A Pilot Study of Short-Duration Sputum Pretreatment Procedures for Optimizing Smear Microscopy for Tuberculosis

Background

Direct sputum smear microscopy for tuberculosis (TB) lacks sensitivity for the detection of acid fast bacilli. Sputum pretreatment procedures may enhance sensitivity. We did a pilot study to compare the diagnostic accuracy and incremental yield of two short-duration (<1 hour) sputum pretreatment procedures to optimize direct smears among patients with suspected TB at a referral hospital in India.

Methodology/Findings

Blinded laboratory comparison of bleach and universal sediment processing (USP) pretreated centrifuged auramine smears to direct Ziehl-Neelsen (ZN) and direct auramine smears and to solid (Loweinstein-Jensen (LJ)) and liquid (BACTEC 460) culture. 178 pulmonary and extrapulmonary TB suspects were prospectively recruited during a one year period. Thirty six (20.2%) were positive by either solid or liquid culture. Direct ZN smear detected 22 of 36 cases and direct auramine smears detected 26 of 36 cases. Bleach and USP centrifugation detected 24 cases each, providing no incremental yield beyond direct smears. When compared to combined culture, pretreated smears were not more sensitive than direct smears (66.6% vs 61.1 (ZN) or 72.2 (auramine)), and were not more specific (92.3% vs 93.0 (ZN) or 97.2 (auramine).

Conclusions/Significance

Short duration sputum pretreatment with bleach and USP centrifugation did not increase yield as compared to direct sputum smears. Further work is needed to confirm this in a larger study and also determine if longer duration pre-treatment might be effective in optimizing smear microscopy for TB.     

Pollen performance of Raphanus sativus (Brassicaceae) declines in response to elevated [CO2]

Although increases in atmospheric [CO₂] are known to affect plant physiology, growth and reproduction, understanding of these effects is limited because most studies of reproductive consequences focus solely on female function. Therefore, we examined the effects of CO₂ enrichment on male function in the annual Raphanus sativus. Pollen donors grown under elevated [CO₂] initially sired a higher proportion of seeds per fruit than ambient [CO₂]-grown plants when each was tested against two different standard competitors; however, by the end of the 5-month experiment, these pollen donors sired fewer seeds than ambient [CO₂]-grown plants and produced a lower proportion of viable pollen grains. The results of this experiment confirm that elevated [CO₂] can alter reproductive success. Additionally, the change in response to elevated [CO₂] over time varied among pollen donor families; thus, changes in [CO₂] could act as a selective force on this species.     

Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos

Inhibition of mango somatic embryo growth was inducedin vitro by treatments for 4 or more weeks with abscisic acid (0–100 M ABA) with and without high osmolarity provided by mannitol (0–10%). High osmolarity and ABA significantly affected somatic embryo length, precocious germination and the production of good quality secondary somatic embryos. High osmolarity also affected root elongation. Abscisic acid was more effective in suppressing growth and development of 0.5 cm-length somatic embryos than smaller somatic embryos. Development beyond the heart stage was significantly inhibited by both ABA and mannitol treatments. The recovery of good quality somatic embryos was enhanced by high levels of ABA (100 M) with and without mannitol (0–5%). Somatic embryos that had been pulsed with ABA were partially desiccated at different relative humidities. Weight loss was affected only by relative humidity; and ABA did not enhance desiccation tolerance.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - MM1 Mango maturation medium - RH Relative humidity     

Ganglioside GM1-mediated Transcytosis of Cholera Toxin Bypasses the Retrograde Pathway and Depends on the Structure of the Ceramide Domain

Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.     

The PathoChip,a functional gene array for assessing pathogenic properties of diverse microbial communities

Pathogens present in the environment pose a serious threat to human, plant and animal health as evidenced by recent outbreaks. As many pathogens can survive and proliferate in the environment, it is important to understand their population dynamics and pathogenic potential in the environment. To assess pathogenic potential in diverse habitats, we developed a functional gene array, the PathoChip, constructed with key virulence genes related to major virulence factors, such as adherence, colonization, motility, invasion, toxin, immune evasion and iron uptake. A total of 3715 best probes were selected from 13 virulence factors, covering 7417 coding sequences from 1397 microbial species (2336 strains). The specificity of the PathoChip was computationally verified, and approximately 98% of the probes provided specificity at or below the species level, proving its excellent capability for the detection of target sequences with high discrimination power. We applied this array to community samples from soil, seawater and human saliva to assess the occurrence of virulence genes in natural environments. Both the abundance and diversity of virulence genes increased in stressed conditions compared with their corresponding controls, indicating a possible increase in abundance of pathogenic bacteria under environmental perturbations such as warming or oil spills. Statistical analyses showed that microbial communities harboring virulence genes were responsive to environmental perturbations, which drove changes in abundance and distribution of virulence genes. The PathoChip provides a useful tool to identify virulence genes in microbial populations, examine the dynamics of virulence genes in response to environmental perturbations and determine the pathogenic potential of microbial communities.     

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.     

Effects of prostaglandin F2alpha and progesterone on the ability of bovine luteal cells to stimulate T lymphocyte proliferation

Bovine luteal cells express class I and II major histocompatibility complex molecules and stimulate T lymphocyte proliferation in vitro. Proliferation of T lymphocytes is greater in cocultures of luteal cells and T lymphocytes collected following administration of a luteolytic dose of prostaglandin (PG) F2alpha to the cow. Whether this results from changes in luteal cells that increase their ability to stimulate T lymphocyte proliferation or from changes in T lymphocytes that enhance their ability to respond to luteal cells is unclear. To determine which is the case, luteal cell-T lymphocyte cocultures were performed using luteal cells and T lymphocytes isolated from the same animals before and 8 h after administration of PGF2alpha. In the presence of T lymphocytes collected before PGF2alpha administration, luteal cells isolated after PGF2alpha were more potent stimulators of T lymphocyte proliferation than were luteal cells collected before PGF2alpha (P<0.05). The effect of progesterone on luteal cell-stimulated T lymphocyte proliferation was also evaluated. Proliferation of T lymphocytes was greater (P<0.05) in cultures containing the cytochrome P450 side-chain cleavage enzyme-inhibitor aminoglutethimide. Exogenous progesterone caused a dose-dependent inhibition of luteal cell-stimulated T lymphocyte proliferation (P<0.05). Progesterone-receptor mRNA was undetectable in peripheral blood mononuclear cells collected before and after PGF2alpha administration, indicating that the effect of progesterone was not mediated via progesterone receptors in lymphocytes. These results imply that specific changes in luteal cells in response to PGF2alpha enhance the ability of these cells to stimulate T lymphocyte proliferation. These results also demonstrate that progesterone can suppress luteal cell-stimulated T lymphocyte proliferation.     

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatin's antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.