Anolis carolinensis sampled from two geographically and genetically distinct populations in the Eastern Florida and the Gulf Atlantic regions of the United States. We used the Mobile Element Locator Tool to identify and genotype polymorphic insertions from five major clades of LINEs  and the 41 subfamilies that constitute them. Across these groups we found large variation in the frequency of polymorphic insertions and the observed length distributions of these insertions, suggesting these groups vary in their activity and how frequently they successfully generate full-length, potentially active copies. Though we found an abundance of polymorphic insertions  most of these were observed at low frequencies and typically appeared as singletons. Site frequency spectra for most LINEs showed a significant shift toward low frequency alleles compared to the spectra observed for total genomic single nucleotide polymorphisms. Using Tajima’s D, FST and the mean number of pairwise differences in LINE insertion polymorphisms, we found evidence that negative selection is acting on LINE families in a length-dependent manner, its effects being stronger in the larger Eastern Florida population. Our results suggest that a large effective population size and negative selection limit the expansion of polymorphic LINE insertions across these populations and that the probability of LINE polymorphisms reaching fixation is extremely low.Vertebrate genomes differ considerably in size and structure. Among the features that show the most variation is the abundance of Long Interspersed Nuclear Elements (LINEs). Mammalian genomes contain 100,000s LINEs that belong to a single clade, L1, and in most species a single family is usually active at a time. In contrast, non-mammalian vertebrates  contain multiple active families, belonging to several clades, but each of them is represented by a small number of recently inserted copies. It is unclear why vertebrate genomes harbor such drastic differences in LINE composition. To address this issue, we conducted whole genome resequencing to investigate the population genomics of LINEs across 13 genomes of the lizard  Among teference , the repeference .Long Interspersed Nuclear Elements are ubiquitous in vertebrates and constitute the dominant category of autonomously replicating retroelements in most vertebrate genomes . They haIn mammals, the evolutionary dynamics of LINEs is relatively well understood. Population genetics and genomics studies in humans have shown that the majority of L1 elements behave as neutral alleles and accumulate readily in the genome of their host . This doAnolis carolinensis, using a complete genome re-sequencing approach. The anole genome is a particularly good model because it is among the most diverse vertebrate genomes in terms of LINE diversity (Table 1). For example, the L1 and the L2 clades contain 20 and 17 highly divergent families, respectively, whereas the CR1 clade is represented by only 4 closely related families. Since these clades and families coexist within the same genome, they are equally affected by the demography of their host. It is thus possible to assess their relative impact on fitness and to infer the evolutionary processes determining their diversification and replicative success.At this point, our understanding of LINE population dynamics is heavily biased toward their dynamics in humans. However, the extreme abundance and low diversity of LINEs in mammals constitute a derived state relative to other vertebrates. Thus, inferences drawn from studies in mammals are unlikely to apply to other vertebrates. In addition, results obtained from previous studies in non-mammalian vertebrates provide only a partial picture since those studies relied on a relatively small number of polymorphisms, principally collected from the published reference genomes . Thus, wiversity . Five LIA. carolinensis and of purifying selection against those insertions.In this article we present the first population genetic analysis of LINEs using re-sequencing data in a non-mammalian vertebrate. We sequenced thirteen individuals, from two populations with different demographic histories, at a depth of coverage ranging from 8 to 16×. For each resequenced genome we then characterized the single nucleotide polymorphisms (SNPs) and polymorphic sites containing LINE insertions not found in the reference genome. We determined that the number of insertion polymorphisms generated by LINEs in this species is large, exceeding 45,000 insertions, with substantial differences in replicative success among clades. We also determined that the vast majority of these insertions exist at very low frequency in natural populations as a result of the very large effective population size of Table 2). The