast majority of these studies have only determined microparticle expression patterns with respect to the cell type of origin, without addressing the possibility that microparticle composition—even when generated from the same leukocyte subset—might differ in relation to disease status and/or mode of cell activation. Of note recent work has also demonstrated that the production of neutrophil microparticles during self-limited inflammation is temporally regulated suggesting that these microparticles are important in orchestrating inflammation-resolution (1).Recent work has established that microparticles can elicit a variety of biological processes ranging from angiogenesis to anti-inflammation; so that it is very unlikely they can continue to be considered “cell debris,” as initially postulated. The following are some examples, relevant to the present study. Ingestion of platelet microparticles alters the phenotype of macrophages, leading to the false identification of endothelial cell progenitor cells in culture (19). Likewise, sonic hedgehog can be transferred, via microparticles, to dysfunctional endothelial cells, restoring the activity of nitric oxide synthase with downstream production of nitric oxide (20). Microparticles can carry functionally active receptor proteins to target cells (21, 22). Finally, in vivo generation of microparticles has been observed within the inflamed microcirculation. Real time analysis of leukocyte recruitment has visualized microparticle release from leukocytes squeezing through an endothelial barrier, providing evidence for their formation in vivo together with potential functional relevance in relation to cell migration (23).On stimulation, neutrophils produce microparticles with rapid and nongenomic anti-inflammatory properties, in vitro and in vivo, reliant on their expression of ANXA1 (12). Whereas these findings are consistent with those obtained by Gasser and colleagues (24) who described inhibitory properties of neutrophil microparticles, other studies have suggested that the same cell type can produce microparticles that elicit activating properties, for instance upon incubation with endothelial cells or monocytes for longer time-points (25, 26). Thus, to gain further insight into the potential mechanisms involved in mediating such distinct effects, we deemed it important to determine the total proteome of neutrophil microparticles. Having established that different stimulation conditions yield microparticle populations with distinct protein profiles, we corroborated our observations in two distinct clinical scenarios, characterizing neutrophil microparticles from skin blister exudates and plasma samples from sepsis patients using a select group of proteins identified in our proteomic profile. We also established that the two microparticles subsets differentially modulate endothelial cell gene expression profile and thereby function, as determined by connectivity map analysis.     

The effects of staged intra-articular injection of cultured autologous mesenchymal stromal cells on the repair of damaged cartilage: a pilot study in caprine model

Introduction

Treatment of chondral injuries remains a major issue despite the many advances made in cartilage repair techniques. Although it has been postulated that the use of marrow stimulation in combination with cell-based therapy may provide superior outcome, this has yet to be demonstrated. A pilot study was thus conducted to determine if bone marrow derived mesenchymal stromal cells (BM-MSCs) have modulatory effects on the repair outcomes of bone marrow stimulation (BMS) techniques.

Methods

Two full-thickness chondral 5 mm diameter defects were created in tandem on the medial condyle of left stifle joints of 18 Boer caprine (N = 18). Goats were then divided equally into three groups. Simultaneously, bone marrow aspirates were taken from the iliac crests from the goats in Group 1 and were sent for BM-MSC isolation and expansion in vitro. Six weeks later, BMS surgery, which involves subchondral drilling at the defect sites, was performed. After two weeks, the knees in Group 1 were given autologous intra-articular BM-MSCs (N = 6). In Group 2, although BMS was performed there were no supplementations provided. In Group 3, no intervention was administered. The caprines were sacrificed after six months. Repairs were evaluated using macroscopic assessment through the International Cartilage Repair Society (ICRS) scoring, histologic grading by O’Driscoll score, biochemical assays for glycosaminoglycans (GAGs) and gene expressions for aggrecan, collagen II and Sox9.

Results

Histological and immunohistochemical analyses demonstrated hyaline-like cartilage regeneration in the transplanted sites particularly in Group 1. In contrast, tissues in Groups 2 and 3 demonstrated mainly fibrocartilage. The highest ICRS and O’Driscoll scorings was also observed in Group 1, while the lowest score was seen in Group 3. Similarly, the total GAG/total protein as well as chondrogenic gene levels were expressed in the same order, that is highest in Group 1 while the lowest in Group three. Significant differences between these 3 groups were observed (P <0.05).

Conclusions

This study suggests that supplementing intra-articular injections of BM-MSCs following BMS knee surgery provides superior cartilage repair outcomes.     

