Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target.New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode  Nematode parasites are a major source of disease in both humans and livestock and are a significant crop pest. According to a 2014 report from the World Health Organization, over 1.5 billion people are infected with nematode parasites worldwide . While aMany anthelmintic drugs have been identified in whole organism screens of compound libraries using death, paralysis, or developmental arrest as endpoints –10. ThesAmong the criteria for an ideal anthelmintic drug target are: 1) that it be absent from host organisms, 2) that it is not the target of an existing compound and therefore not subject to cross-resistance with existing drugs, 3) that it belongs to a multi-gene family which would increase the likelihood of identifying a drug with multiple targets, thereby potentially increasing the efficacy and possibly slowing the evolution of resistance, and 4) that the target is essential to the life-cycle of the organism. The first three criteria can be evaluated using bioinformatics and available genome databases –22. Howein vivo [Genetic strategies have the potential to validate a target in vivo –26. If tC. elegans and comprises eight subunit genes: acc-1, acc-2, acc-3, acc-4, lgc-46, lgc-47, lgc-48, and lgc-49 [The problem of validating targets for which an agonist is required is particularly acute in the case of anthelmintics. Most available anthelmintics, such as levamisole, pyrantel, ivermectin, and monepantel, are agonists that activate members of the pentameric ligand-gated ion channel (pLGIC) superfamily . The supd lgc-49 . Featured lgc-49 . It remaHere, we take a genetic approach to the validation of the ACC channels as suitable anthelmintic drug targets. By analyzing the phenotypes of ACC knockouts, which should mimic the effects of ACC channel antagonists, we show that antagonists of these channels would likely not be sufficiently deleterious to the health or behavior of the worm to be suitable anthelmintics. By contrast, ivermectin-induced activation of ectopically expressed chloride channels (ivermectin receptors) in ACC-expressing tissues, which should mimic the effect of an ACC channel agonist, resulted in paralysis and developmental arrest. We therefore conclude ACC channels would be suitable targets for anthelmintic channel agonists. This strategy, which has been previously used to evaluate the behavioral effects of inhibiting neural circuits , has genXenopus laevis oocytes to express ion channels. Tricaine methanesulfonate (MS222) was used an anesthetic administered to the frogs for oocyte extraction. Frogs were monitored daily for two weeks for appetite as well as for any complications such as dehiscence or infection of the surgical site. All animal care protocols are in compliance with McGill's animal care Standard Operating Procedures; protocol number 2006–5284. All animal care procedures were approved by the McGill Facility Animal Care Committee (FACC).We have used C. elegans ACC protein sequences were used to query the NCBI database (http://blast.ncbi.nlm.nih.gov/) of nematode sequences. The orthologous sequences from parasites  was used as a starting point, which has an SL2 transplicing domain cloned into the multiple cloning site along with the AVR-15::YFP. This vector was then cut with BamHI and MscI (New England Biolabs) and the promoters for the ACC genes were cloned into the vector using the Infusion cloning system (Clontech).We used PCR to amplify regions of the ACC promoters from PCR-amplified GFP variants with Kpn I sites encoded in the primers were cloned into the Kpn I site of AVR-15 cDNA constructs in the pT7 vector . The vecin vitro synthesized RNA (50nL at 500ng/μl). Injections were performed using the Nanoject system  and injected oocytes were incubated at 15°C for approximately 40h before measurements were taken.Oocytes were harvested according to McGill Standard Operating Procedures for amphibian surgery. Oocytes were kept in standard ND96 solution unless otherwise noted and the solution was changed at least once a day while the oocytes were in culture. Oocytes were injected with 25ng of Unless otherwise indicated all Two-Electrode Voltage Clamp (TEVC) experiments were performed using a Maltese Cross chamber  as described in Purtrenko et al. Recordings were done using the AxoClamp 2B amplifier . Data were acquired at 1 kHz using the Clampex 8.1 software  and analyzed using the Clampfit 8.1 software . Hill curves were fit to the concentration-response data using the IGOR Pro 6.0.2.4 Software .acc-1 deletion strain carries the allele tm3268. This knockout was generated by the National BioResource Project , which is part of the International C. elegans Gene Knockout Consortium. The Δacc-2 strain VC1757 acc-2(ok2216) and the Δlgc-49 strain VC40013 lgc-49(gk246966) were made by the C. elegans Reverse Genetics Core Facility at the University of British Columbia, which is part of the international C. elegans Gene Knockout Consortium. The Δlgc-47 strain RB2187 lgc-47(ok296) was made by the C. elegans Gene Knockout Project at OMRF, which is part of the international C. elegans Gene Knockout Consortium. These strains were outcrossed at least seven times to wild type N2 worms and the respective deletions were confirmed using primers described in acc gene deletions, which we predict to be loss-of-functions alleles.A complete list of strains used is found in Pacc::AVR-15::YFP strains were made by injecting plasmids into JD369  worms. Constructs were microinjected into worms along with a transformation marker, rol-6, as described by Mello et al. [Pacc:: AVR-15::YFP strain and the extrachromosomal arrays were integrated into the genome by gamma-irradiation [All of the o et al. . All ACCadiation .Worm culture dishes were made from standard NGM media . Before C. elegans eggs were harvested by standard alkaline-bleach techniques [chniques  and the chniques .50, paralysis or survival rate for each acc promoter::AVR-15 fusion combination. Hill curves were fit to the IVM L1 escape data using the IGOR Pro 6.0.2.4 Software  to obtain EC50 values. The base and maximum values were manually set to 0 and 1, respectively. Statistical analysis of the various acc mutant phenotypes was done in SigmaPlot 12.0 (Systat Software Inc.). A t-test was used to compare N2 values to the mutant strain values.For each transgene, two transgenic strains representing independent genome integration events were tested , pharyngeal pumping (macrocyclic lactones) and egg-laying (macrocyclic lactones) [acc-1 but not acc-2 mutant worms showed slightly slower pharyngeal pumping rates, 244.8 ± 5.8 and 273.1 ± 7.4 pumps per minute respectively, when compared to N2 with 262.2 ± 4.6 pumps per minute  ,41. acc-r minute . acc-1 bh for N2 . acc-2 aed to N2 . None ofC. elegans, ivermectin is permeable to the C. elegans cuticle and AVR-15 belongs to the same pentameric receptor superfamily [Most existing anthelmintics that act on pLGICs are agonists . We therefore wanted to determine if agonists of ACC channels could be expected to exhibit toxicity to nematodes. We reasoned that an ACC agonist would have the same effect on the cells that express them as an agonist of the structurally similar glutamate-gated chloride channels (GluCls) ectopically expressed in those same cells . Ivermecerfamily  as the AXenopus oocytes, the homomeric channels formed by the tagged AVR-15 behaved electrophysiologically like the untagged AVR-15, demonstrating that the fluorescent protein tag does not affect channel assembly or overall function , which display resistance to ivermectin up to 50μg/mL. The resulting transgenic strains express AVR-15 exclusively in ACC-expressing tissues, as determined by the different ACC promoters. IVM exposure activates the AVR-15 chloride channels in tissues that endogenously express the ACC channels, thus mimicking the effects of a direct ACC agonist. Because JD369 worms lack the IVM targets and thus survive exposure to IVM, a return of IVM sensitivity in the Pacc::avr-15 strains indicates that the ACCs are expressed in critical tissues and could be good targets for new anthelmintics.To confirm that the AVR-15 GluCl channel is expressed under the control of the heterologous ACC promoters, we created an AVR-15 cDNA construct with a fluorescent protein tag in the intracellular loop. When expressed in function . We thenPacc-1, Pacc-2, Pacc-3, Plgc-47, Plgc-49, and Plgc-48, and injected them into JD369 worms. Expression of AVR-15::YFP in all strains appeared to be restricted to the nervous system. The YFP appeared to be localized to the plasma membrane, consistent with proper folding and trafficking of the AVR-15::YFP fusion protein. The LGC-48 ACC promoter (Plgc-48) drove expression only in two pairs of non-essential neurons, making it a suitable negative control for non-specific effects of the AVR-15::YFP transgene . The other strains had slightly higher recovery rates with 25.9 ± 5.1% of Pacc-2::AVR-15::YFP adults, 44.5 ± 7.5% of Pacc-3::AVR-15::YFP adults, 39.3 ± 4.0% of Plgc-49::AVR-15::YFP adults and 28.7 ± 10.4% of Plgc-48:: AVR-15::YFP adults recovering the ability to move after being left to recover overnight from 4h IVM exposure  after five days on IVM plates  after five-day exposure to 500ng/mL IVM, and they were severely paralyzed.Since successful treatment of nematode parasitic infections requires killing or clearing animals of various developmental stages, we were also interested in whether an ACC agonist would be likely to have adulticidal activity. To test this, we placed IVM-naïve adults of the M plates . Plgc-48acc gene family in C. elegans as appropriate targets for the development of new anthelmintics and believe this approach can be used to validate ion channel targets in any species that is amenable to transgenic manipulation. Our assay is an enabling technology that provides direct validation to support the development of novel ion channel-based drug screens.We developed a novel validation strategy for potential ion channel targets of anthelmintic drugs. We used the IVM-gated AVR-15 channel to validate five members of the in vivo effect. Genetic approaches to target validation have demonstrated their utility in systems as diverse as cholesterol metabolism [Target validation