Eastern Florida population is restricted to a ∼50 Km band along the eastern coast, extending from Jacksonville in the north to West Palm Beach in the south. Demographically, this population has remained relatively stable during the Pleistocene, with a slight signature of expansion  and once to generate a broader sampling  at lower depth of coverage. Quality assessment was conducted using FastQCv0.11.52 followed by quality trimming. We used Trimmomatic , requiring a high confidence collection of SNPs. SNPs were filtered using VCFTOOLS , by appl5; Anolis reference genome and exhibit low divergence , indicative of recent activity by members of these groups  only the longest was kept in the VCF file. This study focused exclusively on the presence and predicted length of polymorphic LINE insertions and at no point do we analyze or discuss mutations occurring within these insertions since it is nearly impossible to match a SNP within a LINE with its specific genomic location.Mobile Element Locator Tool operates on BWA-aligned re-sequenced genomes, so for each approach . Each BWapproach . The MELFST between populations for each category of markers. These statistics were calculated using VCFTOOLS  by simulating coalescence under the best model for the same number of SNPs as in the original dataset. We performed parameter estimation for 150 of these pseudo-observed datasets to infer CI. Coalescence simulations were performed using fastsimcoal2.5 . We furtp-values from the comparison between the observed Tajima’s D or FST value to the distribution obtained under a neutral model. We also performed a non-parametric bootstrap on the actual SNP dataset and extracted random sets of 100–500 SNPs along each chromosome, computing Tajima’s D and comparing the resulting distribution to the values observed for LINEs.To estimate if the LINE SFS deviated significantly from neutral expectations, we simulated for each family the derived allele frequency spectrum in fastsimcoal2.5. Parameters were sampled from the CI obtained for SNPs. We performed 5,000 simulations for each dataset, assuming unlinked LINE insertion sites, and obtained A. carolinensis genomes from the Gulf-Atlantic population and seven from the East Florida population with a sequencing depth of coverage ranging from 8 to 16× after alignment to the reference genome (Table 2). We detected extensive LINE insertion polymorphism in both populations  with a total of 46,616 polymorphic insertions across the 13 individuals. The East Florida population appears to maintain a greater total number of LINE polymorphisms, with a mean of 10,022 polymorphic LINE insertions per individual . In the Gulf Atlantic population the mean number of insertions per individual was substantially lower at 6,489 . Across all genomes roughly 10% of all polymorphic insertions approximated their full length, though individual populations varied: for individuals from the Gulf-Atlantic, 10.5% of polymorphic LINE insertions were full length , while in the East Florida population only 8.7%  were full length.We sequenced six Tables 1, 3). The most successful group was the CR1 clade, for which we found 27,802 polymorphic insertions. The L2 clade also has a large number of insertions: 11,210. Far fewer polymorphisms were found for the remaining families: the RTE clade had 3,367 polymorphisms, the L1 clade 2,508, and the R4 clade 1,729. Within each clade we also found substantial differences in the success of active families (Table 4). The L1 clade consists of 20 highly divergent families (Table 4). Most families had polymorphic insertions numbering less than 100, however, two families appeared at much higher numbers: L1AC07, which had 532 polymorphic insertions, and L1AC17, which had 763 polymorphic insertions. Together, these two families account for the majority (52%) of all L1 polymorphic insertions we identified. The L2 Clade has 17 known families in the Anolis genome but their differences in replicative success were not as large as those in the L1 clade. All L2 families exhibit polymorphisms and the most frequent group, L2AC09, only constitute 14% of L2 insertions. The RTE clade has only two representatives, RTE-1 and the ancient RTEBovB family. There are nearly six times more RTE-1 polymorphisms than RTEBovB , which is consistent with the idea that RTEBovB may be extinct in Anolis. The two R4 and the four CR1 families previously described  exhibited notable variation in their success at generating new insertions (families . We usedescribed  are nearAnolis genome assembly using GPS-RT (Table 1). This discrepancy is best explained by the large number of severely truncated insertions (<50 bp) that could have been missed by the BLAST search (which used the entire 3′UTR). The number of polymorphic insertions