Progress in malaria control is under threat by wide-scale insecticide resistance in malaria vectors. Two recent vector control products have been developed: a long-lasting insecticidal net that incorporates a synergist piperonyl butoxide (PBO) and a long-lasting indoor residual spraying formulation of the insecticide pirimiphos-methyl. We evaluated the effectiveness of PBO long-lasting insecticidal nets versus standard long-lasting insecticidal nets as single interventions and in combination with the indoor residual spraying of pirimiphos-methyl.ClinicalTrials.gov, number NCT02288637.We did a four-group cluster randomised controlled trial using a two-by-two factorial design of 48 clusters derived from 40 villages in Muleba . We randomly assigned these clusters using restricted randomisation to four groups: standard long-lasting insecticidal nets, PBO long-lasting insecticidal nets, standard long-lasting insecticidal nets plus indoor residual spraying, or PBO long-lasting insecticidal nets plus indoor residual spraying. Both standard and PBO nets were distributed in 2015. Indoor residual spraying was applied only once in 2015. We masked the inhabitants of each cluster to the type of nets received, as well as field staff who took blood samples. Neither the investigators nor the participants were masked to indoor residual spraying. The primary outcome was the prevalence of malaria infection in children aged 6 months to 14 years assessed by cross-sectional surveys at 4, 9, 16, and 21 months after intervention. The endpoint for assessment of indoor residual spraying was 9 months and PBO long-lasting insecticidal nets was 21 months. This trial is registered with vs 767 [42%] of 1809; odds ratio [OR] 0·37, 95% CI 0·21–0·65; p=0·0011). At the same timepoint, malaria prevalence in the two groups that received indoor residual spraying was lower than in groups that did not receive indoor residual spraying  and there was evidence of an interaction between PBO long-lasting insecticidal nets and indoor residual spraying , indicating redundancy when combined. The PBO long-lasting insecticidal net effect was sustained after 21 months with a lower malaria prevalence than the standard long-lasting insecticidal net .7184 (68·0%) of 10 560 households were selected for post-intervention survey, and 15 469 (89·0%) of 17 377 eligible children from the four surveys were included in the intention-to-treat analysis. Of the 878 households visited in the two indoor residual spraying groups, 827 (94%) had been sprayed. Reported use of long-lasting insecticidal nets, across all groups, was 15 341 (77·3%) of 19 852 residents after 1 year, decreasing to 12 503 (59·2%) of 21 105 in the second year. Malaria infection prevalence after 9 months was lower in the two groups that received PBO long-lasting insecticidal nets than in the two groups that received standard long-lasting insecticidal nets (531 [29%] of 1852 children The PBO long-lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent and either intervention could be deployed to good effect. As a result, WHO has since recommended to increase coverage of PBO long-lasting insecticidal nets. Combining indoor residual spraying with pirimiphos-methyl and PBO long-lasting insecticidal nets provided no additional benefit compared with PBO long-lasting insecticidal nets alone or standard long-lasting insecticidal nets plus indoor residual spraying.UK Department for International Development, Medical Research Council, and Wellcome Trust. Evidence before this studyWe did two searches in PubMed with no language restrictions or specified dates. In the first search on long-lasting insecticidal nets treated with piperonyl butoxide (PBO), we used the search terms “malaria” and “long lasting insecticidal net” in combination with “piperonyl butoxide”, “Olyset Plus”, or “PermaNet 3.0”, which produced no references that were randomised controlled trials (RCTs). In a 2015 review of PBO long-lasting insecticidal nets, WHO concluded that although they appeared to have an increased efficacy the evidence was too inadequate to justify a switch from pyrethroid only to PBO nets across all settings. Because of the potential for an antagonistic effect between PBO and organophosphates, WHO also recommended that PBO nets should not be used in areas programmed for indoor residual spraying with pirimiphos-methyl capsule suspension.In the second search on combined vector control interventions, we included the search term “malaria” with one or more of the following: “long lasting insecticidal net” or “insecticide treated net”, “indoor residual spraying”, “vector control”, “pirimiphos methyl”, and “combined interventions”. We identified three other RCTs that have examined combined intervention of long-lasting insecticidal nets and indoor residual spraying. The Gambian RCT, which used DDT (dichlorodiphenyltrichloroethane) as the indoor residual spraying component showed no additional protection compared with long-lasting insecticidal nets alone. This finding could be explained by the high usage of nets or the properties of the sprayed insecticide used. In Benin, there was no advantage to combining indoor residual spraying and long-lasting insecticidal nets; however, suboptimal coverage of nets and the short residual effect of the spray used (bendiocarb) might have affected the effectiveness of the combination intervention. In an earlier Tanzanian study, where coverage of long-lasting insecticidal nets was moderate and pyrethroid resistance present, two rounds of indoor residual spraying with bendiocarb combined with long-lasting insecticidal nets were more effective than long-lasting insecticidal nets alone.Added value of this studyOur study is the first RCT to report that PBO-treated long-lasting insecticidal nets were more effective than standard pyrethroid long-lasting insecticidal nets against malaria infection and transmission. It also provides the strongest evidence to date of the negative effect of high-level pyrethroid resistance on the use and efficacy of standard nets. This study is also the first RCT to provide evidence for the effect of long-term malaria control of the first long-lasting organophosphate formulation to be developed specifically for indoor residual spraying. The RCT provides new evidence on the added value and risks of combining indoor residual spraying and long-lasting insecticidal nets, particularly PBO nets.Implications of all the available evidenceThis study provides justification for the increase in deployment and use of PBO long-lasting insecticidal nets. As a direct consequence of this trial, WHO revised its policy on long-lasting insecticidal nets in September, 2017, gave interim endorsement to pyrethroid-PBO nets as a new WHO class of vector control product, and recommended that PBO nets be deployed for prevention of malaria where vectors are resistant to pyrethroids provided that vector control coverage is not compromised. This endorsement would include many endemic areas in Africa where standard long-lasting insecticidal nets are currently used. The demonstration that long-lasting insecticidal nets with an appropriate change of active ingredient can continue to tackle transmission by pyrethroid-resistant vector populations will ensure the viability of this approach as well as justifies the continued investment and search for alternative insecticides for use on nets.Finally, the organophosphate indoor residual spraying formulation is the first long-lasting, non-pyrethroid insecticide to provide malaria control for at least 9 months over two transmission seasons in the same year. This finding justifies the scale up and use of indoor residual spraying in sub-Saharan Africa and the 12-year investment into long-lasting alternatives to pyrethroid and DDT for indoor spraying between private and public sector organisations.Long-lasting insecticidal nets and indoor residual spraying are the cornerstones of malaria control in sub-Saharan Africa. Together with effective treatment, these interventions are estimated to have globally reduced malaria morbidity by 41% and mortality by 62% between 2000 and 2015.The range of insecticides available for indoor residual spraying is limited. For long-lasting insecticidal nets, the range is particularly restricted because pyrethroids are the only class of insecticides recommended by WHO for nets. Evidence from indoor residual spraying programmes suggests that pyrethroid resistance can contribute to operational control failure—eg, in South Africa, control was only restored once the pyrethroid was replaced by an insecticide to which vectors were susceptible.Anticipating the possible failure of current control tools due to resistance, WHO has encouraged the industry to develop new types of long-lasting insecticidal nets and new insecticides for indoor residual spraying. One of these developments is a new long-lasting insecticidal net that uses piperonyl butoxide (PBO). PBO is a chemical synergist that acts by inhibiting enzymes involved in the natural defense mechanisms of insects, which results in pyrethroid not being detoxified in the insect and the pyrethroid on the long-lasting insecticidal net remaining potent against mosquitoes despite resistance. Such PBO-pyrethroid-treated long-lasting