Ioana Nicolau

Are peripheral microscopy centres ready for next generation molecular tuberculosis diagnostics

To the Editor: Sputum smear microscopy is the primary test for tuberculosis (TB) in most high-burden countries. Direct Ziehl–Neelsen (ZN) microscopy is routinely implemented in these countries via a vast network of decentralised, peripheral microscopy centres (as opposed to centralised reference laboratories), often located within primary or community health centres. This decentralised approach increases access in primary care settings and may help reduce diagnostic delays [1]. However, microscopy has limitations and novel diagnostics are urgently needed, particularly in settings with high prevalence of drug resistance and HIV [1, 2]. While Xpert® MTB/RIF (Cepheid, Sunnyvale, CA, USA), a World Health Organization-endorsed test, is already being rolled out in many countries, it is intended for district or sub-district laboratories [3], and not peripheral microscopy centres. In contrast, at least four next-generation nucleic-acid amplification tests (NAATs) are now on the market, with the goal of point-of-care (POC) use in peripheral laboratories [4, 5]. Can these so-called “POC-NAATs” actually be implemented in peripheral microscopy centres? Is there sufficient expertise ( e.g . to extract DNA) and biosafety ( e.g . to process sputum)? Will the necessary infrastructure ( e.g . stable power supply) be present? Will environmental conditions ( e.g . high temperature) limit their use? The introduction of novel diagnostics that aim to replace smear microscopy in peripheral laboratories is more likely to succeed if the new tests are designed with real world conditions in mind. However, there are limited data on landscape of microscopy centres in high-burden countries. We addressed this gap with a survey of 22 high-burden countries. We designed a one-page, 12-question survey. We focused on the most important requirements and considerations for the next generation of TB diagnostics. We included questions about ambient temperature and humidity conditions, safety, infrastructure, availability of equipment and …

Research Questions and Priorities for Tuberculosis: A Survey of Published Systematic Reviews and Meta-Analyses

Background Systematic reviews are increasingly informing policies in tuberculosis (TB) care and control. They may also be a source of questions for future research. As part of the process of developing the International Roadmap for TB Research, we did a systematic review of published systematic reviews on TB, to identify research priorities that are most frequently suggested in reviews. Methodology/Principal Findings We searched EMBASE, MEDLINE, Web of Science, and the Cochrane Library for systematic reviews and meta-analyses on any aspect of TB published between 2005 and 2010. One reviewer extracted data and a second reviewer independently extracted data from a random subset of included studies. In total, 137 systematic reviews, with 141 research questions, were included in this review. We used the UK Health Research Classification System (HRCS) to help us classify the research questions and priorities. The three most common research topics were in the area of detection, screening and diagnosis of TB (32.6%), development and evaluation of treatments and therapeutic interventions (23.4%), and TB aetiology and risk factors (19.9%). The research priorities determined were mainly focused on the discovery and evaluation of bacteriological TB tests and drug-resistant TB tests and immunological tests. Other important topics of future research were genetic susceptibility linked to TB and disease determinants attributed to HIV/TB. Evaluation of drug treatments for TB, drug-resistant TB and HIV/TB were also frequently proposed research topics. Conclusions Systematic reviews are a good source of key research priorities. Findings from our survey have informed the development of the International Roadmap for TB Research by the TB Research Movement.

Calcium signaling mediates the response to cadmium toxicity in Saccharomyces cerevisiae cells

The involvement of Ca2+ in the response to high Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+ was investigated in Saccharomyces cerevisiae. The yeast cells responded through a sharp increase in cytosolic Ca2+ when exposed to Cd2+, and to a lesser extent to Cu2+, but not to Mn2+, Co2+, Ni2+, Zn2+, or Hg2+. The response to high Cd2+ depended mainly on external Ca2+ (transported through the Cch1p/Mid1p channel) but also on vacuolar Ca2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd2+ was influenced by perturbations in Ca2+ homeostasis. Thus, the tolerance to Cd2+ often correlated with sharp Cd2+‐induced cytosolic Ca2+ pulses, while the Cd2+ sensitivity was accompanied by the incapacity to rapidly restore the low cytosolic Ca2+.