from the L2 clade is slightly less than threefold the number of insertions in the published genome. This is similar to L1, which has just over threefold more polymorphic insertions than insertions in the reference genome, though L1 has far fewer total insertions than L2 . Roughly the same number  of polymorphic RTE insertions were found as were previously detected by BLAST, and the R4 clade was found to have less than half as many polymorphic insertions as insertions identified by BLAST. These differences among clades possibly reflect differences in the fractions of fixed insertions relative to polymorphic ones among clades, which could be due to differential chance of fixation or to different timing of amplification of the LINE clades during the evolution of A. carolinensis. The number of RT hits detected by GPS are 3–5 times higher than the number of polymorphisms but the numbers are roughly proportional in the sense that the clades with the largest number of polymorphisms (CR1 and L2) are also the clades with the most RT hits. This difference in the total number of counts probably reflects the ability of GPS to identify the entire complement of RT-containing elements, including ancient elements that have long been fixed in the Anolis genome.Our prior expectations for the complement of LINE insertions have been shaped in part by published analyses conducted on the g GPS-RT  and by Bg GPS-RT . Those tFigure 1 depicts the length distribution of the different clades. It shows that truncation in R4 and RTE1 can occur anywhere along the length of the element but a large fraction of the elements are transposed all the way to their 5′ end. The probability of truncation in CR1 and L2 decreases proportionally to the distance to the 3′ end and a minority of the elements insert as full-length. L1 elements either truncate early on during transposition (and don’t reach 1 Kb), or if they do, they tend to be complete, hence the large fraction of elements longer than 5 Kb. It should be noted that complete elements fall into two length categories: elements between 5 and 5.5 Kb and elements longer than 6 Kb. These two types correspond to two sub-clades of L1, the families with short (∼230 bp long) 5′UTR (families L1AC16 to 20) and the families with long  5′UTRs (families L1AC1 to 15)  more truncated than full-length elements were found. All the truncated elements had their 3′ extremity and were truncated in 5′. This pattern is typical of LINEs and is caused by premature termination of the reverse-transcription reaction at the site of insertion . The CR11 to 15) . FinallyFigure 2A). We found 16 LINE insertions that were fixed in the East Florida population but absent in the reference genome , and 28 LINE insertions that were fixed in the Gulf population but absent in the reference genome . Only two insertions were found to be fixed across all the genomes sequenced here but absent in the reference sequence and both were from the CR1 clade. The site frequency spectrum (SFS) of insertions is consistently skewed toward low frequencies when compared to SNPs’ minor allele frequencies , which we used as a proxy for the “neutral” demographic history of the two populations. The only exception to this pattern is RTEBovB, where insertions at intermediate and high frequencies were more common in both populations. The skew in SFS was captured by Tajima’s D, which takes negative values for all categories of LINEs and for both populations, and average pairwise differences over the two populations, which were almost always lower for LINE insertions than for SNPs (Table 3). These two statistics are consistent with there being an excess of singletons and rare variants. This pattern was especially strong for RTE-1 and R4 clades in the Gulf population , with a significant reduction in the mean number of pairwise differences even compared with other LINE clades . This reduced polymorphism was also reflected by the lower FST values observed for insertions when compared to SNPs . The proportion of alleles found exclusively in Florida  was higher than the proportion of private alleles in the Gulf-Atlantic population , suggesting a reduced genetic diversity in the Gulf population. Similarly, Tajima’s D was consistently higher in the Gulf population . This pattern was, however, not observed for RTEBovB, which displayed a higher proportion of shared alleles between populations than the other LINEs analyzed here.Strikingly few insertions occurred at high allelic frequencies or are fixed in either population (Table 5 for more details). This pattern is consistent with the higher number of polymorphic sites observed in Florida for all markers. Simulated joint SFS based on the demographic model inferred from SNPs were consistent with the observed SFS , suggesting