prior to undertaking high-throughput drug screens ensures that hit compounds will have the desired tabolism  and HIV tabolism . The undC. elegans to validate ligand-gated ion channels as suitable targets for novel anthelmintic agonists or antagonists. Most successful anthelmintics are channel agonists, and the genetic evidence suggests that antagonists of ivermectin receptors and monepantel receptors would not be efficacious anthelmintics. Ivermectin is an agonist/activator of GluCls, which are themselves, non-essential targets; avr-14, avr-15, glc-1, glc-3 quadruple mutants are insensitive to ivermectin, are viable and only display subtle behavioral phenotypes [C. elegans acr-23 knockout strains have no obvious phenotype [C. elegans and therefore an ACC antagonist is less likely to be an effective anti-parasitic drug. It is possible, however, that unlike the GluCls, the ACCs have redundant essential functions and that a drug that inactivated many ACC channels simultaneously would have deleterious effects on the worm.We were interested in developing a genetic strategy in enotypes ,46–53. Shenotype , while mhenotype  consistehenotype  and the henotype . PresumaHypermorphic or gain-of-function alleles can, in principle, reveal the effect of an agonist, however the technology for generating hypermorphs, specifically the ability to predict what mutations will be hypermorphic, is often inaccessible. In addition, a mutation present from birth raises issues of adaptation or developmental effects that can affect the interpretation of phenotypes, leading to the development of conditional mutations. We have ectopically expressed the IVM-activated chloride channel AVR-15 under control of ACC promoters and treated the worms with IVM, allowing precise temporal and spatial control of tissue inactivation. This approach was used to infer the effects of an ACC-agonist on a model nematode, and could be used to validate other ion channels as suitable targets of agonist drugs.C. elegans larvae. These ACC targets therefore have potential as larvicides for treatment of filariases and microfilariae. Treatment of infections with gastrointestinal nematodes requires expulsion of the adults from the gastrointestinal tract, which can be achieved by affecting the motility of the worms. We found that ivermectin-induced inhibition of tissues expressing ACC-1, or LGC-47, resulted in rapid and severe paralysis. Based on the AVR-15::YFP expression patterns, both of these subunits appear to be expressed in neurons innervating the ventral nerve cord, a key circuit in coordination of locomotion. Thus, we predict agonists of ACC-1 and/or LGC-47 would be efficacious against infections with gastrointestinal nematodes. It is also important to note that we validated individual ACC subunits. A drug that acted on many members of the structurally similar ACC family would likely exhibit greater efficacy and would be less susceptible to the evolution of receptor-mediated drug resistance, although ultimately, the efficacy of an ACC agonist would depend on the physiological roles of the tissues in which the ACCs are expressed.Our results indicate that that the ACCs are promising targets for anthelmintic discovery. Constitutive activation of a chloride channel in ACC-2-, ACC-3-, LGC-47- and LGC-49- expressing tissues was lethal or resulted in arrested or severely delayed development of Interestingly, the of potency of the AVR-15 expressing strains varied significantly, even though the potency might be expected to reflect the affinity of ivermectin for AVR-15, which should be the same in all strains. Instead, the variation in potency, as measured by whole organism phenotypes, may reflect the pharmacokinetics of ivermectin–internal tissues may experience different ivermectin concentrations at a given extra-cuticular ivermectin exposure. Similarly, the recovery from ivermectin treatment  may be aAdulticidal compounds that are not potent larvicides are also of interest for the control of filariases as part of mass drug administration programs. Ivermectin, which is currently the cornerstone of mass drug administration programs for the control of onchocerciasis, only kills microfilaria (larvae). A compound that would also kill adult parasites, which can live up to a decade, would obviate the need for repeat treatments to control symptoms caused by microfilaria. Our results indicate that an LGC-47 agonist would be the most potent adulticide, albeit after prolonged drug exposure. However, the efficacy of inhibition of larval growth was still much greater than the adulticidal activity, indicating that ACC agonists would likely be more effective larvicides than adulticdes.C. elegans as a model for parasitic nematodes is subject to several caveats. First, the target must be widely conserved among nematodes. The conservation of homologous genes among C. elegans and parasitic nematodes varies across the diverse phylum, ranging from 35% -70% [Trichinella spiralis). Second, the anatomy among nematodes should be conserved. Neural anatomy of parasites has not been sufficiently well-characterized to make a clear determination in this regard, although, Haemonchus contortus amphid neurons have been reconstructed by electron microscopy and appear to have close correspondence with C. elegans amphid neurons [Ascaris suum has been characterized electrophysiologically and is analogous to that of C. elegans [Haemonchus contortus and C. elegans, their expression in essential tissues and overall functions appear to be conserved [C. elegans and H. contortus both show inhibition of pharyngeal pumping at comparable, physiologically-relevant, ivermectin concentrations in vitro [C. elegans appears more sensitive to the locomotor effects [C. elegans and the filarial species to the effects of IVM on locomotion in microfilaria and adults as well as effects on larval development in vitro [C. elegans, but not adult filaria, it does so preventing pharyngeal pumping resulting in starvation [C. elegans males by IVM may reflect the conservation of GluCl expression in the male mating/locomotor circuit. Finally, to the extent that an anthelmintic exerts its antiparasitic effect by altering the interaction of the parasite with its host or with a symbiont, those mechanisms will not be reflected in the C. elegans model [The choice of 35% -70% . Importa neurons . Similar elegans –62. Finaonserved ,64. Morein vitro –67, alth effects . The siz effects ,47. Chanin vitro –72. Altharvation , a mechaarvation , 72, thens model ,71,73.C. elegans neurons are thought to be nearly isopotential, and have high enough input resistance, that a single ion channel is sufficient to change the membrane potential of a neuron [Plgc-48::avr-15::YFP strain allowed us to estimate an upper limit on the lethality attributable to general, low-level, leaky expression of the AVR-15::YFP construct, and we propose that the added lethality seen in our other Pacc::avr-15::YFP strains is exclusively due to inactivation of those ACC-expressing tissues. Overexpression of the transgenes, and the associated potentially negative consequences, could be addressed by creating strains with targeted, single-copy insertions of the transgenes, especially now that the technology for such fine-tuned genetic manipulation has become available in many different species [The use of the AVR-15 channel to mimic activation of an endogenous chloride channel also has associated caveats. For instance, the transgenic strains we used contain multiple copies of the transgene , and therefore the transgene is typically overexpressed, possibly resulting in an overestimate of the severity of the phenotype when compared to an agonist of the endogenous channel. However, a neuron . Thus, e species –80.In order to use IVM in our assay, we had to perform all the experiments in an IVM-resistant quadruple GluCl mutant background (strain JD369). As previously mentioned, JD369 worms are viable, fertile and only have very subtle phenotypes. Despite this fact, the IVM resistant background necessary for these experiments may complicate the translation of this technique for validation of chloride channel targets in other species with endogenous IVM targets. While these factors will tend to result in less conservative estimates of target sensitivity to agonists, we are reassured by the similarity of the ACC neuronal expression patterns to endogenous anthelmintic targets such as AVR-14, another pLGIC subunit targeted by IVM .There remain many uncharacterized pLGICs in nematodes and, along with the subunits that have been characterized but are not currently targets of anthelmintics, it is clear that many novel nematode pLGIC subunits could be validated as suitable drug targets. In principle the technique that we presented here to validate the ACCs could be used to validate agonists of other chloride-selective ion channels in any organism or strain amenable to creation of transgenics and that is not otherwise ivermectin sensitive. The validation strategy we describe expands opportunities to employ target-selective screening of ion channels.S1 FigΔacc-2), vc40013 (Δlgc-49), rb2187 (Δlgc-47), and tm3268 (Δacc-1).Depiction of the deleted genomic regions in the strains vc1757 ((DOCX)Click here for additional data file.S2 Figacc-2, acc-3, lgc-47, and lgc-49-promoters fail to develop on 50ng/mL IVM. Worms with AVR-15::YFP expressed under control of lgc-48-promoter develop to adulthood, while worms with AVR-15::YFP expressed exclusively in ACC-1-expressing tissues exhibit delayed growth on 50ng/mL IVM, but do reach adulthood.Worms with AVR-15::YFP expressed under control of (DOCX)Click here for additional data file.S1 TableTable listing all genes used in constructing the ACC phylogeny . Include(DOCX)Click here for additional data file.S2 TableTable describing all primers used to clone all the acc promoters used in the acc::AVR-15 constructs. Gene-specific sequence in lowerase, vector-specific sequence in uppercase.(DOCX)Click here for additional data file.S3 TableΔacc-2), vc40013 (Δlgc-49), rb2187 (Δlgc-47), and tm3268 (Δacc-1).Primers used to screen for deletions in strains vc1757 ((DOCX)Click here for additional data file.S4 TableTable listing all the worm strains used, including a list of the alleles each strain is carrying.(DOCX)Click here for additional data file.