Point-of-Care Urine Tests for Smoking Status and Isoniazid Treatment Monitoring in Adult Patients

Background Poor adherence to isoniazid (INH) preventive therapy (IPT) is an impediment to effective control of latent tuberculosis (TB) infection. TB patients who smoke are at higher risk of latent TB infection, active disease, and TB mortality, and may have lower adherence to their TB medications. The objective of our study was to validate IsoScreen and SmokeScreen (GFC Diagnostics, UK), two point-of-care tests for monitoring INH intake and determining smoking status. The tests could be used together in the same individual to help identify patients with a high-risk profile and provide a tailored treatment plan that includes medication management, adherence interventions, and smoking cessation programs. Methodology/Principal Findings 200 adult outpatients attending the TB and/or the smoking cessation clinic were recruited at the Montreal Chest Institute. Sensitivity and specificity were measured for each test against the corresponding composite reference standard. Test reliability was measured using kappa statistic for intra-rater and inter-rater agreement. Univariate and multivariate logistic regression models were used to explore possible covariates that might be related to false-positive and false-negative test results. IsoScreen had a sensitivity of 93.2% (95% confidence interval [CI] 80.3, 98.2) and specificity of 98.7% (94.8, 99.8). IsoScreen had intra-rater agreement (kappa) of 0.75 (0.48, 0.94) and inter-rater agreement of 0.61 (0.27, 0.90). SmokeScreen had a sensitivity of 69.2% (56.4, 79.8), specificity of 81.6% (73.0, 88.0), intra-rater agreement of 0.77 (0.56, 0.94), and inter-rater agreement of 0.66 (0.42, 0.88). False-positive SmokeScreen tests were strongly associated with INH treatment. Conclusions IsoScreen had high validity and reliability, whereas SmokeScreen had modest validity and reliability. SmokeScreen tests did not perform well in a population receiving INH due to the association between INH treatment and false-positive SmokeScreen test results. Development of the next generation SmokeScreen assay should account for this potential interference.

Vaccinium corymbosum L. (blueberry) extracts exhibit protective action against cadmium toxicity in Saccharomyces cerevisiae cells.

Blueberries (Vaccinium corymbosum L.) are a rich source of antioxidants and their consumption is believed to contribute to food-related protection against oxidative stress. In the present study, the chemoprotective action of blueberry extracts against cadmium toxicity was investigated using a cadmium-hypersensitive strain of Saccharomyces cerevisiae. Four varieties of blueberries were used in the study, and it was found that the extracts with high content of total anthocyanidins exhibited significant protective effect against the toxicity of cadmium and H2O2. Both the blueberry extracts and pure cyanidin exhibited protective effects against cadmium in a dose-dependent manner, but without significantly interfering with the cadmium accumulation by the yeast cells. The results imply that the blueberry extracts might be a potentially valuable food supplement for individuals exposed to high cadmium.

Calcium signaling and copper toxicity in Saccharomyces cerevisiae cells.

To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca2+ in mediating the cell response to high Cu2+ was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu2+ was accompanied by elevations in cytosolic Ca2+ with patterns that were influenced not only by Cu2+ concentration but also by the oxidative state of the cell. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1, also activated by the Cch1-mediated Ca2+ influx. Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu2+ and H2O2 acted in synergy to induce Ca2+-mediated responses to external stress.

Heavy metal accumulation by Saccharomyces cerevisiae cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.

Optical manipulation of Saccharomyces cerevisiae cells reveals that green light protection against UV irradiation is favored by low Ca2+ and requires intact UPR pathway

Optical manipulation of Saccharomyces cerevisiae cells with high density green photons conferred protection against the deleterious effects of UV radiation. Combining chemical screening with UV irradiation of yeast cells, it was noted that the high density green photons relied on the presence of intact unfolded protein response (UPR) pathway to exert their protective effect and that the low Ca2+ conditions boosted the effect. UPR chemical inducers tunicamycin, dithiotreitol and calcium chelators augmented the green light effect in a synergic action against UV‐induced damage. Photo‐manipulation of cells was a critical factor since the maximum protection was achieved only when cells were pre‐exposed to green light.

Epigallocatechin-3-O-gallate, the main green tea component, is toxic to Saccharomyces cerevisiae cells lacking the Fet3/Ftr1

Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.

Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.