Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study

Background

The Xpert MTB/RIF (Cepheid) non-laboratory-based molecular assay has potential to improve the diagnosis of tuberculosis (TB), especially in HIV-infected populations, through increased sensitivity, reduced turnaround time (2 h), and immediate identification of rifampicin (RIF) resistance. In a prospective clinical validation study we compared the performance of Xpert MTB/RIF, MTBDRplus (Hain Lifescience), LightCycler Mycobacterium Detection (LCTB) (Roche), with acid fast bacilli (AFB) smear microscopy and liquid culture on a single sputum specimen.

Methods and Findings

Consecutive adults with suspected TB attending a primary health care clinic in Johannesburg, South Africa, were prospectively enrolled and evaluated for TB according to the guidelines of the National TB Control Programme, including assessment for smear-negative TB by chest X-ray, clinical evaluation, and HIV testing. A single sputum sample underwent routine decontamination, AFB smear microscopy, liquid culture, and phenotypic drug susceptibility testing. Residual sample was batched for molecular testing. For the 311 participants, the HIV prevalence was 70% (n = 215), with 120 (38.5%) culture-positive TB cases. Compared to liquid culture, the sensitivities of all the test methodologies, determined with a limited and potentially underpowered sample size (n = 177), were 59% (47%–71%) for smear microscopy, 76% (64%–85%) for MTBDRplus, 76% (64%–85%) for LCTB, and 86% (76%–93%) for Xpert MTB/RIF, with specificities all >97%. Among HIV+ individuals, the sensitivity of the Xpert MTB/RIF test was 84% (69%–93%), while the other molecular tests had sensitivities reduced by 6%. TB detection among smear-negative, culture-positive samples was 28% (5/18) for MTBDRplus, 22% (4/18) for LCTB, and 61% (11/18) for Xpert MTB/RIF. A few (n = 5) RIF-resistant cases were detected using the phenotypic drug susceptibility testing methodology. Xpert MTB/RIF detected four of these five cases (fifth case not tested) and two additional phenotypically sensitive cases.

Conclusions

The Xpert MTB/RIF test has superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy and other molecular methodologies in an HIV- and TB-endemic region. Its place in the clinical diagnostic algorithm in national health programs needs exploration. Please see later in the article for the Editors'' Summary     

Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi

A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca²⁺]_i and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca²⁺-sensitive dye to quantify and image [Ca²⁺]_i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca²⁺ signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p < 0.001) in the relative fluorescence levels corresponding to [Ca²⁺]_i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L^?1) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data.     

Evidence-based practices for thromboembolism prevention: summary of the ASPS Venous Thromboembolism Task Force Report

In July of 2011, the American Society of Plastic Surgeons Executive Committee approved the Venous Thromboembolism Task Force Report. The report includes a summary of the scientific literature relevant to venous thromboembolism and plastic surgery along with five evidence-based recommendations. The recommendations are divided into two sections: risk stratification and prevention. The risk stratification recommendations are based on the 2005 Caprini Risk Assessment Module, which has been validated in the scientific literature as an effective tool for risk-stratifying plastic and reconstructive surgery patients based on individual risk factors for 60-day venous thromboembolism. The three prophylaxis recommendations are dependent on an individual patient's 2005 Caprini Risk Assessment Module score.     

Where is the light? Bayesian perceptual priors for lighting direction

Perception of shaded three-dimensional figures is inherently ambiguous, but this ambiguity can be resolved if the brain assumes that figures are lit from a specific direction. Under the Bayesian framework, the visual system assigns a weighting to each possible direction, and these weightings define a prior probability distribution for light-source direction. Here, we describe a non-parametric maximum-likelihood estimation method for finding the prior distribution for lighting direction. Our results suggest that each observer has a distinct prior distribution, with non-zero values in all directions, but with a peak which indicates observers are biased to expect light to come from above left. The implications of these results for estimating general perceptual priors are discussed.     

Nitro and amino substitution within the A-ring of 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: influence on topoisomerase I-targeting activity and cytotoxicity

Recently, 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one, 1, was identified as a TOP1-targeting agent with pronounced antitumor activity. In the present study, the effect on activity of substituting a single nitro or amino group in the A-ring in lieu of the methylenedioxy moiety of 1 was evaluated. The presence of either a nitro or amino substituent at the 4-position had a pronounced adverse affect on both TOP1-targeting activity and cytotoxicity. To a lesser extent, derivatives with a nitro or amino substituent at the 1-position were also less active than 1. Replacement of the methylenedioxy moiety of 1 with either a nitro or amino substituent at either the 2- and 3-position did result in analogues with potent TOP1-targeting activity and cytotoxicity.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Surgeon perspectives on surgical options for early-stage breast cancer

To evaluate the practice patterns of general and plastic surgeons regarding patients with early-stage breast cancer, all general and plastic surgeons in Quebec and Maryland were mailed self-administered questionnaires evaluating surgeon demographics, practice patterns, treatment preferences, and satisfaction with the results of lumpectomy and radiation therapy or breast reconstruction. Response rates of 38.3 percent and 26.7 percent were obtained for general surgeons in Quebec and Maryland, respectively. The ratio of reported mastectomies to lumpectomies was 1:2 in Maryland and 1:5 in Quebec. All general surgeons considered lumpectomy an important option. Ninety percent of Maryland surgeons versus 44 percent of Quebec surgeons considered mastectomy important. A total of 53.6 percent versus 24.9 percent of general surgeons in Maryland and Quebec, respectively, considered delayed reconstruction an important option. Additionally, 81.3 percent of Maryland surgeons considered immediate reconstruction important, and 79.6 percent discussed it with all stage I or II patients. More than 75 percent of Quebec general surgeons reported discussing immediate or delayed reconstruction with < or =50 percent of these women. Response rates of 53.6 percent and 48.8 percent were obtained for plastic surgeons in Quebec and Maryland, respectively. In one year Quebec plastic surgeons reported that they performed less than half the number of reconstructions performed by Maryland plastic surgeons (7.2 versus 17.3). In Quebec, 82.3 percent of surgeons reported that they frequently discuss delayed reconstruction, 25.1 percent immediate, 62.5 percent pedicled TRAM, and 51.7 percent nonautogenous options. In Maryland, 74.3 percent of plastic surgeons frequently discuss delayed reconstruction, 95.7 percent immediate, 89.9 percent pedicled TRAM, and 85.9 percent nonautogenous options. For women with early-stage breast cancer, regional variations exist in the surgical options discussed and provided.     

Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.