a good fit of the model. Summary statistics obtained from simulations displayed more negative values for Tajima’s D than the ones obtained from random sampling of 100–500 SNPs across the genome. This suggests that our model is conservative for detecting signatures of purifying selection under insertion/drift equilibrium. Nonetheless, observed SFS for LINE insertions never matched the simulations  and the simulated summary statistics such as FST or Tajima’s D were generally larger than the observed ones (Table 3). Again, the only exception to this pattern was RTEBovB, which even displayed a higher Tajima’s D than expected in Florida.Estimates of current effective population sizes assessed using the SNP dataset confirmed a large Florida population (diploid population size), and a smaller (but still large) population in Gulf-Atlantic . In Florida, Tajima’s D was significantly skewed toward more negative values for complete elements than for truncated ones . The average pairwise differences were consistent with this pattern, being always significantly lower for complete elements than for truncated elements in Florida (Table 6). In the Gulf-Atlantic population, the values for Tajima’s D tend to be lower for full-length CR1, R4 and RTE1 than for truncated ones, but those differences are not significant. However, the average pairwise differences were significantly different between full-length and truncated elements RTE-1, R4 and CR1 but not for L1 and L2 (Table 6).Since previous studies in other organisms have determined that complete elements are found at lower frequencies than truncated ones, we compared the frequency of these two types of elements. We assessed whether there was any difference between these two categories by comparing Tajima’s D, A. carolinensis. We found that LINEs generate a considerable amount of structural polymorphism in this species, in excess of 45,000 insertions, including close to 7,000 full-length elements. This is considerably more than the 998 polymorphic L1 insertions identified by the 1,000 genomes project in the global human population . CR1 accounts for more than half of these insertions, followed in abundance by L2, RTE, L1 and R4. Interestingly the total number of insertion generated by a specific clade is not related to the number of potential progenitors. For instance 62% of the ∼2,850 RTE1 insertions, 41% of the ∼1,730 R4 and 43% of the ∼2,500 L1 are complete whereas only 7% of the ∼11,210 L2 and 9% of the ∼27,800 CR1 are complete. This pattern is clearly related to the probability of truncation of LINEs . These different patterns of truncation are indicative of variations in the processivity of the reverse-transcription reaction among clades that will need to be further explored experimentally. The inverse relation between copy number and fraction of complete elements suggests that clades are using different strategies to ensure their long-term success. Elements that have a low probability of generating full-length copies (CR1 and L2) tend to generate a much larger number of insertions, increasing the odds that some of these insertions are full-length and potential progenitors. By analogy with the field of ecology, this strategy would be similar to a species with an r reproductive strategy, i.e., a strategy where many offspring are produced thus compensating for the low survival to adulthood. In contrast, there is no pressure for L1, RTE1 and R4 to produce a large number of copies since many of the new insertions will be full-length and capable of further transpositions. This is similar to the K strategy where the number of offspring is limited but their chance to propagate the species is high.We report substantial differences in the replicative success of LINEs in anoles (Table 5). Under such demographic conditions, the chance of fixation of a novel insertion, deleterious or neutral, is very low , polymorphic LINE insertions were found at very low frequency and the vast majority were observed from only a single chromosome in our sample. We also showed that the frequency distribution of LINE polymorphisms is significantly skewed toward lower values than the SNP distribution, which presumably reflects the effect of purifying selection acting on LINEs. In addition, we found this skew to be more pronounced for the Floridian population than for the Gulf-Atlantic population and for long elements than for the truncated ones. Purifying selection efficiently prevents the fixation of LINE insertions in anoles because the effective population size of extant and ancestral anole populations is large, ranging from ∼236,000 individuals for the extant Gulf-Atlantic population to ∼3,332,000 for Florida  suggesting that they are negatively selected. This result contrasts with studies in humans