insecticidal nets appear to have similar or better efficacy against resistant mosquitoes under controlled household conditions than standard long-lasting insecticidal nets that do not have PBO.Although the range of insecticide classes suitable for indoor residual spraying use is wider than long-lasting insecticidal nets, few insecticides are effective for more than a few months when sprayed onto walls and this limitation has been a constraint on their adoption and use. The organophosphate pirimiphos-methyl is an exception, and the recently developed long-lasting formulation, Actellic 300CS , is effective for up to 10 months when used for indoor residual spraying.In attempts to accelerate malaria control progress, long-lasting insecticidal nets and indoor residual spraying have been deployed together in several countries.To develop an improved strategy for control of malaria transmitted by pyrethroid-resistant mosquito vectors, we aimed to compare the effectiveness of PBO long-lasting insecticidal nets with standard long-lasting insecticidal nets as single interventions and in combination with the long-lasting indoor residual spraying of pirimiphos-methyl.We did a cluster RCT of four groups using a two-by-two factorial design. The RCT started on March 1, 2014. The post-intervention assessment period was initially planned for 18 months  and was subsequently extended on our request to the funding agency to 24 months  to enable further assessment of the PBO long-lasting insecticidal net .Figure 1Anopheles gambiae and Anopheles arabiensis were the only vectors found in 2012. High levels of resistance to pyrethroids have been reported in A gambiae in the study area, and synergy bioassay tests done with PBO and pyrethroid together partially restored the toxicity of pyrethroids.The study area was Muleba district of the Kagera region in northwest Tanzania, and comprised 40 villages. In 2011, malaria infection prevalence in children was 23%.The trial was approved by the ethics review committees of the Kilimanjaro Christian Medical University College, the London School of Hygiene & Tropical Medicine, and the Tanzanian Medical Research Coordinating Committee (NIMR/HQ/R.8a/VolIX/1803). A trial steering committee reviewed progress. Written informed consent from parents or guardians was obtained for each survey and entomology collection.We used restricted randomisation to allocate the 48 clusters to the four study groups: standard long-lasting insecticidal nets, PBO long-lasting insecticidal nets, standard long-lasting insecticidal nets plus indoor residual spraying, and PBO long-lasting insecticidal nets plus indoor residual spraying. We limited potential imbalance using three restriction variables: malaria infection prevalence in children aged 6 months to 14 years, usage of long-lasting insecticidal nets, and socioeconomic status, as recorded in the baseline survey between September and October, 2014. Of the 200 000 random allocations, 29 478 met the restriction criteria of no more than 7% difference in mean malaria prevalence, 10% in mean usage of long-lasting insecticidal nets, and 10% of households in the lowest socioeconomic status tertile between study groups. After verifying that clusters were independently allocated to study groups, we randomly chose one of the eligible allocations.We masked the inhabitants of each cluster to the type of long-lasting insecticidal nets received. The two types of nets were of similar colour and shape, and only distinguishable by label codes and coloured thread inserted during manufacture. Additionally, we masked field staff, who took blood samples in the cross-sectional surveys, to the study groups the clusters were assigned to. It was not possible to mask either the investigators or the participants to the treatment allocation of indoor residual spraying.We used the following vector control products: Olyset Net  containing 2% permethrin , Olyset Plus  containing 2% permethrin and 1% PBO , and Actellic 300CS containing microencapsulated pirimiphos-methyl .2. The residual decay of Actellic 300CS was monitored by a laboratory technician every 3 months on representative wall surfaces in several houses using WHO Cone bioassay tests  and a reference strain of susceptible A gambiae. The permethrin and PBO contents of the long-lasting insecticidal nets were determined by high-performance liquid chromatography at yearly intervals for 2 years.We georeferenced all houses in the study using hand-held global positioning system units . The indoor residual spraying campaign was done once only in February, 2015, by the Research Triangle Institute funded by the President's Malaria Initiative. In the two groups assigned to indoor residual spraying intervention, Actellic 300CS was sprayed to the interior walls and ceilings of each dwelling at the recommended dosage of 1 g/mDistribution of long-lasting insecticidal nets and health education communication on net usage were done in each cluster by the Tanzania Communication and Development Centre. On the basis of census data, each household received one net per two people. Altogether, 45 000 standard long-lasting insecticidal nets and 45 000 PBO long-lasting insecticidal nets were distributed in February, 2015. Nets already owned were not removed but householders were requested to use the study nets provided.Cross-sectional household and malaria infection prevalence surveys were done by project field assistants and nurses at baseline in September and October, 2014, and after intervention at the end of each malaria transmission season (June to July and November to December) in 2015 and 2016 . During Plasmodium falciparum circumsporozoite protein.A gambiae and A arabiensis) and to identify mutation in the voltage-gated sodium channel (Vgsc-1014F and Vgsc-1014S) associated with resistance to pyrethroids.A gambiae and Anopheles funestus of unknown age, the frequency of pyrethroid resistance was determined using 0·75% permethrin papers in WHO cylinder tests. We determined resistance intensity using CDC bottle bioassays and probit analysis to estimate the ratio of the permethrin concentration needed to kill 50% of wild mosquitoes relative to the susceptible strain.Mosquito surveillance was done from March, 2015, to December, 2016, in each cluster by a project field assistant for one night per month in seven randomly selected houses per cluster using CDC Miniature Light Trap Model 512  as a proxy for human biting rates.Plasmodium spp infection measured by the rapid diagnostic test in children aged 6 months to 14 years assessed by the cross-sectional surveys. The trial was initially funded for 18 months after intervention. Although this period could have been chosen as the endpoint, it was not known for how long the PBO and pyrethroid active ingredients in the long-lasting insecticidal nets would last. This effect needed to be monitored every transmission season. We subsequently secured extension from the funding agency for 24 months. WHO then reset the policy agenda declaring that new types of long-lasting insecticidal nets (such as the PBO net) should be effective for at least two transmission seasons or 2 years. The primary endpoint for the indoor residual spraying was 1 year, based on reports of duration of residual activity. Because the two intervention products were being assessed separately and in combination, the main endpoint for assessment of the indoor residual spraying was 9 months and the PBO long-lasting insecticidal nets was 9 months and then 21 months.The primary outcome was the prevalence of The secondary main outcome was malaria transmission or entomological inoculation rate, defined as the mean number of infective mosquito bites per household per month, during the first year and second year after intervention. Other secondary endpoints were the proportion of children with moderate-to-severe anaemia (defined as haemoglobin <8 g/dL), the sporozoite rate , and anopheline population density.vs no indoor residual spraying, and PBO long-lasting insecticidal nets vs no PBO long-lasting insecticidal nets) and a 40% difference between any of the individual groups, with 24 clusters of 80 individuals per cluster being tested in each of these comparisons, and assuming a mean prevalence of 20% in the reference groups and a coefficient of variation of 0·3 (based on data from the earlier study).This study had 80% powervs standard long-lasting insecticidal nets, and indoor residual spraying vs no indoor residual spraying) on prevalence of infection and prevalence of anaemia. We estimated interaction between the two main effects by including an appropriate term in the model. We also examined the effect of each intervention  compared with the control group . Effects were interpreted in relation to a postulated minimum difference of 28% for factorial analysis and 40% for the analysis of each intervention. Analysis of anaemia was restricted to children aged 6 months to 4 years. The per-protocol analysis is available in the Statistical analysis was done using Stata (version 12). All statistical inferences allowed for within-cluster correlation of responses by use of a robust variance estimator to calculate SEs. No allowance was made for multiplicity of testing in the analyses. In the intention-to-treat