Data collected from 1288 patients in The Finnish Cardiovascular Study (FINCAVAS), who satisfied the inclusion criteria, was used for the analyses. HRV parameters  were calculated from 2-min segment in the rest phase before exercise and 2-min recovery period immediately after peak exercise. Area under the receiver operating characteristic curve (AUC) was used to determine the predictive performance for each parameter with and without HR corrections in rest and recovery phases. The division of HRV parameters by segment's RRavg to the power 2 (HRVDIV-2) showed the highest predictive performance under the rest phase  with minimum correlation to HR (r = −0.15 to 0.15). In the recovery phase, Kaplan-Meier (KM) survival analysis revealed good risk stratification capacity at HRVDIV-2 in both groups . Although higher powers of correction (HRVDIV-4and HRVDIV-8) improved predictive performance during recovery, they induced an increased positive correlation to HR. Thus, we inferred that predictive capacity of HRV during rest and recovery is augmented when its dependence on HR is weakened by applying appropriate correction procedures.The non-linear inverse relationship between RR-intervals and heart rate (HR) contributes significantly to the heart rate variability (HRV) parameters and their performance in mortality prediction. To determine the level of influence HR exerts over HRV parameters' prognostic power, we studied the predictive performance for different HR levels by applying eight correction procedures, multiplying or dividing HRV parameters by the mean RR-interval (RR Heart rate (HR) recovery and heart rate variability (HRV) have been used by researchers for assessing the role of autonomic regulation in predicting all-cause and cardiovascular mortality , descriptive information, medical history and habitual lifestyle of each patient were recorded. More detailed information regarding the patient population and sample size determination is described elsewhere  were calculated from each segment individually for the HR correction procedure.ECG was recorded at a sampling frequency of 500 Hz using CardioSoft exercise ECG system  and was analyzed using Modified CASE software . After producing the RR-interval tachogram, the data was preprocessed to remove abnormal intervals and artifacts before they were divided into shorter segments based on the stages of rest and recovery. HRV parameters were determined using the Kubios HRV analysis software  power has been found to increase during exercise in normal subjects and reflects both sympathetic and vagal influences  curve. Spearman's rank correlation was performed to determine the degree of correspondence to HR. The cut-off points for Kaplan-Meier (KM) survival analyses were defined from the ROC analyses for each segment. The point of highest overall predictive performance (average of sensitivity and specificity) was chosen as the cut-off to distinguish mortality and survival groups based on HRV observed in the patient population. It has to be noted that these cut-off points were not optimized in order to preserve uniformity during comparisons. The Log-rank chi-square estimates were then used to evaluate the significance of the correction methods based on this classification.During the follow-up of the patients who satisfied the inclusion criteria, 66 cardiac deaths were recorded, which included 31 sudden cardiac deaths, with a mean follow-up time of 54 months . 94 patients died of non-cardiovascular causes between 1.2 and 110.7 months of follow-up (mean: 60.2 months). The baseline characteristics, clinical conditions and medications used by patients who suffered cardiac and non-cardiac deaths are listed in Table RR = 2.27, 95% confidence interval (CI) = 1.26–4.09, p < 0.05]. Age ≥ 60 years was a risk factor for cardiac  and non-cardiac  mortality. Clinical conditions were significantly associated with risk of cardiac death. Medication such as ACE inhibitors  and beta blockers  were significantly associated with increased risk of cardiac mortality (p < 0.001).The univariate Cox regression results for various factors associated with cardiac and non-cardiac mortality are presented in Table r, presented in Figure DIV-2, which revealed minimum correlation to HR, was the best predictor for both outcomes  in the rest phase. However, during recovery, higher standard HRV  was associated with worse prognosis (AUC < 0.5), as seen in Figure avg . Conversely, after division by higher powers of RRavg , higher HRV was associated with better prognosis (AUC > 0.5). Though higher orders of correction resulted in better predictive capacity, it also induced moderate/strong positive correlation to HR .The area under the ROC curve (AUC) for HR was found to be 0.57/0.70 (rest/recovery) for cardiac mortality and 0.53/0.64 for non-cardiac mortality, implying that HR is a better predictor during recovery than during rest phase. The AUC for RMSSD, VLF and LF power, calculated under different correction methods during rest and recovery phases are presented in Figures MUL-4 and HRVDIV-16 were excluded due to their very strong correlation to HR. Mortality prediction was most significant for HRVDIV-2 in the rest phase. During recovery, the division of HRV by higher powers of RRavg resulted in better risk stratification for cardiac and non-cardiac deaths. Although HRVDIV-4 and HRVDIV-8 exhibited better predictive powers during recovery, the HRV indices exhibited strong positive correlation to HR  and non-cardiac (p < 0.05) mortality during recovery, with minimum influence of HR (r = −0.15 to 0.15). Figures DIV-2 during rest and recovery.These results were further corroborated by KM survival analysis. Log-rank estimates at different degrees of correction for both cardiac and non-cardiac mortality are presented in Table DIV-2, r = −0.15 to 0.15), suggesting that exclusion of HR influence on resting HRV improved prognostic capacity for cardiac and non-cardiac mortality. Since HR is a poor predictor at rest, removal of HR influence perchance resulted in improved prognostic capacity. On the contrary, HR during recovery phase exhibited significant risk stratification for both outcomes. Thus, increasing HRV's dependence on HR enhanced its predictive capacity (observed in HRVDIV-4 and HRVDIV-8). However, higher degrees of correction produced moderate/strong positive correlation to HR, similar to observations made by Sacha et al. (DIV-2 demonstrated improvement in predictability of mortality risk during recovery phase with minimum dependence on HR.The HRV indices computed from RR-interval measurements correlated with HR as a result of the non-linear relationship between the RR-interval and instantaneous HR (Chiu et al., a et al. , Sacha (a et al. . To attaHowever, conclusive evidence could not be established to distinguish between cardiac and non-cardiac related deaths. This is in contrast to findings by Sacha et al. , who sugThis study suffered certain limitations. First, the risk factors for individual, clinical conditions and medication were not modeled to determine their contribution toward mortality prediction. By including these variables to the analyses, a more definite conclusion on the cause of mortality could have been established. Second, the patients were not controlled for the type of medication prescribed. The effects of beta blockers and nitrates have been known to affect HR, which could have an effect on the results of HR correction. However, the purpose of the current study was to evaluate the effects of HR correction methods in mortality prediction and therefore, these issues need to be considered in future studies.The findings of this study indicate that the predictive power of HRV parameters for both cardiac and non-cardiac mortality is augmented when its dependence on HR is weakened during rest and recovery. In addition, when HR is a good predictor, increasing HRV's dependence on HR further enhances the risk stratification for both modes of death.The study was conceptualized by Tuomo Nieminen, Kjell Nikus, Terho Lehtimäki, Mika Kähönen, and Jari Viik. Data acquisition and analysis was performed by Paruthi Pradhapan, Mika P. Tarvainen, Rami Lehtinen, and Jari Viik. All authors contributed equally in drafting and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Small- and medium-sized enterprises (SMEs) generate nearly 80% of the jobs in China, but the dangerous work environment often found in these enterprises poses a major concern for public health. Psychosocial pressure and mental health problems among the workers are also common in SMEs. However, mental health of workers in SMEs is largely neglected in occupational health research and practice in China. The purpose of this study is to assess mental health of the workers and to explore the associations between physical and psychosocial work environment and workers’ mental health in SMEs in South China.Data were collected in 2012 through a cross-sectional survey among 1200 workers working in small- and medium-sized enterprises (SMEs) in Guangdong, China. Mental health was measured by psychological well-being in the current study. Job Demand-Control-Support (JDCS) model was used as a theoretical framework to examine the psychosocial factors associated with workers’ psychological well-being. Data were analyzed using SPSS 20.0 and analysis was performed using bivariate analyses and multivariate logistic regression.About three in ten workers (35.3%) in the sample had poor psychological well-being. Those who were men, younger in age, or migrant workers had worse psychological outcome in bivariate analyses. After controlling for individual variables , we found that longer weekly work hours , more exposure to hazardous work environment , higher job demands , and lower job autonomy  were significant associated with worse psychological well-being. The results were consistent with predictions of the JDCS model.The results indicate that the JDCS model is a useful framework in predicting psychological well-being among Chinese workers in SMEs. Future mental health promotion should focus on young migrant male workers as they appear to be most vulnerable in their psychological well-being. Both physical and psychosocial aspects of the work environment should be taken into account in policy making to prevent mental disorder and promote psychological well-being among workers in SMEs. China has experienced dramatic industrialization, urbanization, and economic growth over the last three decades. Small- and medium-sized enterprises (SMEs) play a crucial role in the Chinese economy, providing about 80% of the jobs in urban China . CompareIt is estimated that the burden of mental illness will account for 1/4 of the total disease burden in 2020 globally . Mental Working conditions are key to understanding psychological well-being of workers who are frequently exposed to hazardous environment. Both physical and psychosocial work environment are important. According to previous studies, shift work, long work hours, hazardous work environment  might be potential risk factors for poor mental health of the Chinese workers –17. ReseOver the past several decades, one of the most influential models in examining the relationship between work environment and mental health is the Job Demand-Control-Support (JDCS) model. The JDCS model identifies three critical aspects at work: job demands, job autonomy and worksite support . A recenThe purposes of the current study are to assess the mental health status of the SMEs workers, and to explore the associated work-related factors  with the mental health status of the SMEs workers in South China.This cross-sectional study was conducted between September and October in 2012 in Guangdong Province, China. A two-stage stratified random sampling method was carried out. First, two developed cities (Guangzhou and Foshan) and two less-developed cities (Zhaoqing and Qingyuan) in Guangdong Province were selected. Second, based on the final sample size requirement (see next section for detail), we randomly selected SMEs