The use of predator-exclusion fencing as a management tool improves the breeding success of waders on lowland wet grassland

Waders breeding on lowland wet grassland have undergone dramatic declines across Europe in recent decades. Few species now achieve the levels of breeding success required for population stability and recovery, with predation from large mammals acting as a key compounding factor limiting nest survival and productivity. Predator management through lethal control is often controversial, yet alternative non-lethal methods are little tested in the context of grassland breeding waders. Excluding predators through the use of electric fences has led to improvements in nest and chick survival in other habitats. To test the applicability of this method to lowland wet grassland we constructed predator-exclusion fences on sites across the UK and, with Lapwing Vanellus vanellus as a study species, used historical and contemporary data to test whether excluding large mammalian predators leads to an increase in wader nest survival and productivity, and whether effects differ between fence designs. Lapwing nest survival was significantly higher in the presence of any type of predator-exclusion fence, with significantly fewer nests predated each day. Overall productivity also improved, with significantly higher numbers of chicks fledged per pair in years when fences were operational. Different designs and methods of powering fences resulted in different levels of success, with combination design fences and those powered by mains electricity performing best. Excluding large mammalian predators from areas of lowland wet grassland with predator-exclusion fencing successfully improves Lapwing nest survival and productivity, allowing breeding success to exceed the levels required for population recovery. Other wader species breeding in the same habitat are also likely to benefit from the increased protection from predation provided by fences. Predator-exclusion fencing is therefore an effective management tool for protecting restricted and declining populations of breeding waders on lowland wet grassland.     

Contextualising the Last Survivors: Population Structure of Marine Turtles in the Dominican Republic

Nesting by three species of marine turtles persists in the Dominican Republic, despite historic threats and long-term population decline. We conducted a genetic survey of marine turtles in the Dominican Republic in order to link them with other rookeries around the Caribbean. We sequenced a 740_bp fragment of the control region of the mitochondrial DNA of 92 samples from three marine turtle species [hawksbill (n = 48), green (n = 2) and leatherback (n = 42)], and incorporated published data from other nesting populations and foraging grounds. The leatherback turtle (Dermochelys coriacea) in the Dominican Republic appeared to be isolated from Awala-Yalimapo, Cayenne, Trinidad and St. Croix but connected with other Caribbean populations. Two distinct nesting populations of hawksbill turtles (Eremochelys imbricata) were detected in the Dominican Republic and exhibited interesting patterns of connectivity with other nesting sites and juvenile and adult male foraging aggregations. The green sea turtle (Chelonia mydas) has almost been extirpated from the Dominican Republic and limited inference could be made from our samples. Finally, results were compared with Lagrangian drifting buoys and published Lagrangian virtual particles that travelled through the Dominican Republic and Caribbean waters. Conservation implications of sink-source effects or genetic isolation derived from these complex inter-connections are discussed for each species and population.     

Defining the Vulnerable Period for Re-Establishment of Clostridium difficile Colonization after Treatment of C. difficile Infection with Oral Vancomycin or Metronidazole

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. A significant proportion of patients receiving oral vancomycin or metronidazole for treatment of Clostridium difficile infection (CDI) develop recurrences. However, the period of vulnerability to re-establishment of colonization by C. difficile after therapy is not well defined.

Principal Findings

In a prospective study of CDI patients, we demonstrated that most vancomycin-treated patients maintained inhibitory concentrations of vancomycin in stool for 4 to 5 days after therapy, whereas metronidazole was only detectable during therapy. From the time of elimination of the antibiotics to 14 to 21 days after therapy, a majority of stool suspensions supported growth of C. difficile and deep 16S rRNA sequencing demonstrated persistent marked alteration of the indigenous microbiota. By 21 to 28 days after completion of CDI treatment, a majority of stool suspensions inhibited growth of C. difficile and there was evidence of some recovery of the microbiota.

Conclusions

These data demonstrate that there is a vulnerable period for re-establishment of C. difficile colonization after CDI treatment that begins within a few days after discontinuation of treatment and extends for about 3 weeks in most patients.     

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.     

Reproductive success of Woodlarks Lullula arborea in traditional and recently colonized habitats

Capsule Set-aside schemes have allowed breeding Woodlarks to colonize farmland, but heathland and forestry habitats remain the stronghold.

Aims To determine which habitats provide the best conditions for breeding Woodlarks and whether a buffer effect is operating, with density increasing faster in the poorer quality habitat as the population grows.

Methods Habitat colonization was examined and breeding success compared among heathland, clear-felled and young conifer plantations, and farmland set-aside.

Results Woodlarks on heathland and forestry habitats had similar clutch sizes and nesting success, but clutch sizes may be lower on farmland. Heathland was recolonized when population density was low in forest habitats, while farmland was colonized when density was increasing, and areas close to forest were preferentially occupied. Woodlarks breeding on farmland preferred set-aside stubbles to other field types.

Conclusion Forestry and heathland habitats are similar in quality for breeding Woodlarks, with no evidence for a buffer effect. Farmland set-aside may be suboptimal but the area available is much greater than the area of forest or heathland, and could therefore make a significant contribution to the conservation of the Woodlark population. However, set-aside should not be seen as an alternative to the conservation of forest and heathland.