where truncated insertions were shown to behave like neutral alleles (Anolis, although those seem to be more deleterious. It was proposed that the deleterious effect of LINEs in vertebrates result mostly from their ability to mediate ectopic recombination leading to chromosomal rearrangements (The excess of singletons and the general lower frequency of LINE polymorphisms than SNPs suggest that LINEs are negatively selected and constitute a genetic load for their host. This pattern is consistent with the very low divergence calculated between elements from the same family  and supp alleles . Thus, tngements , and ourngements , and thaA. carolinensis populations should help characterize the extent of shared polymorphism at the species scale, allowing us to better evaluate the likelihood of recent bursts of activity in distinct populations.An alternative explanation for the observed excess of singletons is a departure from transposition-selection equilibrium. Our coalescence simulations implicitly assume a constant mutation/transposition rate. However, it has been shown that transposable elements can go through bursts of transposition, leading to an excess of insertions having the same age. Thus, a recent burst of transposition can also lead to an excess of recent insertions compared to the expectation under equilibrium, even if LINEs are not under purifying selection . HoweverAnolis and vertebrates in general.Even if non-equilibrium explanations for the excess of rare insertions are considered unlikely , neutralRR and SB designed the project. RR and YB analyzed the data. SB and YB prepared the original artwork. RR, YB, and SB wrote the manuscript. All authors have made substantial intellectual contributions to the research project and approved the final 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.


The approval of psychotropic drugs with novel mechanisms of action has been rare in recent years. To address this issue, further analysis of the pathophysiology of neuropsychiatric disorders is essential for identifying new pharmacological targets for psychotropic medications. In this report, we detail drug candidates being examined as treatments for psychiatric disorders. Particular emphasis is placed on agents with novel mechanisms of action that are being tested as therapies for depression, schizophrenia, or Alzheimer’s disease. All of the compounds considered were recently approved for human use or are in advanced clinical trials. Drugs included here are new antipsychotic medications endowed with a preferential affinity at dopamine D3 receptor (cariprazine) or at glutamatergic or cannabinoid receptors, as well as vortioxetine, a drug approved for managing the cognitive deficits associated with major depression. New mechanistic approaches for the treatment of depression include intravenous ketamine or esketamine or intranasal esketamine. As for Alzheimer’s disease, the possible value of passive immunotherapy with agents such as aducanumab is considered to be a potential disease-modifying approach that could slow or halt the progressive decline associated with this devastating disorder. To achieve this goal, it is essential to focus on developing drugs that target the pathophysiology underlying the disease, which increases the likelihood of identifying efficacious agents rather than symptomatic treatments. The pathology-to-drug discovery approach specifies that an understanding of the pathophysiology of neuropsychiatric disorders is the required initial step in identifying disease pathways and for validating new pharmacological targets1. A more complete understanding of the disease pathways will facilitate both the selection of therapeutic targets and the development of relevant models for screening drug candidates2. The pathology-to-drug discovery approach inspired the creation of NEWMEDS (Novel Methods Leading to New Medications in Depression and Schizophrenia), a European project designed to identify specific brain circuits, particularly those involving the prefrontal cortex, that are involved in the pathophysiology and treatment of major depression and schizophrenia3. On the other hand, a better knowledge of the mechanism of action of older drugs  has permitted, in part, a better understanding of the pathophysiology of neuropsychiatric diseases, enabling drug design according to the pathology-to-drug discovery approach.Epidemiological data indicate neuropsychiatric disorders as being some of the most prevalent, devastating, and yet poorly treated illnesses. As the approval of central nervous system drugs with novel mechanisms of action has been rare in recent years, there is a critical need to enhance drug discovery in neuropsychopharmacologyAs the neuropsychopharmacology community has only recently adopted