analysis, logistic regression was used to estimate odds ratios (ORs) of the effect of each of the two interventions  were A funestus. Of the 990 A gambiae sensu lato identified to species, 946 (95·6%) were A gambiae sensu stricto and 44 (4·4%) were A arabiensis.The study area comprised 29 365 households and a population of 135 900. Of the 10 560 households selected for post-intervention survey, 7184 (68·0%) were included whereas 1127 (10·7%) were ineligible (no children younger than 15 years), 150 (1·4%) refused, 1543 (14·6%) were absent, and 556 (5·3%) were unvisited. Of the 17 377 eligible children selected, 15 492 (89·2%) attended for testing . Pre-intBetween baseline and the first cross-sectional survey 4 months after intervention, long-lasting insecticidal net ownership (≥one net per household) increased to 1690 (97·6%) of 1732 households, access  increased to 1550 (89·6%) of 1730, and long-lasting insecticidal net use increased to 7807 (76·9%) of 10 152 . Long-la827 (94%) of 878 households selected for the survey received indoor residual spraying in the two groups assigned to this intervention. The insecticide residues on sprayed walls decayed gradually over the year; mosquito mortality in WHO cone bioassays was 99%  shortly after spraying, 82%  after 9 months, and 59%  after 12 months.In the intention-to-treat factorial analysis for the prevalence of malaria infection, the effect of indoor residual spraying versus no indoor residual spraying was evident at 4 months  whereas there was no evidence of a difference between PBO long-lasting insecticidal nets and standard long-lasting insecticidal nets  and the PBO long-lasting insecticidal net group or the combination of the standard net plus indoor residual spraying group was greater than that observed in the factorial analysis at every timepoint between PBO nets and non-PBO nets or between indoor residual spraying and no indoor spraying . The indPrevalence of severe-to-moderate anaemia was lower for the groups receiving PBO long-lasting insecticidal net compared with their standard long-lasting insecticidal net reference groups, and was also lower in the groups receiving indoor residual spraying than in the non-indoor residual spraying reference groups in the surveys after 9 months and 16 months intervention . ResultsA total of 16 371 vector mosquitoes were collected in 5756 indoor light-trap collections over the 2 years. In the first year, vector densities, sporozoite rates, and entomological inoculation rates were lower in the PBO long-lasting insecticidal net groups than in the standard long-lasting insecticidal net groups , but onlA gambiae sensu lato and 54·5%  for A funestus. The lethal concentration required to kill 50% of the wild A gambiae sensu lato was 38-times higher and of wild A funestus was 34-times higher than for the susceptible reference mosquitoes. The Vgsc gene mutation was found in all tested A gambiae with co-occurrence of Vgsc-1014F and Vgsc-1014S in 22 (9%) of 234 A gambiae mosquitoes. No mutation was found in the 247 A arabiensis tested.The mortality of mosquitoes exposed to permethrin for resistance determination in the WHO cylinder tests was 8·8%  for This trial showed that long-lasting insecticidal nets incorporating the synergist PBO (Olyset Plus) were more effective than the standard pyrethroid long-lasting insecticidal net (Olyset Net) in reducing malaria infection prevalence in an area of high usage of these nets and high pyrethroid resistance in the primary vectors. The additional effect of the PBO long-lasting insecticidal nets on malaria prevalence was evident at the end of the first year with a 44% protective efficacy and at the end of the second year with a 33% protective efficacy compared with the standard long-lasting insecticidal nets. These findings were supported by the entomological outcomes, which showed a significant reduction in malaria transmission, with entomological inoculation rates being reduced by 87% during the first year and 67% during the second year in areas receiving PBO long-lasting insecticidal nets compared with standard long-lasting insecticidal nets. At 9 months, the addition of pirimiphos-methyl indoor residual spraying to the standard long-lasting insecticidal nets provided similar protection against malaria (44% protective efficacy) relative to the standard nets alone, whereas the addition of indoor residual spraying to PBO long-lasting insecticidal nets did not significantly improve protection based on the interaction observed when both indoor spraying and PBO long-lasting insecticidal nets effect were at their strongest. The impact of indoor residual spraying on the entomological inoculation rates was more than 95% in the first year. This effect on malaria transmission occurred shortly after implementation of indoor residual spraying whereas the effect of PBO long-lasting insecticidal nets took longer. This rapid impact of indoor residual spraying is one reason why this intervention is sometimes more favoured than long-lasting insecticidal net distribution during malaria epidemics, although there has been a paucity of evidence to justify this advice.A gambiae, both before and after multiple washing of the nets.A gambiae was still susceptible to pyrethroids, the differential effect between Olyset Plus and standard Olyset Net was less evident.This trial is the first to provide evidence to suggest that incorporation of the synergist PBO to long-lasting insecticidal nets provides improved community protection compared with standard pyrethroid-only nets against malaria transmission by pyrethroid-resistant vector populations. Previous small-scale experimental hut studies of PBO long-lasting insecticidal nets measured entomological outcomes such as mosquito mortality and biting rates. In Benin, these studies showed that Olyset Plus was more effective than standard Olyset Net against pyrethroid-resistant A gambiae and A funestus vectors in the Muleba area are highly resistant to pyrethroid and any difference in surface permethrin between Olyset Plus and Olyset Net in our trial is unlikely to result in differential mortality rate. A study has shown that under household conditions a 20-times increase in the surface content of permethrin on hand-treated nets causes no increase in mortality to free-flying pyrethroid-resistant A gambiae.A gambiae from our study area are killed by a permethrin concentration they would normally survive if it were not mixed with PBO.Despite the 83% loss in PBO content after 21 months, the PBO and permethrin retained on the net remained highly effective against malaria infection and entomological inoculation rates throughout. By contrast, the loss of residual activity of the single round of indoor residual spraying of Actellic 300CS led to resumption of transmission and to increasing entomological inoculation rates and malaria prevalence in the second year. The PBO nets will be monitored during a third year to assess whether effectiveness is maintained at low PBO content. There was also a 42% loss of permethrin content in Olyset Plus and 22% in Olyset Net over the two years. The differential release rate of permethrin in the two nets has been observed in other studies, and it has been suggested in an earlier WHO review of Olyset Plus that the more effective performance of the PBO net is due to the higher release rate and surface concentration of permethrin in this net compared with Olyset Net.A funestus was the main vector.The more effective performance of PBO long-lasting insecticidal nets compared with standard long-lasting insecticidal nets in reducing the prevalence of malaria infection, together with no change in prevalence following the initial distribution and high usage of standard nets, suggests that insecticide resistance of the magnitude reported is compromising the effectiveness of standard pyrethroid nets in northwest Tanzania. A recent study in neighbouring Uganda reported no change in incidence of malaria before and after the distribution of standard long-lasting insecticidal nets.Our study provides further insight into the question of whether indoor residual spraying and long-lasting insecticidal nets should be combined to accelerate the control of malaria. In a previous cluster RCT in Muleba, where conditions of high pyrethroid resistance and moderate usage of long-lasting insecticidal nets (50%), indoor residual spraying with the carbamate bendiocarb provided an added benefit (OR 0·43).The combination of indoor residual spraying of pirimiphos-methyl and PBO long-last insecticidal nets have been suggested to be antagonistic.This trial has several potential limitations. Buffer areas of 300 m were small compared with what has been used in other trials,In conclusion, this trial shows the residual efficacy of indoor residual spraying with pirimiphos-methyl for malaria control of over 1 year, and provides strong evidence for increasing the coverage of PBO long-lasting insecticidal nets over standard long-lasting insecticidal nets of pyrethroid to meet the increasing challenge of pyrethroid resistance and to improve personal and community protection from malaria, particularly in areas of intense pyrethroid resistance. As a consequence of the trial, WHO has made this policy recommendation.


Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and ESBL-producing E. coli were demonstrated to have been advanced by favorable fitness balance associated with high-level resistance to fluoroquinolones. The paper shows that favorable fitness in the major STs/lineages of these pathogens was principally attained by the capacity of evolving mutations in the fluoroquinolone-binding serine residues of both the DNA gyrase and topoisomerase IV enzymes. The available information on fitness balance incurred by individual and various combinations of mutations in the enzymes is reviewed in multiple species. Moreover, strong circumstantial evidence is presented that major STs/lineages of other multi-drug resistant bacteria, primarily vancomycin-resistant Enterococcus faecium (VRE), emerged by a similar mechanism. The reason(s) why the major ST/lineage strains of various pathogens proved more adept at evolving favorable mutations than most isolates of the same species remains to be elucidated.The major international sequence types/lineages of methicillin-resistant  Staphylococcus aureus (MRSA)   and topoisomerase IV  genes impact pathogens is of utmost importance.Researchers investigating the clonal dynamics of the above pathogens are in concordance that the crucial difference between the major and minor clone strains lies in the formers' ability (or the inability of the latter) to develop favorable but not detrimental genetic alterations mutations in the DNA gyrase and topoisomerase IV genes -MRSA in Europe and the United States, the ST22 and the new lineage of ST8, both characteristically carry two identical mutations: he Table , while mhe Table .Interestingly these double-serine mutations were also observed in rapidly expanding local strains (sublineages) of other sequence types of MRSA  though will often be complemented with additional QRDR mutations . Furthermore, they harbor usually three or sometimes two supplementary mutations; typically: gyrA Asp87Asn, parC Glu84Val, and parE Ile529Leu  and an acidic residue four amino acids downstream of both respective positions  was observed to be associated with some fitness cost  the molecule's physiology remains intact  characteristically carries four or rather five well-defined mutations  have not been individually tested the global success of the ST131 H30 isolates carrying these genetic traits  incur higher level resistance to fluoroquinolones in S. pneumoniae  conferred high-level resistance to an isolate and were observed to be associated with just a very small fitness cost  also incurred high-level resistance and were associated with some loss of vitality in a strain-specific fashion  differences exert on the evolution of favorable QRDR mutations in P. aeruginosa.Further studies could unequivocally establish whether or not the double-threonine/serine mutations contributed to the clonal dynamics in B. cepacia the gyrA Thr83Ile and parC Ser80Leu double mutations were reported to have conferred high level resistance to fluoroquinolones but were associated with significant fitness cost.In C. difficile and C. jejuni—can-not evolve genetic combinations equivalent to the double mutations mentioned above. In the major international clones of C. difficile a single QRDR mutation—gyrA Thr82Ile—proved optimal, concurrently ensuring proper fluoroquinolone MIC value and adequate fitness balance . Some mC. difficile a single mutation—gyrA Thr86Ile—may confer the adequate level of resistance to C. jejuni  was not restricted to a few STs/lineages. To our understanding C. jejuni may be the single pathogen among bacteria reviewed in this paper that has efficiently adapted to resistance against fluoroquinolones “as a species.” In this bacterium a multitude of genetic variants - not just a few clones—have been able to develope favorable mutational resistance . Moreover, there is some circumstantial evidence that a similar mechanism may have contributed to the dissemination of fluoroquinolone resistance in S. pneumoniae and P. aeruginosa (see above).It is well-established that C. difficile and C. jejuni lacking the topoisomerase IV gene had to embrace different strategies. However, even in these species the ability to evolve an energetically favorable gyrA threonine mutation proved crucial for dissemination—for major clones (C. difficile) or for the whole species (C. jejuni)—in a fluoroquinolone environment.Slow-growing bacteria like As we have seen individual STs/lineages of various species differ in their ability to develop favorable QRDR mutations. This trait, however, has been shown to be amenable to change. Clones/lineages are capable of undergoing a remarkable metamorphosis and improve their ability of evolving QRDR mutations.ST239 and ST8 MRSAs remain minor clones in Hungary and the latter was observed to lack the favorable double-serine genetic combination  is associated with a slight fitness advantage. The question arises, if these amino acids are not favorable why have they been evolutionarily preserved across species?The double-serine residues confer susceptibility to an important group of antibiotics, fluoroquinolones. In addition, the replacement of the gyrase serine residue  were all susceptible to a natural antibiotic  produced by several streptomyces species. Conversely fluoroquinolone susceptible wild type isolates harboring both serine residues proved resistant. The parC serine mutation proved pivotal in incurring resistance -Ib-cr- conferring reduced susceptibility to ciprofloxacin by N-acetylation of its piperazinyl amine. Both mechanisms confer low-level resistance to fluoroquinolones -Ib-cr gene is common in both major ST/lineage ESBL-producing K. pneumoniae and ESBL-producing E. coli isolates  have been mentioned as potentially essential clonal enhancers, however, the incidence of these virulence factors remains at variance with the observed clonal shifts in HA-MRSA. The issue was recently reviewed Fuzi, .In summary, virulence factors seem to play only a secondary role compared with diverse fitness cost associated with fluoroquinolone resistance in the selection and promotion of international ST/lineage isolates in the multi-drug resistant variants of at least three species.S. pneumoniae and P. aeruginosa hint at a possible role for fluoroquinolones in the dissemination of these pathogens. However, in an additional species—vancomycin-resistant E. faecium (VRE)—both the genetic and epidemiological evidence for the involvement of a similar process remains much stronger.As we have shown data available on It is well-established that a few lineages related to a single international clone of VRE (CC17) dominate the hospital setting worldwide .Though resistance to ampicillin has been a feature of CC17-related lineages for a long time  why these “competent” pathogens are more adept to favorably mutate than most strains of the same species and how previously “non-mutating” strains develop the capacity to mutate remain to be elucidated. Furthermore, our better understanding of the mechanism of clonal dynamics permitting a more judicious use of fluoroquinolone type antibiotics would allow for the amelioration of the general antibiotic resistance situation.MF devised and wrote the manuscript. DS and RC helped with their advice and provided relevant literature.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.


Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the number of proteins analyzed, the accuracy of spot quantification and reproducibility. In this review article, the efficiency of the most widely used dyes was investigated. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes  and cyanine labeled methods were compared. The next typical steps of the flow of gel-based proteomics are spots visualization and evaluation , expression analysis, and finally protein identification by mass spectrometry . In order to take advantage of the high resolution capacity of 2DE, proteins have to be completely denatured, disaggregated, reduced and solubilized  to disrupt molecular interactions and to ensure that each spot represents an individual polypeptide. Proteins can be stained before the 2DE separation (pre-electrophoretic protein stain), or after 2DE separation (post-electrophoretic protein stain).Protein separation by two-dimensional electrophoresis 2DE) is largely used in proteomic approaches because of both high resolution and the availability of powerful image analysis software for gel comparison and compatibility with subsequent protein characterization by mass spectrometry . For theDE is larClassically, Coomassie blue was the most widely used non-covalent dye for post-electrophoretic protein staining . HoweverCovalent protein labeling, called ‘‘difference gel electrophoresis’’ (DIGE) is nowadays an interesting alternative, which allowes multiplexing of samples and the use of an internal standard . This prThe present review compares several non-covalent and covalent protein dyes for large-scale comparison of 2D gel electrophoresis patterns by image analysis in order to select the best method according to the biological question and sample type.Figure 2). R stands for reddish hue and G for greenish hue, the number 250 is an indicator number for dye strength. Typically, R-250 is used to stain SDS polyacrylamide gels and G-250 in the Bradford assay. The G-250 form allows an intensification of the stain with a low background in comparison with the R-250 form, thanks to an apparent conversion into a colloidal state in 12.5% TCA [Two forms of Coomassie brilliant blue are available, R-250 and G-250  and aromatic residues  give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Such non-covalent binding of the dye allowed an excellent compatibility with MALDI-TOF mass spectrometry .) contributed to a lack of information certainly due to a decrease of precision during spot picking. Moreover, an inconsistent mass compatibility was observed essentially for the weak concentration [Colloidal Coomassie blue was observed as a judicious dye for large scale proteomic analysis . It allontration .Two categories of silver staining were used to visualize proteins in gel: the acidic silver nitrate and the alkaline silver diamine procedure, which differed on binding specificity, sensitivity, cost and safety risk . Numberset al. [) [The “Vorum silver staining protocol” of Mortz et al.  allowed t al. [) . Acidic t al. [) . In our t al. [) , silver During the last decade, different fluorescent dyes were introduced and proved to combine high sensitivity and compatibility with mass spectrometry. These encompass both commercially available stains, such as the series of Sypros , and Rut) and the good properties of classical organic stains such as Coomassie blue [Sypro Ruby was described to combine sensitivity close to that of silver staining  [Deep Purple is a sensitive fluorescent-based stain  based onemission . It was emission . The gression [) . A littlssion [) . This wassion [) .Difference gel electrophoresis (DIGE) takes advantages of structurally similar cyanine-based dyes to label different pools of protein samples, which are then co-separated on a single 2DE gel .Figure 4). Since the same proteins present in two different samples were pre-labeled with two different dyes , they could be combined and separated on the same 2DE without the loss of the relative protein abundance in the original samples [The biggest advantage of DIGE over other 2DE-based technologies is that it enables the analysis of two or more protein samples simultaneously on a single 2DE . For differential 2DE analysis it is important to obtain maximal information by combining non-covalent and covalent staining techniques. DIGE allows identification of faint spots from several samples, but in the future, the detection range of proteins needs to be considerably lowered to allow identification by mass spectrometry of such low abundance proteins.Several dyes are available to label proteins, either before or after electrophoresis. Some are quite expensive  while others are rather economical  and affordable, as well as useful in protein identification by mass spectrometry (


Most impressive was the significant improvement in the mini mental state examination [MMSE (p < 0.001)]. Some metabolic parameters such as plasma malondialdehyde, markers of insulin metabolism and serum triglycerides were also significantly different in the AD patients compared with the control group, and probably caused by probiotic treatment. Additionally, serum high sensitive C-reactive protein (hs-CRP) significantly changed after probiotic supplementation (p < 0.001). However, changes in other biomarkers of oxidative stress and inflammation were negligible. The authors concluded that evaluation of other biomarkers of inflammation and oxidative stress would be informative.Akbari and colleagues .In an earlier study , with Akkermansia muciniphila (p < 0.01) and with serum neopterin (p < 0.05). Further, a strong correlation was found between anti-inflammatory α1-antitrypsin and pro-inflammatory S100A12 (p < 0.001) in the fecal specimens of our cognitively impaired patients. The anti-inflammatory action of α1-antiptrypsin on microglial mediated neuroinflammation could be shown in vitro , a protein modulating tight junction permeability between cells of the digestive tract. All these findings together may indicate changes in the microbiota-gut-brain-axis correlated to neuroinflammation during cognitive decline. Because neuroinflammation is an early event in the pathogenesis of dementia (Caracciolo et al., In our series The increased immune activation and inflammation in AD could indeed relate to the age-related changes of gut microbiota as is indicated by the close relationship between fecal S100A12 and serum neopterin concentrations (Leblhuber et al., Overall, the role of probiotics in preventing dementia seems promising and should be further elucidated in future studies. These investigations should include the bacterial taxa as well as the serum and intestinal inflammation markers mentioned above together with the metabolic parameters mentioned by Akbari et al. . A possiAll authors have made substantial contribution and critical revision to this 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.