in shoe making, electronics, and plastics industries from each city by applying computer-generated random numbers from the list of enterprises in these cities. The reason to choose industries in shoe making, electronics, and plastics is that workers in these industries were exposed to the same occupational hazard, organic solvent, which causes about 50% of all cases related to occupational diseases in Guangdong Province . The couAccording to literature review, the rate of depression among occupational group was 50% in China . AssuminThe recruitment criteria for workers included: 1) front-line workers in assembly lines who were exposed to occupational hazards ; 2) workers who had resided in the study area for at least six months. Exclusion criteria included team leaders or management personnel who differ in their work environmental exposure . WorkersThe study was approved by the Institutional Review Board (IRB) of the School of Public Health, Sun Yat-sen University in China. Written informed consents were obtained from all study participants.We employed psychological well-being to examine mental health status of workers in SMEs in China. The World Health Organization (WHO) (five) Well-Being Index, 1998 version (WHO-5)  was desiWHO-5 is a uni-dimensional measure that asks the feelings of participants in the previous two weeks and contains five positively-worded items: "I have been cheerful and in good spirits"; "I have been calm and relaxed"; "I have been active and vigorous"; "When I wake up, I feel fleshed and rested"; and "My daily life is filled with things that interest me". The options of the answers regarding their feelings are on a 6-point likert scale ranging from 0  to 5 (constantly present). The possible total scores vary from 0 to 25, with higher scores indicating better well-being. Scores of 13 and below indicate poor well-being and warrant follow-up diagnostic procedures , 29, 30.Physical work conditions were measured by whether workers had employment contract (yes/no) and shift work schedule (yes/no), how many work hours per week, and physical work environment which was assessed by two questions:" How often are you exposed to disturbing noise?" "How often are you exposed to excessive toxic chemicals?" Responses for the two exposure questions were on a five-point scale ranging from 1 to 5 .According to the Job Demand-Control-Support (JDCS) model , and ChiParticipants also provided individual characteristics on gender, age, educational attainment , individual monthly income, household registration (permanent residents or migrant workers).A database was constructed using Epidata 3.0 and statistical analyses were performed using SPSS version 20.0. First, as part of a test of reliability, internal consistency was examined using Cronbach’s alpha. WHO (five) Well-Being Index, extent of exposure to hazardous environment, job demands, job autonomy, and worksite support demonstrated a reliability alpha coefficient of 0.91, 0.70, 0.70, 0.73, 0.61, respectively, suggesting that all of the measures had acceptable to satisfactory internal reliability .P < 0.05. Odds ratios (ORs) with 95% confidence interval (CI) were presented. Two-sided tests of significance and confidence intervals were based on the 0.05 level.The WHO (five) Well Being Index scale scores were calculated according to the established algorithms with higher scores indicating better mental health status. Well-being scores were categorized into two categories (1 ~ 13/14 ~ 25). The distribution of individual characteristics and work-related factors were illustrated by numbers and percentages, means and standard deviations as appropriate. Bivariate analyses were employed to analyze the associations between factors and mental health. Multivariate logistic regressions were used to simultaneously identify work-related variables associated with mental health, while adjusting for individual characters significant in bivariate analyses at Table Summaries of the bivariate analyses are presented in Table P < 0.05, longer weekly work hours , more exposure to hazardous work environment  were significantly and negatively associated with poor psychological well-being. Higher job demands , and lower job autonomy  were significantly and negatively associated with poor psychological well-being. The results were consistent with the JDCS model as the model predicts psychosocial factors associated with mental health status of workers in SMEs [Table  in SMEs .Table 4The current study intended to assess mental health of workers in SMEs in Guangdong, China and to explore the related physical and psychosocial factors of the work environment. 35.3% of the workers were found poor in their psychological well-being status. Those who were men, younger in age, or migrant workers were more likely to have worse psychological well-being. Our study found significant associations between physical work-related factors  and mental health. In addition, we also found significant associations between psychosocial work factors  and mental health among workers in SMEs, guided by the Job Demand-Control-Support (JDCS) model .Results of the current study are consistent with the predictions of the JDCS model . Such model has been widely applied to mental health and psychological well-being research , 35. TheEven though the New Labor Law was passed in 2008 in China that regulated no more than 40 hours a week among workers, such regulation was not strictly enforced. In the current study, the average weekly work hours were 55.4 hours. Previous study suggests a negative association between work hours and workers’ mental status, with odds ratio being 2-4 for depression for those working more than 40 hours per week . In our Workers’ psychosocial well-being is not only associated with physical work environment , but also with psychosocial work environment. In the present study, high job demands and low job autonomy were associated with poor psychological well-being, which is consistent with the prediction of the Demand-Control model (JDC) and other studies , 39. ThePrevious study found that the depressive rates of the Chinese working population in mild, moderate, and severe status were 25.6%, 23.5%, and 1.52%, respectively . Our stuThere are several limitations of this study that should be acknowledged. First, the cross-sectional research design in the current study does not allow causal analyses, only associations. It is plausible that poor mental health may have preceded hazardous work environment . Associations between physical and psychosocial work-related factors and poor mental health should be further explored in longitudinal studies among SMEs workers in China. Second, we didn’t have objective measurements of work environment. All measurements are self-report which may subject to recall bias or socially desired preferences. Third, a wide range of factors may be related to mental health of workers, our study mainly included work-related factors and individual social demographic factors whereas other important factors  that may potentially influence mental health are not included. Fourth, we used a few key indicators  to determine work-related factors based on the Job Demand-Control-Support (JDCS) model, and Chinese Version of Job Content Questionnaire. Even though all the Cronbach’s alpha of work-related factors is above 0.6 in our study, the validity of using such scale still needs to be further confirmed.Our study is one of the first to assess psychological well-being and examine the associated work environment factors among workers in SMEs in China. The results indicate that the JDCS model is a useful framework in examining psychological well-being among Chinese workers in SMEs. The primary policy implication is that young migrant male workers are a key population for mental health promotion. Both physical and psychosocial work environment should be taken into account in policy making to prevent mental illness and promote psychological well-being among workers in SMEs in China.Zhi Zeng and Yan Guo: co-first authors.


Studies with greater reduction in UFP levels and larger sample sizes are needed. There also needs to be more complete assessment of resident time activity and of outdoor vs. indoor source contributions to UFP exposure. HEPA filtration remains a promising, but not fully realized intervention.Exposure to traffic-generated ultrafine particles  is likely a risk factor for cardiovascular disease. We conducted a trial of high-efficiency particulate arrestance (HEPA) filtration in public housing near a highway. Twenty residents in 19 apartments living <200 m from the highway participated in a randomized, double-blind crossover trial. A HEPA filter unit and a particle counter (measuring particle number concentration (PNC), a proxy for UFP) were installed in living rooms. Participants were exposed to filtered air for 21 days and unfiltered air for 21 days. Blood samples were collected and blood pressure measured at days 0, 21 and 42 after a 12-hour fasting period. Plasma was analyzed for high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), tumor necrosis factor alpha-receptor II (TNF-RII) and fibrinogen. PNC reductions ranging from 21% to 68% were recorded in 15 of the apartments. We observed no significant differences in blood pressure or three of the four biomarkers  measured in participants after 21-day exposure to HEPA-filtered air compared to measurements after 21-day exposure to sham-filtered air. In contrast, IL-6 concentrations were significantly higher following HEPA filtration  compared to sham filtration. Likewise, PNC adjusted for time activity were associated with increasing IL-6 in 14- and 21-day moving averages, and PNC was associated with decreasing blood pressure in Lags 0, 1 and 2, and in a 3-day moving average. These negative associations were unexpected and could be due to a combination of factors including exposure misclassification, unsuccessful randomization ( Thus2.5 and PM10. However, the sham/HEPA comparison would test for effects from all PM filtration, not only the PNC we measured. We also lacked outdoor measurements of air pollution, but we were able to discern spikes of indoor sources that were probably from cooking and estimate that they contributed only a small (median < 5%) amount to total PNC. Our study had a number of limitations. Our sample size was small, limiting statistical power to see associations. We had a study population that was relatively heterogeneous with a range of co-morbidities and levels of BMI, including two cases of extreme obesity. If these factors are associated with sensitivity or resistance to the effects of UFP, then it is possible that our effective sample size was smaller than 20. In addition, we did not include a washout period in the study design, our time-activity adjustments may have led to underestimation or overestimation of some exposures, and our randomization was not ideal for IL-6 at baseline (higher for sham-first group) or anti-inflammatory use (more frequent for HEPA-first group). Our air pollution data also had limitations. It was restricted to one pollutant, PNC, while the HEPA units would be expected to filter larger particles as well, including PMWe developed, implemented and evaluated a small-scale trial of an intervention designed to reduce traffic-related PNC in public housing near a highway. Reductions in PNC were smaller than we had hoped in most apartments and were actually the same or higher than during sham filtration in some apartments. We found no evidence that the filtration improved blood pressure or blood markers of inflammation or coagulation. However, given the broad evidence for PM affecting cardiovascular health, our small sample size, and the lessons learned from this study, we suggest there is a need for further research that seeks to find effective interventions for near-highway residents. Key elements to future interventions include supplying HEPA filtration in multiple rooms, a larger sample size, and finer grain tracking of resident time-activity patterns.