the pathology-to-drug discovery approach, it remains unknown whether agents discovered in this way are currently available for the clinical management of psychiatric disorders and whether such drugs display significant advantages over conventional therapies with respect to efficacy and tolerability.Summarized below are some of the more exciting and relevant advances in the field of neuropsychopharmacology as they pertain to the design and development of novel psychotherapeutics. Highlighted are molecules displaying novel mechanisms of action that were recently approved for human use or that are now undergoing phase II/III clinical trials. Particular emphasis is placed on the identification of new drugs and drug candidates for the treatment of schizophrenia and major depression. Finally, a specific section is dedicated to neurodegenerative disorders such as Alzheimer’s disease (AD), where pharmacological strategies can significantly differ from the approaches currently adopted in psychotic and affective disorders.4. The development of second-generation antipsychotics that began 25 years ago has yielded some advances in terms of efficacy, with some modest improvement in addressing the negative symptoms of this condition, and in tolerability, particularly with regard to extrapyramidal side effects5. However, no antipsychotics display robust effects on the cognitive deficits or impaired social processing that are important components of this disorder4. For years, the limited efficacy of conventional and second-generation agents has led to theories about whether the manipulation of brain targets other than, or in addition to, the dopamine D2 receptor (D2R) may be necessary for treating this disorder and to significantly improve safety and tolerability.At a mechanistic level, drug treatments for schizophrenia are presently based on the dopamine hypothesis concerning the symptoms of this disorder6. According to this theory, the excessive dopamine release in the mesolimbic pathway and the decrease in dopamine release from the mesocortical pathway in the prefrontal cortex, which are responsible for some of the symptoms of schizophrenia, are secondary to a decrease in NMDA receptor control of inhibitory GABAergic neurons. No drugs capable of selectively enhancing NMDA receptor activity in this key brain region have yet been approved for human use. Studies indicate that prodromal and early-in-disease schizophrenic patients have elevated brain glutamate levels compared to healthy controls and ultra-high-risk patients who do not become psychotic7. Alterations in NMDA receptor-mediated excitation of GABAergic neurons indicate that schizophrenia is associated with dysfunctional glutamatergic systems in the prefrontal cortex and limbic regions of the brain. One approach has been to target the glutamatergic system using pomaglumetad methionil, a potent and highly selective orthosteric metabotropic glutamate receptor (mGluR) 2/3 agonist8, but alternative approaches directed at group III mGluRs are also currently studied in preclinical models of schizophrenia9. Preclinical research suggests that by reducing glutamate release, pomaglumetad methionil normalizes heightened glutamate activity in cortical pyramidal neurons8. While pomaglumetad methionil was reported to display beneficial effects on both the positive and the negative symptoms of schizophrenia in initial clinical trials, positive results were not obtained in phase III studies. Analysis of the clinical data suggests that pomaglumetad methionil is most efficacious in early-in-disease schizophrenics (<3 years’ duration) with a known hyperactive glutamatergic pathophysiology8. It is anticipated, therefore, that new antipsychotics acting on the mGluR2/3 will be developed to treat schizophrenic patients in an early phase of the illness with the hope of slowing disease progression and improving prognosis.In recent years, the N-methyl-D-aspartate (NMDA) receptor hypothesis of schizophrenia has been validated in preclinical animal models and humans10 and identity in the transmembrane regions, including the binding site11. Such structural similarities make it difficult to design ligands that selectively interact with D3R. This, in turn, has compromised the ability to fully define the localization and functions of this site. A number of approved drugs that were believed to act primarily at the D2R recognition site have now been found to interact with the D3R as well (see14. Cariprazine displays higher affinity at D3R and a similar affinity at D2 and serotonin 2B (5-HT2B) receptors13. In rodents, cariprazine reverses the deficit in novel object recognition that is caused by neonatal administration of phencyclidine14 or by subchronic phencyclidine exposure in adults16. Preclinical and