T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role in the development of autoimmune diseases. Novel insights into this mechanism are crucial to understanding disease development and establishing new treatment strategies. MHC multimers have been used for detection of antigen-responsive T cells since the first report by Altman et al. showed that tetramerization of pMHC class I molecules provided sufficient stability to T cell receptor (TCR)-pMHC interactions, allowing detection of MHC multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for expanding the capacity of MHC multimer-based detection technologies to facilitate large-scale epitope discovery and immune monitoring in limited biological material. Screening of T cell specificity using large libraries of pMHC molecules is suitable for analyses of T cell recognition potentially at genome-wide levels rather than analyses restricted to a selection of model antigens. Such strategies provide novel insights into the immune specificities involved in disease development and response to immunotherapy, and extend fundamental knowledge related to T cell recognition patterns and cross-recognition by TCRs. MHC multimer-based technologies have now evolved from detection of 1–2 different T cell specificities per cell sample, to include more than 1000 evaluable pMHC molecules using novel technologies. Here, we provide an overview of MHC multimer-based detection technologies developed over two decades, focusing primarily on MHC class I interactions. Techniques for the detection of antigen-responsive T cells exploit the interaction between a given TCR and its pMHC recognition motif. T cell detection using multimerized pMHC molecules has evolved into the preferred method for detecting antigen-specific T cells. Over the past 20 years, since the first multimerizations of pMHC molecules , severalMHC multimer technologies have primarily been developed and applied for analyses of CD8 T cell responses, because MHC class I molecules have proven easier to handle in terms of protein folding and expression. Additionally, the MHC class I binding groove is more restricted in terms of the length of peptide bound—hence, it is more straightforward to predict MHC class I binding peptides. Most of the technologies described in this review relate to detection of specific CD8 T cell responses, but they are in principle also applicable to MHC class II multimers and detection of CD4 T cell responses. Specific challenges associated with the production and use of MHC class II multimers are addressed in the final section.b molecules. The di-peptide exchange technology has not yet been applied in larger T cell epitopes mapping strategies. Together, these technologies have enabled efficient production of large libraries of pMHC molecules, and consequently high-throughput detection of CD8 T cell recognition using pMHC-based reagents.MHC molecules are largely unstable when they are not part of a complex with peptide. For this reason, pMHC-based technologies were initially restricted by the tedious production of pMHC molecules, where each peptide required an individual folding and purification procedure , 3. ThusAll the MHC-based strategies described throughout this report are summarized in Table Early attempts to improve the complexity of MHC multimer-based detection of antigen-responsive T cells were based on the success of gene-expression arrays, including the availability of efficient systems capable of quantifying fluorescent probes when spatially separated as spots on a microarray . ProteinIn early studies by Soen et al.  and ChenA general limitation with such array-based strategies is the propensity of a given T cell to pursue all potential pMHC interactions displayed on a given array. As a consequence, the frequency of antigen-responsive T cells in the cell preparations should be >0.1% to allow a robust readout. Deviren et al.  particulAn alternative approach to pMHC microarrays is to utilize the increasing number of fluorescent labels available for flow cytometry. Two such parallel approaches were described in 2009, using combinatorial encoding of fluorescently labeled MHC multimers. Both of these approaches take advantage of the assumption that any given T cell in the cell samples will only bind one of the pMHC molecules in a given library. Thus, each distinct pMHC multimer can be assigned with a unique dual color code , which iWith the emergence of mass cytometry that uses heavy metal ions as tags, there has been a dramatic leap in the number of labels that can be applied in parallel –24. CombMore recently, a matrix-based approach has been developed for T cell identification. In this method, a set of MHC multimers are split into matrix-defined pools, in such a way that no specific MHC multimer are present in all pools. The multimer pools are used to stain an equal number of cell aliquots derived from a given sample. The approach uniquely combines antigen recognition with sequencing of the TCRβ chain in that the MHC multimer positive and negative populations of each reagent pool are sorted and clonotypes enriched within the positive population are identified . The advThis forms an attractive tool for detecting many antigen-responsive T cells simultaneously, especially since it also enables the identification of the corresponding TCRβ clonotypes. However, the necessity to split samples into a number of pools represents an inherent drawback in terms of detecting rare T cell populations especially in patient material that is often of limited access.Thus, until recently no technology has been available that simultaneously permits high-throughput analyses of T cells while tolerating the large diversity of T cell antigen specificity, thereby allowing detection of epitopes at potentially genome wide levels even in limited biological samples, and recovery the positively interacting T cells for gene expression analysis.10 different sequences, each forming a unique tag   .The DNA barcode-based approach offers the advantage that it is less dependent on stringent fluorescent-based separation of multimer-positive from multimer-negative events, because even events that may not be fluorescently separated due to low level pMHC multimer interactions will still bind enough DNA barcode-labeled MHC multimers to allow recovery of their specific DNA barcode element, thus enabling the identification of the TCR specificity. The technology therefore represents an attractive tool for detecting low avidity TCR:pMHC interactions such as shared tumor-associated antigens. When applying cytometry-based approaches, the signal intensity associated with a given pMHC is the only measure distinguishing those T cells that interact specifically with MHC multimers and those that do not. Therefore, it is essential that a given T cell binds numerous MHC multimers, to obtain sufficient fluorescence intensity and thereby enable unambiguous identification of their specificity. A T cell that has low affinity TCR:pMHC interactions or low surface TCR expression may not bind sufficient MHC multimers to obtain fluorescent separation, but will still provide a signal from the attached DNA barcode. We have demonstrated that the antigen-responsive composition of T cells in a sample can be determined independent of any fluorescent label, by sorting the whole CD8 T cell population as opposed to only the multimer-positive T cells .In line with the reasoning that antigen recognition can be determined from the number of DNA barcode reads irrespective of the fluorescent-based separation of multimer-positive cells, other criteria can be applied to determine which cell populations to sort. Throughout the workflow of the DNA barcode-based strategy only a few different fluorescent labels are applied to determine the viable CD8 positive population, leaving a considerable number of fluorescent labels to stain for other cell properties. Thus, one can distinguish between CD8 T cells with certain phenotypic characteristics or of a certain activation status, and retrospectively determine the composition of antigen-responsive T cells among the different populations through sequencing of the associated DNA barcodes. This strategy was illustrated with the activation of antigen-responsive T cells present in healthy donor blood or tumor-infiltrating lymphocytes (TILs), through stimulation with a pool of common virus-derived peptides or autologous tumor cell line, respectively. Subsequently, samples are stained with collections of DNA barcode-labeled MHC multimers and intracellular activation markers. The fluorescent signal from the MHC multimers was disregarded and cells were sorted exclusively based on the cytokine secretion profile . Thus, w10; thus the technology has the potential for analyzing T cell reactivity at close to genome-wide levels. Using earlier high-throughput methodologies it has not been possible to analyze for T cell reactivity of structurally related pMHC epitopes in the same sample, e.g., peptides differing only at a single amino-acid position or 9-, 10- or 11-mer versions of the same peptide with a given HLA restriction. However, this issue could be circumvented without much difficulty since these strategies, being only semi-high-throughput, have often required the splitting of samples into several tubes to analyze all potential epitopes of interest. Users simply have to ensure that different versions of a pMHC epitope were represented in different MHC reagent mixtures. However, with the possibility of screening, e.g., a full cancer-mutagenome in one sample, it is essential that structurally related pMHC epitopes can be identified in parallel using only one mixture of MHC reagents. The DNA barcode-based approach provides information on recognition of a given pMHC molecule, but such recognition may