Perfusion abnormalities are frequently seen in Single Photon Emission Computed Tomography (SPECT) when a left bundle branch block is present. A few studies have shown decreased coronary flow reserve in the left anterior descending territory, regardless of the presence of coronary artery disease.82Rb) positron emission tomography imaging in the assessment of myocardial blood flow and coronary flow reserve in patients with left bundle branch block.We sought to investigate rubidium-82 (82Rb-positron emission tomography with absolute quantitative flow measurements using Cedars-Sinai software (mL/min/g). The relative myocardial perfusion and left ventricular ejection fraction were assessed in 17 segments. These parameters were compared with those obtained from 30 patients with normal 82Rb-positron emission tomography studies and without left bundle branch block (GII).Thirty-eight patients with left bundle branch block (GI), median age 63.5 years, 22 (58%) female, 12 with coronary artery disease  and 26 with no evidence of significant coronary artery disease (GI-B), underwent rest-dipyridamole stress p<0.05). The comparison of coronary flow reserve between GI-A and GI-B showed that it was different from the global coronary flow reserve (p<0.05) and the stress flow was significantly lower in the anterior than in the septal wall for both groups. Perfusion abnormalities were more prevalent in GI-A (p=0.06) and the left ventricular ejection fraction was not different between GI-A and GI-B, whereas it was lower in GI than in GII (p<0.001).Stress myocardial blood flow and coronary flow reserve were significantly lower in GI than in GII (82Rb-positron emission tomography imaging may be useful in identifying coronary artery disease in patients with left bundle branch block.The data confirm that patients with left bundle branch block had decreased myocardial blood flow and coronary flow reserve and coronary flow reserve assessed by  Noninvasive diagnosis of coronary artery disease (CAD) in left bundle branch block (LBBB) patients is a clinical and methodological dilemma because septal perfusion abnormalities have been demonstrated by myocardial perfusion scintigraphy (gated-SPECT) even in the absence of CAD 82Rb) positron emission tomography (PET) stress myocardial perfusion has emerged in the clinical setting as a noninvasive imaging method for diagnosis and risk-stratification that has several advantages compared to Single Photon Emission Computed Tomography (SPECT). In addition to assessing myocardial perfusion, wall motion and left ventricular function, 82Rb-PET can quantify global and regional myocardial blood flow (MBF) during both rest and stress, and can also measure CFR, which is important for the clinical management of patients with CAD Rubidium-82 , 38 were selected (Group I - GI) who met the inclusion criteria: LBBB and sinus rhythm. The median age was 63.5 years, range 58-68 years and 22 were women (58%). Patients with unstable angina, recent myocardial infarction, pacemaker rhythm and atrial fibrillation or flutter were excluded from GI. In addition, we selected 30 patients without LBBB (Group II - GII), who were free from known CAD and had normal 82Rb-PET studies , and matched them based on the same clinical risk factors that are associated with reduced absolute quantitative flow measurements From February to October 2013, 665 consecutive patients, who were referred for dipyridamole-stress gated-SPECT for the evaluation of known or suspected CAD, underwent 82Rb-PET perfusion study, patients were categorized into a subgroup with LBBB and no CAD .In a subset of patients, a request to perform a coronary anatomy assessment was made by the patient's doctor. Therefore, a cardiac catheterization or coronary computed tomography angiography (CTA) The patients were instructed to fast for 4 h, abstain from caffeine and cigarettes for 24 h and theophyllines for 36 h, and discontinue beta-blocker or calcium-channel blocker medications for 3 days and long-acting nitrates for 6 hours before the study.82Rb  over a 60-second interval.Cardiac PET was performed using a Gemini-TOF 64-slice system . Resting images were acquired using a 3-D list mode acquisition over 8 min, after a square wave intravenous injection of 82Rb activity was administered four minutes later. Stress images were acquired and processed for the resting scan. The symptoms were treated with intravenous aminophylline after the end of image acquisition.Dipyridamole was infused intravenously (0.56 mg/kg) over four minutes and the same 82Rb-PET image acquisitions with two low-dose CT-based attenuation corrections for PET transmission scans were performed , after a normal end-expiration. The estimated effective radiation dose from the complete PET study (rest/dipyridamole 82Rb and 2 CT attenuation corrections) was 3.0 mSv Before rest and after stress, Fused CT and emission images were visually evaluated for alignment by an experienced technologist and, if necessary, corrected by manual 3-D translation. The images were reconstructed using a 3-dimensional row-action maximum likelihood algorithm (3-D-RAMLA), with 3 iterations/33 subsets and a medium filter. The entire 8 min of emission data was binned to form a dynamic image sequence  for MBF quantification and the last 6 min to form myocardial uptake and ECG-gated images. The images were semi-automatically reoriented to generate short-axis and vertical long-axis slices. The left ventricle ejection fraction (LVEF) was calculated from the rest and stress images using 4D QGS The studies were processed in batch mode using QPET  to quantify MBF and CFR. In brief, left ventricle (LV) contours were positioned automatically with an algorithm that determines the LV contours from the summed dynamic images data. The 3-D cylindrical region  for the LV input function was automatically placed in the middle of the valve plane and oriented along the long axis of the heart. Dynamic myocardial samples were obtained from the polar map by analyzing all of the time frames within the fixed LV contour boundaries 1K [mL/min/g of myocardial tissue] and 2k [min−1]), blood-to-myocardial spillover fraction and myocardial partial-volume corrections. A previously calibrated 82Rb extraction fraction was used to estimate MBF from 1KA standard 1-tissue compartment model was used to quantify MBF, which includes regional uptake and clearance parameters , according to the standard 17 segments of the American Heart Association model Rest and stress uptake images were visually analyzed using a 5-point score  for relative myocardial perfusion in 17 segments t or Mann-Whitney tests conditioned by the normality distribution assumption, which was checked using the Anderson-Darling test and the assumption of homogeneity of variances, which was established by the Levene test. In turn, discrepancies among GI-A, GI-B and GII were verified by ANOVA or Kruskal-Wallis tests, supported by the assumptions of normality and homogeneity of variances. Next, multiple comparisons relative to GI-A vs. GI-B and GI-B vs. GII were performed using the parametric or nonparametric Dunnett test Quantitative variables were expressed in medians (25%-75% quartile), while qualitative variables were expressed in percentages. Differences in the quantitative variables between GI and GII were verified by R package, version 3.1.1. Comparisons of the anterior or septal wall with the other walls were evaluated using the paired nonparametric Dunnett test All of the procedures that were performed in studies that involved human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.82Rb-PET perfusion. The median ages were not significantly different . The female gender was predominant in all groups. Furthermore, the main clinical risk factors, such as hypertension, dyslipidemia and diabetes, were also not significantly different between the groups.The patient demographics are summarized in p<0.001), whereas the rest flow was lower only in the septal and apical walls . A comparison of the anterior or septal walls with the other walls in GI revealed that the rest flow, stress flow and CFR were significantly lower in the anterior wall and less so in the septal wall. However, the rest and stress flows were also significantly lower in the septal than in the inferior wall (p=0.05) and could identify CAD in LBBB . We found statistically significant differences between the groups when we considered perfusion abnormalities in the anterior and/or septal walls (p=0.04) as well as in the inferior and/or lateral walls (p=0.003) (Twenty-four patients in GI (63%) had no significant perfusion abnormalities and all patients in GII had normal perfusion. Moreover, the perfusion abnormalities (SSS ≥4 and/or SDS ≥2) tended to be more prevalent in the presence of CAD (GI-A p=0.003) .p<0.001), but no significant differences between GI-A and GI-B (p=ns). However, GI-A had a high prevalence of LVEF dysfunction (<45%) only during stress (p=0.02).LVEF values and LV cavity volumes are summarized in 82Rb-PET, beyond relative myocardial perfusion image interpretation, has already been established. Worse patient outcomes and a higher incidence of cardiac events  have been demonstrated in cases with global CFR <2 mL/min/g in patients with normal and abnormal relative myocardial perfusion An evolution in cardiovascular imaging with PET-CT systems being incorporated into the clinical setting has been occurring since the early 2000s. This trend is partially driven by the fact that cardiac PET-CT offers a noninvasive method to assess relative myocardial perfusion, LV function, MBF and calcium score (15). The importance and independent prognostic values of MBF and CFR using 15O-water PET and the study results showed that the septal/lateral MBF ratio was 19% lower than that in the controls, as a result of functional alterations. Our study assessed LBBB patients and showed that stress MBF as well as CFR were significantly lower in all of the walls in a larger LBBB population compared to the controls. We were careful to compare 2 homogeneous populations that were matched for the presence of primary clinical risk factors, which are associated with reduced stress flow and CFR 82Rb-PET that could aid in the identification of CAD and risk-stratification of LBBB patients.Despite the low prevalence of LBBB in the general population Our results suggest that resting MBF in the inferior wall may not be influenced by the presence of LBBB . In GI-B patients, stress and rest flow were also lower in the anterior than in the septal and inferior walls. It appears that LBBB did not interfere with the evaluation of MBF and CFR in the inferior wall or the abnormal activation of the LV because LBBB is more proximal (anterior and high septum areas), sparing the inferior or even lateral walls, which are opposite and more distal. In the presence of LBBB (both with and without CAD), stress MBF was also lower in the anterior than in the septal wall. The septal wall showed a smaller MBF impairment than that in the anterior wall, which could be explained by the fact that the septum is a less extensive area compared to others and thus, MBF may have a lower expression or be underestimated in a quantitative evaluation.82Rb-PET relative myocardial perfusion could discriminate between LBBB patients with and without CAD, even when considering the LAD territory alone. Perfusion abnormalities were more prevalent in the presence of CAD. Although anterior and septal abnormalities have been described in most similar studies in the absence of CAD, perfusion abnormalities may also result from technical imaging issues , functional ischemia, CFR alterations caused by the LV dynamic asynchrony, or because of subclinical myocardial disease Our study also suggests that Rest and stress LVEFs in LBBB patients were also lower than those in GII. As expected, GI-B had less LV dysfunction than GI-A at stress and it was associated with CAD. Furthermore, GI-A on average had a lower LVEF response to stress compared to GI-B. Nevertheless, even in the absence of established CAD, patients with LBBB demonstrated a decreased LV reserve and might not respond appropriately to stress The mechanisms that underlie the reduced MBF and CFR in nonischemic cardiomyopathy can include endothelial dysfunction, macro- and microvascular obstruction, vascular remodeling and extravascular compressive forces 82Rb-PET is that it could provide additional information for the assessment of CAD in LBBB patients.Thus, the presence of CAD had a profound effect on CFR and also on traditional relative myocardial perfusion and LV function measures, with a higher prevalence of perfusion and LVEF abnormalities in GI-A. There was a detrimental influence of LBBB over ventricular dynamics, independent of the presence of CAD. These functional abnormalities may be associated with abnormal MBF, perfusion, and function. According to our data, an advantage of using 82Rb-PET has several advantages compared with SPECT studies, one disadvantage is the inability to perform 82Rb-PET in association with exercise stress tests, due to the short half-life of the tracer. However, in LBBB, pharmacological stress is preferable to exercise perfusion imaging for both diagnosis and risk stratification Our study has some limitations. Despite the fact that 82Rb-PET in these patients were expected even in the absence of CAD This was a single-center observational study that was performed with a select population of LBBB patients. Thus, its results may not be applicable to all such patients because the great diversity of clinical situations in which LBBB can be present may yield different results. Moreover, cardiac catheterization or CTA were not available in all LBBB patients. Usually, the absence of relative perfusion defects in PET-CT studies is associated with non-significant CAD 82Rb-PET have decreased MBF and CFR in all LV territories. Moreover, these measures are important diagnostic tools for patients with LBBB and suspected CAD, which might be identified with a CFR evaluation.In the present study, we showed that LBBB patients who were assessed by


Biological and medical systems are predominantly irregular, complex and non-linear, since cannot be quantified by classical geometry approach. Novel mathematical algorithms can expand the information content of medical images, providing an objective measurement to reduce subjectivity in the perception and interpretation ,2. The aFractal Dimension (FD) is a statistical quantity that gives an indication of how completely a fractal appears to fill space, zooming down to more finer scales. We proposed a method of medical images analysis obtained from a wide range of sources - radiology imaging. The fractal parameters of these images were calculated for 7 patients with liver lesions for ultrasound, CT, MRI images and generated 3D vector and voxel models by patented method, "covering" the parts of these expertly segmented images by two-dimensional geometric shapes  and three-dimensional  with applying iteration method, which involves finding the appropriate (i-th) value approaching the value of FD.FD was estimated as 1.67 for hepatocellular carcinoma case; 1.72 - for cholangiocarcinoma; 1.45-1.56 for complex cysts; and 1.15-1.35 for metastases. We consider that only three-dimensional reconstruction from expertly segmented images allows to perform accurate analysis. The most informative description of self-similarity is fractal analysis, conducted with the maximum number of steps. However, objective analysis is limited by resolution of diagnostic equipment, is possible only under visual control by expert. The application of automated and semi-automated image analysis leads to control the process, correctly selecting the areas for research, preselecting a suppositive fractal structure.Fractal analysis of medical images is a promising non-invasive sophisticated approach, it should become an highly informative indicator of pathological formations using nonlinear mathematical parameters of structure, gives insights into tumor morphology and can become a useful tool for analyzing tumor growth patterns for diagnosis, staging and treatment follow up.Further studies on large patient cohorts are recommended to assess different pathological processes to establish scientifically valid standards.