clinical data suggest that by selectively activating D3R, cariprazine may have a positive impact on the cognitive symptoms of schizophrenia. Cariprazine was approved in 2015 by the U.S. Food and Drug Administration for the treatment of schizophrenia17. Further clinical studies with cariprazine and other antipsychotics selectively targeting D3R will be essential to validate the role of D3R as a new pharmacological target for the treatment of cognitive symptoms in schizophrenia.The dopamine D3 receptor (D3R) is another pharmacological target that appears to play a prominent role in the pathogenesis of schizophrenia. Unlike the D2R, which has been extensively studied with respect to the symptoms of schizophrenia, little is known about the extent to which changes in D3R and dopamine D4 receptor (D4R) activity contribute to the symptoms of this disorder. It is known that the molecular structure of D3R is very similar to that of D2R and D4R and that in a variety of animal species D2R and D3R share high homology4. Many questions remain as to whether it will be possible to identify drugs that will directly affect the underlying disease process in a way that would delay or prevent disease progression in schizophrenia.Another new approach proposed for the treatment of schizophrenia is the development of disease-modifying agents to prevent the full onset of schizophrenia and/or to slow its progression18. As a negative allosteric modulator of the cannabinoid 1 (CB1) receptor, CBD ameliorates the psychotogenic effect of THC and may possess antipsychotic properties19. Given these findings, the CB1 receptor has been proposed as a new pharmacological target for the treatment of schizophrenia19. Indeed, there have been reports that 800 to 1,000 mg of cannabidiol per day safely reduces the signs and symptoms of schizophrenia19. However, uncertainty remains about the mechanism of CBD action. Recently, Seeman reported that CBD, like aripiprazole, is a partial agonist at the D2R20. Thus, CBD could be the first molecule of a class of antipsychotics that interact with both the CB1 and the D2R. Ideal candidates for clinical trials with such agents would be high-risk individuals to assess whether CBD dampens the acute psychotic symptoms and cognitive deficits associated with schizophrenia. Three different phase II clinical trials are underway to assess the clinical efficacy of cannabidiol monotherapy in newly diagnosed schizophrenic patients and as adjunctive therapy with conventional second-generation antipsychotics (NCT02088060 and NCT02504151).There are also many challenges to consider for designing clinical trials to prove that a given drug candidate can modify the course of this disorder over time. A first step might be to identify young individuals who are at high risk for psychosis. Particular emphasis could be placed on those who are also heavy users of cannabis, as there is an increased risk for schizophrenia in those who abuse highly potent preparations of cannabis  containing a high percentage of delta-9-tetrahydrocannabinol (THC) (about 15%) with a scarcity of cannabidiol (CBD 0–1%)2. Cognitive dysfunction represents a distinct biological and clinical dimension in MDD21, with evidence suggesting that the presence of cognitive symptoms in depressed patients can predict a low rate of response to antidepressants and reduced remission rates23. Multimodal drugs, such as vortioxetine, represent a new class of antidepressants. These agents display multiple molecular mechanisms of action in addition to inhibition of the serotonin transporter25.While currently available antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs), are effective for most patients, approximately 30% of those with major depressive disorder (MDD) fail to respond to these agents26. Vortioxetine is not the only example of a multimodal antidepressant, with others, such as vilazodone , having been approved for the treatment of major depression28. Other psychotropic drugs developed in the last 30 years can possess a multimodal pharmacodynamic profile (see e.g. trazodone), but the novelty of this approach is to combine multiple pharmacological actions affecting both monoamine targets and other non-monoaminergic systems (e.g. glutamatergic system)24. The multimodal approach seems to be an interesting approach to target the different biological and clinical dimensions of MDD. Current evidence does not suggest a global greater efficacy of multimodal antidepressants compared to SSRIs or SNRIs but an improved efficacy on specific clinical domains where SSRIs or SNRIs are less effective, as observed with vortioxetine, which displays a specific clinical efficacy in the treatment of cognitive deficits associated with MDD25.Vortioxetine is a multimodal antidepressant that