likely be shared among several T cell populations. Thus, cross reactivity to multiple epitopes in a given library will not disrupt analyses. Analyzing the cross-reactive features and relative recognition profile among different pMHC complexes can be accomplished when T cells are assessed at a single cell level.Inarguably, the greatest advantage of using DNA barcode-labeled MHC multimers is the possibility to screen in an unbiased manner for T cell reactivity across large libraries of pMHC epitopes in a single sample. Applying the current DNA barcode design, we can generate unique labels in the order of 10The DNA barcode labeling of MHC multimers provides a new tool to gain insight to T cell recognition in complex disease situations. Nonetheless, the main disadvantage of the methodology is that it merely provides an estimate for T cell frequencies, and does not offer any possibility for direct visual assessment of the findings in terms of T cell recognition. Thus, users rely on good quality assurance of the sequencing procedure and validated programs to translate read number into the presence of antigen-responsive T cells. Furthermore, pMHC binding T cells are lysed in the process of DNA barcode amplification, and are consequently not available for subsequent cell culturing.Due to the limited number of cells available directly from excised tumor fragments, it has for most prior purposes been necessary to expand TILs in vitro to enable analyses of antigen recognition of the relevant pMHC multimer molecules. Previous studies have shown that expansion of T cells may introduce a bias in antigen recognition due to variable growth potential of TILs, and specifically, preferential growth of T cell populations that recognize virus epitopes . ApplicaSince it is no longer necessary to split samples into multiple aliquots to screen for antigen recognition, it is possible to look for T cell recognition in an unbiased fashion in samples with a considerably greater number of precursor cells than what had previously been applied. With increasing cell numbers, the stochastic variation of the compositions of antigen-specific T cell populations is reduced, and the chance for detecting low-frequent T cell populations increases, reflecting a higher absolute number of pMHC responsive T cells in the given sample.6 TILs/MHC reaction mixture (9–13 different reaction mixtures/patient specific neoepitope library). Despite the limited availability of sample, we were able to detect a total of nine different neoepitope-responsive T cells when applying the DNA barcode-based MHC multimer technology. This may partly be explained by the greater number of cells included in the initial binding reactions (4–8 × 106 TILs in one MHC reaction mixture) and due to the possibility for analyzing peripheral blood. In fact, several of the detected responses were predominantly or exclusively found in the peripheral blood sample [The advantages of being able to include more T cells in the initial binding reaction were illustrated in a study where we screened for T cell recognition of predicted HLA-binding peptide sequences containing cancer-specific mutations in two patients with non-small cell lung carcinoma. A large number of personal neoepitope peptides had been predicted for each patient resulting in libraries of 288 and 417 different pMHCs, respectively . In thesE. coli [+ T cell populations.An additional challenge in the field of immune analyses relates to the characterization of antigen-responsive CD4 T cells. Antigen-responsive CD4 T cells are believed to play a significant role in tumor-cell recognition , 34 and E. coli . PeptideE. coli . ImportaE. coli . MoreoveE. coli . This maAlong with the improvements in prediction tools for MHC-II ligands  and the 10, the limitations inherent to combinatorial encoding using fluorescent or metal tags have been removed. However, new bottlenecks may arise with increasing library sizes. Moving the complexity of MHC multimer libraries within the range of 10,000–100,000 specificities will require automation of all aspects of the pMHC multimer production as well as more efficient and cost-effective production of synthetic peptides. Furthermore, it will most likely require the development of microfluidics systems that can ensure efficient pMHC interaction with all T cells in a given sample.At present proof-of-principle have been provided that up to 1031 DNA barcode-labeled MHC multimers can be applied in one reaction mixture to detect several populations of antigen-responsive T cells in parallel. This complexity, though already representing a tenfold leap compared to earlier methodologies, can potentially be increased to >10,000–100,000 differently labeled MHC multimers. Since the potential complexity of the DNA barcode label is in the order of 10In the era of personalized medicine and with accumulating types of immunotherapeutic strategies being employed, it is becoming increasingly important to understand the T cell-mediated recognition of cancer cells, and identify biomarkers predicting the response to therapy—potentially based on experimental analyses of T cell recognition Fig. .Recent data suggest that a substantial fraction of the T cell reactivity induced during checkpoint inhibition is directed towards neoepitopes that are generated through genetic alterations –46. Howe


To investigate atrial flow patterns in the normal adult heart, to explore whether caval vein arrangement and patency of the foramen ovale (PFO) may be associated with flow pattern.Time-resolved, three-dimensional velocity encoded magnetic resonance imaging (4D flow) was employed to assess atrial flow patterns in thirteen healthy subjects  and thirteen subjects  with cryptogenic stroke and patent foramen ovale (CS-PFO). Right atrial flow was defined as vortical, helico-vortical, helical and multiple vortices. Time-averaged and peak systolic and diastolic flows in the caval and pulmonary veins and their anatomical arrangement were compared.A spectrum of right atrial flow was observed across the four defined categories. The right atrial flow patterns were strongly associated with the relative position of the caval veins. Right atrial flow patterns other than vortical were more common (p = 0.015) and the separation between the superior and inferior vena cava greater  in the CS-PFO group. In the left atrium all subjects except one had counter-clockwise vortical flow. Vortex size varied and was associated with left lower pulmonary vein flow . A diastolic vortex was less common and time-averaged left atrial velocity was greater in the CS-PFO group . One CS-PFO subject demonstrated vortical retrograde flow in the descending aortic arch; all other subjects had laminar descending aortic flow.Right atrial flow patterns in the normal heart are heterogeneous and are associated with the relative position of the caval veins. Patterns, other than ‘typical’ vortical flow, are more prevalent in the right atrium of those with cryptogenic stroke in the context of PFO. Left atrial flow patterns are more homogenous in normal hearts and show a relationship with flow arising from the left pulmonary veins. The right atrium is a dynamic, complex structure with multiple functions that evolve during life. In the fetus, it directs nutrient-rich blood to the left heart via the foramen ovale; whilst postnatally it functions as a reservoir, a conduit and a pump to deliver deoxygenated blood via the right ventricle to the pulmonary circulation.The morphology and function of the right atrium is now well described but there is limited literature describing the nature of blood flow within it. The accepted pattern, reported in case studies, is that of a clockwise vortex with a counter-clockwise vortex observed in the left atrium . Time-reThe foramen ovale, which is universally present in fetal life, persists postnatally in a quarter of the population . VariousThe purpose of this study was to investigate atrial flow patterns in the normal adult heart, primarily to establish whether right atrial flow is always vortical and if not, whether absence of vortical flow may be associated with patency of the foramen ovale in the setting of a related clinical syndrome. In addition we report left atrial and aortic flow, as previous studies have suggested they may be influential in the evolution of cryptogenic stroke in the patient with patent foramen ovale –12.Thirteen subjects  with normal trans-thoracic echo were prospectively recruited between May 2013 and May 2015. All control subjects were recruited by advertisement within the Newcastle Hospitals NHS Foundation Trust. Also, thirteen subjects  with cryptogenic stroke or transient ischaemic attack and patent foramen ovale (CS-PFO) on trans-oesophageal echocardiography were recruited. Exclusion criteria included age under 18 or over 55 years or contraindication to magnetic resonance imaging (MRI). Newcastle and North Tyneside 2 Ethics committee approved the study (12/NE/0140). All participants gave informed written consent.CS-PFO subjects had no other identifiable cause for their stroke event and had been referred for consideration of PFO closure and consecutively recruited to this study. Vascular risk factors were assessed at the time of attendance for 4D flow imaging.Trans-oesophageal echocardiography was performed, as per standard clinical protocols in the CS-PFO group, with local anaesthesia under light sedation. The diameter  and length  of the PFO tunnel, presence of an atrial septal aneurysm (excursion >10 mm) and Eustacian valve prominence were recorded .3, temporal resolution: 50-55ms, 20 phases, SENSE factor 2 and turbo factor 2). A respiratory navigator was employed to reduce motion artifact  . Hel. Hel2]. Fig 2). Time-averaged flow was also obtained. Time-averaged velocity within the manually