JAGuaR is an alignment protocol for RNA-seq reads that uses an extended reference to increase alignment sensitivity. It uses BWA to align reads to the genome and reference transcript models (including annotated exon-exon junctions) specifically allowing for the possibility of a single read spanning multiple exons. Reads aligned to the transcript models are then re-mapped on to genomic coordinates, transforming alignments that span multiple exons into large-gapped alignments on the genome. While JAGuaR does not detect novel junctions, we demonstrate how JAGuaR generates fast and accurate transcriptome alignments, which allows for both sensitive and specific SNV calling. Deep sequencing of transcriptomes on high throughput sequencing platforms, also called RNA-seq, is an effective technique for interrogating transcript expressions. The data type also provides nucleotide level sequence information, allowing for variant detection, alternative splicing, and novel transcript discovery, among other uses. In cancer studies variant detection from RNA-seq data is important for identifying potential driver mutations for disease, so there is a need for good quality RNA alignment tools that support sensitive and accurate variant calling.However, with increasing read length, read sequences can often span one or more exon-exon junctions, making it challenging to align them to genomic sequence alone. A number of tools have been developed to address this. TopHat and TopHat2 Most methods combine the alignment of gapped and un-gapped reads, requiring the use of their own particular alignment algorithm, and do not work with different aligners. BWA JAGuaR first uses a modified GTF (Gene Transfer Format) of known splice sites to build the junction reference sequence (Table S1 in http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?study=SRP041367). The reference for the alignments in all cases was based on GRCh37-lite (hg19) with corresponding transcript models from Ensembl61 To demonstrate the performance of JAGuaR, three sets of cell line libraries were analyzed: Universal Human Reference RNA from Agilent Technologies (Sample 1 and 2), and HelaS3 (Sample 3). On the Illumina HiSeq 2000 platform, 100 bp paired end reads were sequenced (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/). These were separated into two VCFs , for variants estimated as homozygous, and heterozygous, respectively. These were each implanted separately into the hg19 reference (GRCh37-lite) using the GATK tool FastaAlternativeReferenceMaker In addition to these real datasets, we generated a simulated RNA-seq dataset (Sample 4) using the Flux Simulator software We compared the performance of JAGuaR (v2.1) with three other popular split read alignment tools, TopHat2 (v2.0.8b), GSNAP (v2012-12-12) and MapSplice (v2.1.5). We also attempted to compare to SpliceMap JAGuaR was run with BWA at default settings where -t (number of threads) is set to 1. TopHat2, was run with -p (number of threads) set to 4, "—no-novel-juncs" set, and the GTF annotation file specified. MapSplice was run with -p (number of threads) set to 4. GNSAP was run with -B (batch mode) set to 5, -t (number of worker threads) set to 8 and the specified GTF annotation file (converted to binary format). As GSNAP ran much slower than the other tools, we increased the number of threads used so the analysis would complete in a reasonable amount of time. The output which includes multiple alignments for a read was filtered for the first two paired ends of the highest quality. This was also done for JAGuaR/BWA-MEM.We compared the performance of the methods on the simulated dataset as well as the cell-line samples. In the absence of ‘truth’ data for the cell-line samples, we used the total reads aligned and the number of unique exon-exon junctions that were covered by at least one read as metrics to estimate alignment accuracy and sensitivity of each tool. In addition, dbSNP The samples used are derived from commercially available cell lines. The work described herein was conducted at the BC Cancer Agency's Michael Smith Genome Sciences Centre, reviewed by the University of British Columbia - BC Cancer Agency Research Ethics Board.JAGuaR performed well when comparing the number of identified exon-exon junctions, sequence coverage of junctions, and the number of dbSNP concordant SNVs called . DespiteThe overlap between SNVs called using JAGuaR (with BWA and BWA-MEM), GSNAP, MapSplice2 and TopHat2 from each of the samples was analyzed. This was done for the subset of known SNVs in dbSNP, known non-synonymous SNVs, and those seen in the COSMIC database .With further filtering based on non-synonymous SNVs and those in the COSMIC database, concordance between all tools is higher. The number of SNVs called in each category is quite similar, showing that the majority of the SNVs are called by all methods.Memory usage and the length of time it took each tool to process a set of paired end RNA-seq reads into a BAM formatted alignment file are reported in We also compared the performance of JAGuaR with BWA-MEM alone using sample 1 by examining the coverage at exon boundaries. The combination of JAGuaR with both BWA and BWA-MEM increases the dbSNP concordance of called SNPs. JAGuaR combined with BWA-MEM also calls more known SNVs than BWA-MEM alone  as provided by modern sequencing platforms. Its computational requirements are comparable to existing methods and fastest when used with BWA-MEM. It offers an improvement in alignment sensitivity over some existing methods while still maintaining a higher specificity over others, as shown by the fact that in all comparisons, SNV calls using JAGuaR alignments provide either a higher dbSNP concordance or a high total number of dbSNP concordant calls over other tools. As variant discovery is an important component of many sequencing projects, as a fast, accurate and sensitive tool JAGuaR offers a valuable functionality to RNA-seq analysis. While JAGuaR is not designed to detect differential gene expression or un-annotated transcripts, novel isoforms may still be reconstructed from JAGuaR-aligned reads provided that such isoforms consist of a new combination of known splice sites. As annotation quality increases in human and other model organisms, an accurate and fast alignment for clinical applications is a priority that JAGuaR satisfies.File S1Table S1, Example Transcript Model. Figure S1, JAGuaR first requires the reference genome of interest and a transcript model in order to build the reference of the genome sequence and exon junctions. Figure S2, Based on a transcript model (Table S1), JAGuaR assesses each exon-exon junction of all available transcripts. Figure S3, SNV concordance between tools for one read set (Sample 1). Figure S4, SNV concordance between tools for one read set (Sample 3). Table S3, SNV Comparison to running of BWA-MEM alone. Figure S5, Comparison of JAGuaR+BWA-MEM/BWA-MEM exon start or stop coverage fraction. Text S1, Parameters used for Flux Simulator.(DOCX)Click here for additional data file.