segmented right atrial volumes was also measured.Planes were positioned, in the angiogram generated from the previous manual segmentation, orthogonal to the caval veins at their junction with the right atrium . A contour was placed in each orthogonal plane and adjusted for vessel motion during the cardiac cycle. The flow waveform at each contour was plotted and peak systolic and diastolic flow measured  and extended to the mitral valve annulus. Left atrial vortex size was measured at peak systole and diastole using the 4D flow MRI data. A plane was placed orthogonal to the vortex core and the radii of the major and minor axes of the ellipsoidal vortex were measured in order to calculate the cross-sectional area. Duration of the vortex was assessed visually: the vortex was defined as being present when the flow pattern formed a complete circle/ellipse. Left atrial area at end systole in the two-chamber cine view was measured as the best surrogate for left atrial volume .Fig 2). Time-averaged flow was also obtained. Time-averaged velocity within the left atrial volumes was also measured.Planes were positioned, using the angiogram generated from the previous manual segmentation, orthogonal to the pulmonary veins at their junction with the left atrium. A contour was placed in each orthogonal plane and adjusted for vessel motion during the cardiac cycle. The flow waveform at each contour was plotted and peak systolic and diastolic flow measured . Age is expressed as mean and range. Continuous variables are expressed as mean ± standard deviation. Normality was assessed for all continuous variables using the Anderson-Darling test. Continuous data were compared using the two-tailed, unpaired Student t-test. Categorical data were compared using Fischer’s Exact test. Pearson correlation was used to identify possible predictors of left atrial vortex cross-sectional area. Significance was set at<0.05. The inter-scan, inter-observer and intra-observer bias and 95% limits of agreement were separately calculated for time-averaged caval flow, time-averaged pulmonary venous flow and time-averaged atrial velocity using the Bland-Altman method .Tables 2. Body mass index, heart rate and systolic BP were comparable between groups. Diastolic BP was higher in the CS-PFO group .Baseline demographics are shown in Of the CS-PFO subjects, six were recruited from the Newcastle Hospitals NHS Foundation Trust and seven subjects from approved participant identification centres in the North of England. Eleven CS-PFO subjects had clinical and radiological evidence of cryptogenic stroke. One subject presented with an ophthalmic stroke and had normal brain imaging but evidence of branch retinal artery thrombosis on ophthalmoscopy. One subject’s neurological symptoms resolved within 24 hours, and were thus classified as a transient ischemic attack, but demonstrated multiple minor abnormalities on imaging.The diameter of the PFO tunnel was 3±2mm and tunnel length was 11±5mm. Six subjects had a concomitant atrial septal aneurysm and five patients had a prominent Eustacian valve. One subject had lipomatous hypertrophy of the inter-atrial septum. Four subjects had a severe shunt at rest. No Chiari networks were seen.Table 3). Five CS-PFO subjects did not have these patterns and exhibited multiple vortices with various patterns observed . Additional small vortices were identified secondary to the primary flow pattern in the first three groups. No more than two vortices were seen in any scan.A spectrum of primary flow patterns were observed and defined as vortical, helico-vortical, helical and multiple vortices as described in the methods (Table 3). Peak systolic and diastolic flow in both SVC and IVC was comparable between CS-PFO subjects and controls. Time to peak systolic and diastolic flow, when adjusted for heart rate also showed no difference between CS-PFO subjects and controls. However, when analysed for flow patterns, non-vortical right atrial flow  showed reduced peak systolic flow  and a trend to reduced peak diastolic flow  in the IVC. No difference in time-averaged caval flow or time-averaged atrial velocity was found between groups or between flow patterns (Table 3).Absence of the ‘standard’ vortex during systole and diastole was more common in the CS-PFO group . The separation of the caval veins in the right left plane also corresponded to systolic flow patterns , S1 Fig.Table 5).The majority of CS-PFO subjects (11 out of 13) and controls (12 out of 13) had four pulmonary veins. There was no statistical difference between the numbers of pulmonary veins seen between groups. There was also no difference in left atrial size between groups, although it was notable that the CS-PFO group exhibited a greater range of left atrial sizes. All CS-PFO subjects and controls, except one, had ‘typical’ vortical flow within the left atrium, described in literature as counter-clockwise .(MP4)Click here for additional data file.


We examined the effectiveness of early rehabilitation for the prevention of postintensive care syndrome (PICS), characterised by an impaired physical, cognitive or mental health status, among survivors of critical illness.We performed a systematic literature search of several databases  and a manual search to identify randomised controlled trials (RCTs) comparing the effectiveness of early rehabilitation versus no early rehabilitation or standard care for the prevention of PICS. The primary outcomes were short-term physical-related, cognitive-related and mental health-related outcomes assessed during hospitalisation. The secondary outcomes were the standardised, long-term health-related quality of life scores (EuroQol 5 Dimension (EQ5D) and the Medical Outcomes Study 36-Item Short Form Health Survey Physical Function Scale (SF-36 PF)). We used the Grading of Recommendations Assessment, Development and Evaluation approach to rate the quality of evidence (QoE).Six RCTs selected from 5105 screened abstracts were included. Early rehabilitation significantly improved short-term physical-related outcomes, as indicated by an increased Medical Research Council scale score (standardised mean difference (SMD): 0.38, 95% CI 0.10 to 0.66, p=0.009) (QoE: low) and a decreased incidence of intensive care unit-acquired weakness , compared with standard care or no early rehabilitation. However, the two groups did not differ in terms of cognitive-related delirium-free days  and the mental health-related Hospital Anxiety and Depression Scale score . Early rehabilitation did not improve the long-term outcomes of PICS as characterised by EQ5D and SF-36 PF.Early rehabilitation improved only short-term physical-related outcomes in patients with critical illness. Additional large RCTs are needed. This meta-analysis is the first meta-analysis of comprehensive postintensive care syndrome (PICS) based on randomised controlled trials (RCTs), in which the study intervention populations were limited to early rehabilitation.This meta-analysis was mainly limited by (1) the small number of patients in the included RCTs, (2) a lack of detailed analysis of the adverse effects and PICS domains after hospital discharge and (3) no confirmation of the exact first day of rehabilitation initiation in one of the six included RCTs.We used the Grading of Recommendations Assessment, Development and Evaluation approach in the review process.Dramatic developments and improvements in the technique, instruments and education systems used in intensive care units (ICUs) have reduced the mortality of patients with critical illness over the past four decades.2 3Postintensive care syndrome (PICS) was established as a new syndrome encompassing new or worsening impairments in physical, cognitive or mental health status that arise after critical illness and persist beyond acute care hospitalisation, with the aim initiating improvements for ICU survivors and their families across the continuum of care.Physiotherapy with early rehabilitation is considered an integral component of the multidisciplinary management of patients in ICUs. In other populations, exercise has been shown to improve strength and function, decrease inflammationhttp://www.crd.york.ac.uk/PROSPERO/) (Registration No. CRD42016039759).18This review protocol was registered in PROSPERO, an International Prospective Register of Systematic Reviews at the National Institute for Health Research and Centre for Reviews and Dissemination at the University of York (We searched the Medline (via PubMed from 1996 to 7 June 2016), Embase (until 7 June 2016) and Cochrane Central Register of Controlled Trials (CENTRAL) databases (until 7 June 2016) for full-text clinical trials conducted in humans to retrieve relevant articles for the literature review. We also performed a manual search to retrieve all potentially relevant articles from 8 June to 31 August 2016. The key search terms used to identify potential studies are listed in online 10.1136/bmjopen-2017-019998.supp1Supplementary dataThree authors  performed the first-line comprehensive literature search and removal of duplicates. Subsequently, two randomly selected authors independently screened the study titles and abstracts for potential relevance. The kappa value for selection bias was 0.396. When disagreements arose between reviewers, the full text of the paper was retrieved, and the disagreements were reconsidered and discussed until a consensus was reached. The full texts of articles included in the final selection were reviewed by two randomly chosen authors. We included trials with the following characteristics:Study types: Randomised controlled trials (RCTs) were included; non-randomised and observational studies were excluded.Population: Adult patients (aged >18 years) admitted to the ICU were included. We excluded patients with traumatic brain injury and stroke who did not fulfil the PICS criteria, specifically those with acquired physical and psychiatric/cognitive dysfunction after ICU admission.Intervention: The intervention was early rehabilitation, defined as (1) starting at an earlier time point than usual care or (2) administration within 7 days of ICU admission. ’Rehabilitation' included all physiotherapy, occupational therapy and palliative care-related support. We excluded RCTs in which rehabilitation was initiated before ICU admission and those comparing early rehabilitation with another intervention.Control: The control group received standard care or no early rehabilitation.Outcome: The primary outcomes  were as follows: (1) physical-related outcomes (incidence of ICU-acquired weakness (AW), Medical Research Council (MRC)To assess the quality of the included studies, we adopted the Cochrane risk of bias tool.25Two authors (RF and TH) independently used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) tool to rate the quality of evidence (QoE). We used the GRADE approach to rate the QoE of early rehabilitation for outcomes. Although the QoE represents a continuum, we assessed the quality of the body of evidence for each outcome categorised as high, moderate, low or very low using the GRADE pro Guideline Development Tool.The author(s), title, journal name, year of publication, website URL and abstract of each included article were identified. Conference abstracts were excluded. Data from each study were extracted independently by two review authors. When faced with insufficient or incomplete data, we contacted the authors of the studies directly. Information related to the study population, sample size, interventions, comparators, potential biases in the conduct of the trial, outcomes (including adverse events), follow-up and statistical analysis methods were extracted from the original reports into specially designed paper forms and then entered into a spreadsheet.As data from several trials were available, we performed a meta-analysis according to the Cochrane Handbook for Systematic Reviews of Interventions and PRISMA guidelines using Review Manager software . We summarised the results of this meta-analysis using the generic inverse variance method to facilitate the pooling of estimates of treatment effects. We used ORs with 95% CIs for dichotomous outcomes, and mean differences or standardised mean differences (SMDs) with 95% CIs for continuous outcomes when appropriate. If a quantitative synthesis was not appropriate for a particular outcome, we devised a qualitative summary of that outcome.2 statistics to quantify inconsistencies. We considered that significant heterogeneity was present when the reason for heterogeneity could not be explained and the I2 was ≥50%. The presence of strong clinical heterogeneity was considered when deciding to perform a quantitative data synthesis or a sensitivity analysis with a special focus.26For each outcome, we assessed heterogeneity between trials using IEstimates were pooled using a random effects model. We performed our analysis based on all published or available data.The PRISMA flow chart of study selection is shown in 10.1136/bmjopen-2017-019998.supp2Supplementary dataThe study characteristics are summarised in The evaluation of the risk of bias in each included RCT is shown in the risk of bias summary for PICS-related outcomes without in The quality assessment and a summary of each outcome measure are shown in GRADE working group grades of evidence: High quality—further research is very unlikely to change our confidence in the estimate of effect. Moderate quality—further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate. Low quality—further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate. Very low quality—we are very uncertain about the estimate.Incidence of ICU-AW: Two RCTs reported the incidence of ICU-AW (2=0%).f ICU-AW . There wThe risk of bias was determined to be serious because all RCTs included a moderate risk of bias. Imprecision was also determined to be serious because of the small number of included patients as per the optimal information size (OIS) criteria. Therefore, the QoE was downgraded from high to low because of these serious risks.MRC: Three RCTs reported MRC scores (2=0%)).C scores . We obseDelirium-free days: Two RCTs reported the incidence of delirium-free days (2=0%). The risk of bias was determined to be serious because all RCTs included a moderate risk of bias, and imprecision was also determined to be serious because of the small number of included patients as per OIS criteria. These serious risks led to the downgrading of the QoE from high to low.ree days . There wIncidence of HAS/HADS: Two RCTs reported the incidence of HAS/HADS (2=0%). The risk of bias was determined to be serious because all RCTs included a moderate risk of bias, and imprecision was also determined to be serious because of the small number of included patients. These serious risks led to the downgrading of the QoE from high to low.HAS/HADS . These gEQ5D: Two RCTs reported the EQ5D scores (2=72%). The risk of bias was deemed serious because all RCTs demonstrated a moderate risk of bias, and inconsistency was deemed serious because of severe heterogeneity . Imprecision was also considered serious because of the small number of included patients. These serious risks led to the downgrading of the QoE from high to very low.D scores . No signSF-36 PF: Two RCTs reported SF-36 PF scale scores (2=98%)). However, the different timings of SF-36 PF scale evaluation among the various studies led to significant heterogeneity. Imprecision was also considered serious because of the small number of included patients as per the OIS criteria. Therefore, these serious risks led to the downgrading of the QoE from high to very low.e scores . HoweverThis meta-analysis revealed that early rehabilitation significantly improved short-term physical function in patients with critical illness, as assessed by the MRC scoring system and the incidence of ICU-AW. However, it did not significantly improve the patients' cognitive and mental status-related outcomes. These results suggest that early rehabilitation has a limited effect on the prevention of PICS in patients with critical illness, and only reflect improvements in short-term physical function.To our knowledge, the present work is the first meta-analysis of comprehensive PICS based on RCTs in which the study intervention populations were limited to early rehabilitation. Additionally, we used the GRADE approach in the review process which allowed us to make judgements about the QoE and strength of recommendations in a systematic and transparent manner. Thus, the present meta-analysis allowed us determine whether critical care physicians should initiate early rehabilitation for patients with critical illness, and provided new evidence about the effectiveness of physiotherapy interventions aimed at early mobilisation.33et al found that physical therapy in the ICU may improve muscle strength, physical function, quality of life, ventilator-free days, ICU stay and hospital stay; however, the authors included one RCT involving patients who underwent laparotomy , and another study initiated physical therapy 30 days after bed rest.et al used the Medical Outcomes Study 36-Item Short Form survey score, whereas the present review used the EQ5D.Four systematic reviews have addressed the effect of rehabilitationet al conducted a systematic review and meta-analysis of the effect of early rehabilitation during an ICU stay on functional statuset al.et al reported a systematic review regarding the effects of active mobilisation and rehabilitation in the ICU on mortality and function,et al.Castro-Avila The initiation of early rehabilitation improved short-term outcomes, such as the physical function score and the incidence of ICU-AW in the present systematic review; however, it did not improve. The tendency to favour early rehabilitation during mechanical ventilation suggests that early rehabilitation could potentially improve short-term survival and maintain and increase muscle strength.However, the initiation of rehabilitation during acute phase may inflict tremendous physical stress and exhaustion on patients in the ICU, thus increasing ICU mortality.meta-analysis had several limitations. First, the analysis of each outcome included only a small number of patients. Although Schweickert et al reported data concerning delirium, we could not extract cognitive-related outcomes because no data about delirium-free days were not addressed directly.et al, and little evidence to support the feasibility of individual patient randomisation across multiple sites using a complex intervention  had been reported before the study conducted by Hodgson et al. Moreover, these two RCTs demonstrated the same level of risk as other RCTs, according to risk of bias summary conducted in this meta-analysis (The current analysis .et al reported that a longer duration of delirium on admission was associated with worse global cognition and executive function scores in the surgical ICU at 3 and 12 months.Fourth, we could not confirm the exact first day of rehabilitation initiation in the three studies, although ’early rehabilitation' was described in the text, and we contacted the authors to acquire additional information.Early rehabilitation has a limited effect on the prevention of PICS, although it led to significant improvements in short-term physical-related outcomes, including MRC scores and the incidence of ICU-AW. However, early rehabilitation had no significant effect on cognitive function and mental health-related outcomes or mortality in patients with critical illness. Additional large-scale, rigorous RCTs are needed to confirm our results.