HIV increases women’s risk for high-risk human papillomavirus (hrHPV) infection and invasive cervical cancer. South Africa has a high HIV prevalence but low cervical cancer screening coverage. Self-collection of cervical specimens and hrHPV testing, including hrHPV messenger-RNA (mRNA) testing, are methods aimed at increasing screening rates. However, data are limited on the acceptability and accuracy of tampon-based self-collection for hrHPV mRNA testing in HIV-infected women.We recruited 325 HIV-infected women seeking care at a government HIV clinic in Pretoria, South Africa. A clinician performed a pelvic examination and obtained an endocervical specimen. Study participants performed self-collection using a tampon. Both clinician- and self-collected specimens were tested for hrHPV mRNA. Acceptability of both collection methods was assessed, the prevalence of hrHPV mRNA in our study population was estimated, test positivity of the two collection methods were compared, and test agreement was assessed by calculating the κ-statistic, sensitivity, and specificity.Over 90% of women reported no difficulties self-collecting specimens and 82% were willing to perform the tampon-collection at home. Based on clinician-collection specimens, the prevalence of hrHPV mRNA in our study population was 36.7% (95% CI: 31.4%– 42.0%). There was no difference in test positivity between clinician-collection, 36.7%, and tampon-collection, 43.5% . Using clinician-collection as the reference test, the sensitivity and specificity for hrHPV mRNA of tampon-collection were 77.4% (95% CI: 69.8–85.0%) and 77.8% (95% CI: 71.9–83.6%), respectively.Tampon-based self-collection is acceptable to women and has similar hrHPV mRNA positivity rates as clinician-collection, but has reduced sensitivity and specificity compared to clinician-collection. The hrHPV mRNA prevalence in our study population is high, but similar to other high-risk populations, and highlights the need for improved cervical cancer screening. Further research into the optimal use of tampon-based collection as a cervical cancer screening tool is warranted. Cervical cancer is caused by persistent infection with high-risk human papillomavirus (hrHPV).  GloballyScreening for hrHPV using self-collected specimens has been suggested as one way to increase cervical cancer screening coverage in low-resource settings.  Some advMolecular diagnostics aimed at detecting hrHPV viral integration, via the production of oncogenic E6 and E7 messenger-RNA (mRNA), might offer an improvement in specificity for predicting precursors to cervical cancer. Initial studies in routine-screening populations reveal a sensitivity and specificity for detecting cervical intraepithelial neoplasia of grade 2 or higher (CIN 2+), to be 94.2–97.5% and 90.2–94.5%, respectively. ,20. WhenOur objectives were to 1) estimate the prevalence of hrHPV mRNA among a cohort of HIV-infected women, 2) compare the test positivity between the two collection methods, 3) assess the accuracy and agreement of self-collected tampons compared to clinician-collected specimens for hrHPV mRNA testing, and 4) assess the acceptability of the self-collected tampon method.We conducted a cross-sectional study from February 2014 through April 2014. We enrolled 325 HIV-infected women seeking care at a government HIV clinic in Tshwane District, Gauteng Province, South Africa.Inclusion criteria were HIV-infected women, 25 years or older, who did not have a cervical cytology test result documented in their chart within the past three years. Women who had a hysterectomy or who were currently menstruating were excluded from the study. The women who were menstruating were invited to come back to the clinic for study enrollment after menstruation.Women who were eligible to participate were seen by a study nurse who obtained informed consent. Participants then were administered a brief questionnaire to collect socio-demographic, reproductive, and health data, as well as an assessment of knowledge regarding cervical cancer. HIV viral load and CD4+ cell count were extracted from the patient’s medical record.After administering the intake questionnaire, the study nurse performed a pelvic examination. Lubricant was not applied to the speculum prior to insertion. After speculum placement, the cervical os was visualized and a cotton-tipped swab was used to remove any excess secretions. The cervical specimen was collected by inserting the broom-like collection device into the cervical os and rotating five times. The collection device was then immediately swirled into a ThinPrep Pap collection container containing PreservCyt solution  to dislodge cervical cells. The collection brush was then discarded.After obtaining the cervical specimen, the study nurse instructed the study participant on how to perform the tampon-based self-collection. Briefly, the woman was instructed to insert the tampon into her vagina, leave it in-place for one to two hours, then to remove and place the tampon into the provided collection tube and return it to the study nurse. Tampon-collection was performed using mini-sized tampons, with light absorpency . Tampon insertion and removal was not witnessed by the study nurse. The tampon collection tube was a 30mL plastic bottle that contained 10mL of saline solution to prevent drying of the sample. At the time of receipt, the nurse verified that the tampon was completely submerged into the saline and the collection bottle was closed correctly.After returning the tampon specimen, a second nurse administered an acceptability questionnaire about the woman’s experiences with the pelvic exam and the tampon-based self-collection. To minimize bias, the study nurse who performed the pelvic examination was in another room and did not participate in the post-test questionnaire administration. The questionnaire used a Likert scale, from 1 (most favorable) to 5 (least favorable), to evaluate experiences related to perception of care, comfort, privacy, embarrassment, and pain for both collection methods individually. The questionnaire also assessed difficulties encountered during tampon-collection and had questions regarding the ability to perform self-collection, including home collection.The ThinPrep collection containers and the tampon collection tubes were stored in a refrigerator (4–8°C) within an hour after collection. The specimens were transported three times per week  in a cold box for processing and testing by Toga Labs .Cytological smears, from clinician-collected specimens only, were prepared on the ThinPrep T2000 slide preparation machine and were read by two cytotechnologists blinded to any results from molecular testing. Smears were classified according to the 2001 Bethesda System for cervical cytology.  Study paWomen were referred for colposcopy if they had abnormal cytology results, including low-grade intraepithelial lesions (LSIL), atypical squamous cells cannot exclude high-grade lesion (ASC-H) and high-grade intraepithelial lesions (HSIL). Women with atypical squamous cells of undetermined significance (ASC-US) were instructed to repeat cytology test in six months. Women with normal cytology results were instructed to follow routine screening recommendations.hrHPV mRNA testing was performed on both the clinician- and self-collected specimens. For the clinician-collected specimens, a 1mL aliquot was transferred from the ThinPrep Pap Test container to an Aptima Specimen Transfer tube within one week of receipt at the laboratory. These specimens were then batched and tested using the Aptima HPV assay, according to manufacturer’s instructions. The Aptima HPV assay detects E6/E7 mRNA of 14 high-risk HPV types .  Each tesUpon delivery to the laboratory, the tampon specimens were removed from the collection container and the liquid was extracted from the tampon. This liquid was spun down to make a cell pellet. The supertanant was aspirated until 2ml of the liquid remained, at which point the cell pellet was resuspended. Then 1ml of this cell-containing liquid was extracted into an Aptima Specimen Transport Tube and the tampons were discarded; the remaining 1mL of liquid was stored in a -80°C freezer. The specimens in the Aptima Specimen Transfer Tube were tested on the Aptima Assay, according to the manufacturer’s instructions.In order to estimate the hrHPV mRNA positivity in our study population, we used published test characteristics and the prevalence of cervical dysplasia as reported in a recent study within an HIV-infected population in South Africa. ,20,26,27Acceptability of the Pap test and the tampon self-collection were assessed using a Likert scale based on different acceptability categories . The mean and standard deviation for Likert scale data were calculated for each category. The mean from each collection method were compared using a student t-test. We compared the overall prevalence of hrHPV mRNA between the two collection methods. The clinician-collected hrHPV mRNA test results were used as the reference standard to estimate the sensitivity and specificity, with corresponding 95% confidence intervals, of the tampon-collection method. We calculated the k-statistic, with corresponding 95% confidence intervals, to assess the agreement between the two collection methods. All statistical analyses were performed in Stata .All study procedures were explained to participants and written informed consent was obtained. All study protocols and documents were reviewed and approved by the institutional review boards at the University of Pretoria, Pretoria, South Africa (#112/2012) and the University of California, San Francisco, San Francisco, CA (#13–11129).In total, 325 women consented and participated in study procedures . The medAll 325 women had paired clinician-collected and self-collected specimens obtained for laboratory testing. Nine results (2.8%) were excluded among the clinician-collected specimens and eight (2.4%) among the tampon specimens due to laboratory processing errors or invalid hrHPV mRNA results.Of the 316 clinician-collected specimens included in the analysis, 116 tested positive for hrHPV mRNA, corresponding to a prevalence of 36.7% (95% CI: 31.4%– 42.0%) . The tesResults from each of the collection method are presented in Participants reported feeling slightly better cared for during the clinician-collection (mean = 1.04) compared to self-collection , but there were no other significant differences in privacy, embarrassment, discomfort or pain between the two collection methods . There wTo our knowledge, this is the first report of hrHPV mRNA prevalence within a primary screening population infected with HIV. Based on clinician-collection, we report the prevalence of hrHPV mRNA to be 36.7%. There are only two reports on the prevalence of hrHPV mRNA in low-resource settings. ,22 The pWe found no difference in the hrHPV mRNA positivity rate between the two collection methods. Furthermore, the κ—statistic (0.54) indicated “moderate” agreement. Few studies have evaluated the performance of hrHPV mRNA testing of self-collected versus clinician-collected specimens, and even fewer studies report using similar study measures. Johnson et al. reported a similar hrHPV mRNA positivity rate between clinician- and self- collected swabs, with a κ—statistic of 0.62,  and CherWe report that out of 115 hrHPV mRNA-positive clinician-collected specimens, 89 tampon-collected specimens were also positive for hrHPV mRNA, corresponding to a sensitivity of 77.4%. Likewise, of 193 clinician-collected specimens negative for hrHPV mRNA, 150 tampon-collected specimens were also negative for hrHPV mRNA, corresponding to a specificity of 77.7%. Data reporting the sensitivity and specificity of tampon-based self-collection in this way are limited, even in the hrHPV DNA testing literature. Jones et al. compared tampon-based self-collection to clinician-collection using two separate HPV DNA tests. They report a wide range of sensitivities for tampon collection, 59.5% and 91.8% for the two different HPV DNA tests performed, and estimate the specificity to be 86.2% and 90.6%.  Our findOne reason for the lower sensitivity of the tampon-collection might be due to sampling location. The clinician-collected specimen, obtained directly from the cervix, preferentially collects cervical cells in the transformation zone, whereas the tampon-method samples a mix of cells from both the cervix and vagina, and therefore might not collect enough cells from the transformation zone. It is possible that extending the time the tampon was held might increase the sensitivity, but might lead to decreased acceptability.  The diffTampon-based self-collection proved to be an acceptable method of cervical cancer screening within our cohort of HIV-infected women. Very few women reported difficulty performing the tampon collection and nearly all women reported positive experiences with collection. Our findings of tampon acceptability are supported by other research among HIV-positive women in South Africa, suggesting that a large proportion of urban women might actually prefer tampon-based self-collection to other self-collection methods.  One advaOur study had several strengths. First, we recruited and screened a large sample of HIV-infected women who represent a high-risk primary screening population. Secondly, both collection methods were performed sequentially on the same day, allowing for direct comparison of the samples collected. Third, we were able to assess both the acceptability as well as the performance of the tampon-collection method for hrHPV mRNA testing. However, our study has limitations. One limitation is that we were not able to directly evaluate which collection method was more favorable. Second, we did not perform cervical biopsies on study participants and do not know the true disease status of these women.In summary, we found that tampon-based self-collection for hrHPV mRNA could function as a viable method for cervical cancer screening among HIV-infected women in low-resource settings, increasing the options available to women and program planners. The collection method and test might be offered as a way to increase primary screening coverage among women with limited access to screening services or who prefer self-collection methods. In light of our findings, one option to improve the utility of the tampon-based self-collection method and mRNA testing might be to screen more frequently using this method, which might increase the probability of detecting cervical infections. Given the lower specificity of the tampon-based self-collection method, women with positive results would need follow-up with a confirmatory test to reduce the need and costs of unnecessary follow-up by colposcopy. Further research is needed to 1) evaluate the tampon-collection and hrHPV mRNA testing against true cervical disease, 2) explore the implementation and uptake of the collection and testing method in a routine screening population, taking into account women’s preferences for screening methods, and 3) optimize the timeline for testing.


In the Microbicide Trial Network MTN-003 (VOICE) study, a Phase IIB pre-exposure prophylaxis trial of daily oral or vaginal tenofovir (TFV), product adherence was poor based on pharmacokinetic (PK) drug detection in a random subsample. Here, we sought to compare behavioural and PK measures of adherence and examined correlates of adherence misreporting.We included participants with PK and behavioural data from VOICE random subsample. Behavioural assessments included face-to-face interviews (FTFI), audio computer-assisted self-interviewing (ACASI) and pharmacy-returned product counts (PC). TFV concentrations <0.31 ng/mL in plasma  and <8.5 ng/swab in vaginal group were defined as “PK non-adherent.” Logistic regression models were fit to calculate the combined predictive ability of the behavioural measures as summarized by area under the curve (AUC). Baseline characteristics associated with over-reporting daily product use relative to PK measures was assessed using a Generalized Linear Mixed Model.N=472), PK non-adherence was 69% in the oral group (N=314) and 65% in the vaginal group (N=158). Behaviourally, ≤10% of the cohort reported low/none use with any behavioural measure and accuracy was low (≤43%). None of the regression models had an AUC >0.65 for any single or combined behavioural measures. Significant (p<0.05) correlates of over-reporting included being very worried about getting HIV and being unmarried for the oral group; whereas for the vaginal group, being somewhat worried about HIV was associated with lower risk of over-reporting.In this random adherence cohort of VOICE participants assigned to active products, (PK measures indicated similarly low adherence for the oral and vaginal groups. No behavioural measure accurately predicted PK non-adherence. Accurate real-time measures to monitor product adherence are urgently needed.Trial registration: ClinicalTrials.gov identifier: NCT00705679 TM (tenofovir (TFV) disoproxil fumarate/emtricitabine) has received regulatory approval for PrEP in the United States [Daily oral pre-exposure prophylaxis (PrEP) is an effective prevention strategy for persons at risk of HIV-1 acquisition, and Truvadad States –4. Recipd States –11. Inded States ,11.Daily TFV gel use was not found effective in preventing HIV-1 acquisition in VOICE either, attributed to poor adherence based on levels of detectable TFV in plasma (25%) and vaginal swabs (49%) . PericoiAccurately measuring adherence to study products has been an on-going challenge in HIV prevention trials. Self-reports typically overestimate adherence because of various biases, including recall and social desirability, yet alternative measures to assess product use are limited ,15–17. HTM was found protective among men who have sex with men and transgender women [Recent PrEP trials have highlighted a biological–behavioural adherence gap. It was small (14 to 16% overestimation by self-report) in the TDF2 and Partners PrEP trials, where oral products were protective and all monitoring modalities confirmed high adherence . Howeverer women ,24.Here, we examined different behavioural measures of adherence in the VOICE study and compared them to biological pharmacokinetic (PK) assessment of TFV in plasma  and vaginal swabs , to explore their accuracy, alone or in combination, for predicting biological use. We also explored correlates of adherence over-reporting to identify those at greater risk of concealing low product use.VOICE was a Phase IIB double-blinded, randomized, placebo-controlled trial that tested three products ; oral emtricitabine (FTC)/TDF; and TFV 1% vaginal gel) among 5029 women in Uganda, Zimbabwe and South Africa. As described in the primary publication, the oral TDF and FTC/TDF tablets had differing appearances (different sizes and shades of blue) so to maintain blinding each oral arm participant was assigned to take two tablets a day, TDF or TDF placebo and FTC/TDF or FTC/TDF placebo . Intent-N=3017), 510 were included in the original PK random subsample [N=472; see Of the participants in the VOICE study who were assigned to active arms , using a cut-off of 0.31 ng/mL ,25. In tAt quarterly visits, frequency of product use in the past seven days, collected both through FTFI and ACASI, was dichotomized into zero versus ≥1 doses in the past week to match the biological measures. During the ACASI, participants were also asked to rate their ability to use the product as instructed in the previous 4 weeks, using a validated six-point rating scale (very low to excellent) .Two bottles of 30 pills or a box containing 30 gel applicators were dispensed to participants at baseline and at monthly follow-up visits scheduled at 28-day intervals. At monthly visits, pharmacists collected the number of products returned and dispensed. The difference between the number of products received at the previous visit and the number of products returned represented the number of products assumed used by PC. This number was compared with the number of days that had elapsed between the previous visit and the current visit. The PC was dichotomized as ≤75% use in the past month to most closely match the estimate for PK non-adherence in the past week. In other words, if a participant did not use tablets or gel in the past week, at most she could be 75% adherent by monthly PC.We defined “obvious” over-reporting when self-report of product used daily in the past seven days by FTFI (or ACASI) occurred with no evidence of use by PK data .We compared characteristics of the adherence subset to the remaining VOICE sample using t-tests for continuous variables and Chi-square tests for categorical variables.For each behavioural measure, we analyzed its predictive properties for biologically measured PK non-adherence. Accuracy was defined as the proportion of concordant results between the biological and behavioural measures, calculated as the per cent of behavioural measures that were correct given the biological measure. We further explored the ability of the self-reported measures to predict biologically defined adherence by combining the measures to predict PK adherence in a logistic regression model. Using predicted adherence from the logistic regression model as a marker of adherence, we calculated the corresponding area under the ROC curve (AUC) to compare the predictive ability of the single or combined self-reported measures.a priori set the significance level at p<0.1  for entering significant factors into a multivariable model. Sensitivity analyses were conducted assessing characteristics of participants associated with over-reporting at first and second quarter with similar findings (data not presented).We assessed the association between baseline characteristics of participants and obvious over-reporting of product use by self-report (FTFI) versus biological data. For each route of administration, we used a Poisson Generalized Linear Mixed Model (GLMM) with random intercept and included all quarterly visits with complete biological and behavioural data, adjusting for time (in months since enrolment) and country. We estimated the relative risk (RR) ratio for the following group of predetermined baseline characteristics: sociodemographic, sexual behaviour, contraception and HIV risk. We used a validated HIV risk assessment tool  that predicts HIV acquisition among African women .For the accuracy assessment, we randomly selected one quarterly visit for each participant . Sixty-nine per cent of those in the oral group and 65% of those in the vaginal group exhibited drug levels below the PK non-adherence cut-off. In contrast, by FTFI, 4% of participants in the oral group and 2% in the gel group reported not taking any dose in the past week. By ACASI, this was 6% in both groups. Based on the PC measurement, 8 and 6% of participants in the oral and the vaginal groups were classified as non-adherent, respectively .When asked by ACASI to rate their adherence in the past 4 weeks from very poor to excellent, the majority of women reported high adherence regardless of biological evidence .Supplementary File 1).Accuracy by FTFI, ACASI or PC was low: in the oral group, of the 11 participants who reported taking no doses in the past week by FTFI, all were PK non-adherent. However, among the 295 (96%) who reported ≥1 dose in the past week by FTFI, 67% were found to be PK non-adherent (36% accuracy). Accuracy was also low by ACASI (38%) and by PC (38%). Accuracy was similarly low in the gel group for the three behavioural measures  the multivariable models were not much better than chance at predicting PK non-adherence. In the multivariable models, the AUC for participants in the oral group was 0.63 and for those in the gel group it was 0.59 .The average proportion of obvious over-reporting in the oral group (across month 3-30 visits) was 66% by FTFI and 67% by ACASI, with respect to assessment of product use in the previous seven days (i.e. self-reported using daily with no evidence of product use by TFV concentration). In the vaginal product group, the average proportion of obvious over-reporting (across month 6–15 visits) was 69% by FTFI and 54% by ACASI.We examined baseline risk factors for over-reporting product use, controlling for time in study and country . In the In this random adherence cohort of 472 VOICE participants assigned to active products, PK thresholds based on route of administration indicated similarly low adherence for women randomized to both the oral and vaginal product arms. This finding is similar to MTN-001, an open-label, cross-over trial of the same products but for a 6-week duration, where adherence estimates differed among US versus African participants, but within sub-Saharan Africa, was similar between products . Among tTo improve on the accuracy of adherence assessed by FTFI, other methods have been evaluated. Minimal misreporting seems to have occurred in the Partners PrEP Study of HIV-serodiscordant couples, where biological evidence, electronic monitoring (MEMS) and other behavioural measures all agreed on high adherence . ACASI dIndeed, findings from two qualitative ancillary studies indicate that participants were motivated to enrol and stay in VOICE for the quality health care, regular HIV testing, and other benefits, but were fearful of using the investigational products, which contained ARVs, so that they likely concealed their poor adherence to remain in the trial ,42,43. TFor our analysis of factors associated with “obvious” over-reporting, we purposefully selected a crude definition  to minimize misclassification because of recall bias, given that plasma and VF PK data provide average estimates of recent use. Importantly, this low threshold of PK adherence (use within the last week) will not provide any expectation of protection and was selected solely to compare methods of adherence and assess their lack of concordance. Biomarkers that may provide more information on average use over longer term periods, or on white-coat compliance to explain recent use, were not available for our analyses ,47. Our In VOICE, “being worried about getting HIV in next year” was the only factor associated with over-reporting both in the oral and vaginal groups; although, the associations differed in each group. However, this variable may not have accurately measured participants’ risk perceptions. True risk perception may only be one of many factor associated with product adherence and with reporting. Other trials have used different measures of risk perception and the lack of standardization hinders comparisons across prevention trials ,50. In iWith more accurate electronic or biological measures of product use increasingly available, real-time feedback on adherence can be provided to trial participants ,52,53. HIn this study, TFV PK measures indicated similarly low adherence among VOICE participants in both the oral and vaginal product groups. Furthermore, no behavioural measure, regardless of data collection mode, accurately predicted non-adherence, defined by PK. Over-reporting of product use was widespread, and no clear characteristics defined participants who grossly over-reported use of tablets or gel. Objective monitoring of product use should begin early in future trials before behavioural patterns related to low product use and over-reporting are established. Most important, accurate real-time measures to monitor product adherence should become a standard of prevention trial implementation and are key to inform the interpretation of trial results.Click here for additional data file.