is thought to act by inhibition of transmitter reuptake and interactions with various 5-HT receptor sites. This pharmacodynamic profile is well described in the Neuroscience-Based Nomenclature, a new pharmacologically driven classification of psychotropic drugs that reflects current knowledge about the underlying neurobiological characteristics of the disorder, an understanding of the neurotransmitter/molecule/system being modified , and the mode/mechanism of drug action3, 5-HT7, and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist, and an inhibitor of the serotonin transporter29. It is thought to activate the glutamatergic system in rat frontal cortex by blockade of 5-HT3 and 5-HT7 receptors. It is reported that, as compared to fluoxetine, vortioxetine displays a superior efficacy in aged mice as a treatment for visuospatial memory and depression-like behavior30. The pharmacodynamic profile is consistent with the results of clinical studies indicating that vortioxetine has antidepressant properties as well as positive effects on cognitive function (e.g. memory and executive functioning)25. The most common side effects associated with this drug are nausea, vomiting, and constipation. Vortioxetine is currently studied as a potential therapeutic alternative for patients who fail to respond to SSRIs or SNRIs31.Vortioxetine is a 5-HT32. Several well-controlled clinical studies indicate that a single ketamine infusion (0.5 mg/kg) induces a rapid, generally transient, antidepressant effect in addition to small, but significant, increases in psychotomimetic and dissociative symptoms33. The ketamine-induced antidepressant effect occurs within 1 to 2 hours following its intravenous administration and may be sustained for up to 2 weeks34. This rapid onset of action has stimulated studies to explore the possibility that ketamine may represent a life-saving drug for TRD patients at imminent risk of suicide35. Twice- and thrice-weekly administration of ketamine at 0.5 mg/kg maintains antidepressant efficacy for over 2 weeks with no signs of tolerance36.Drugs, such as ketamine, that are known to affect descending glutamatergic systems represent a new approach for managing treatment-resistant depression (TRD)32. Preclinical and clinical studies with ketamine have resulted in the identification of new pharmacological targets related to NMDA receptor activation or inhibition, such as the NR2B receptor subunit, and the mammalian target of rapamycin (mTOR), a signaling system that controls synaptic plasticity and appears to be a key element in the antidepressant response to ketamine32. Recent studies also suggest that the pharmacological profile of ketamine is more complex than being a simple NMDA receptor antagonist because this drug also shows a significant affinity for D2R and opioid receptors as well as for monoamine transporters37. Further studies are needed to identify all the molecular mechanisms underlying the rapid-acting antidepressant effects of ketamine. Efforts are now directed towards the development of new antidepressants displaying the efficacy and rapid onset of action of ketamine but lacking its psychotomimetic effects34.It is believed that by blocking NMDA receptors on GABAergic interneurons, ketamine causes a rapid, but transient, increase in extracellular glutamate in the prefrontal cortex. At the molecular level, the ketamine-induced blockade of NMDA receptors results in inhibition of elongation factor 2 (eF2) kinase, de-phosphorylation of eF2, and a consequent augmentation of brain-derived neurotrophic factor (BDNF) synthesis38. Phase III clinical trials are ongoing to assess the safety and clinical efficacy of intranasal esketamine in patients with TRD .Attempts are also underway to exploit the established clinical value of ketamine and its derivatives, such as S(+) ketamine (esketamine), while reducing their side effect potential by administering them intramuscularly or intranasally at doses (e.g. 0.2 mg/kg) lower than those used for the intravenous studies39. This novel approach stems from the evidence that mGluR5s are functionally involved in the mild modulation of NMDA receptor activity and mGluR5 antagonists exert significant antidepressant effects in animal models of depression by acting as mild NMDA receptor negative modulators39. Therefore, mGluR5s have been recently considered as a new target of novel antidepressants, and basimglurant, a negative allosteric modulator of mGluR5, is now in clinical development for the treatment of MDD (NCT00809562 and NCT01437657).An alternative approach to target the glutamatergic system in MDD and mimic ketamine’s effects might be the use of mGluR5-selective antagonists or negative allosteric modulators40. This disorder is associated with the presence of senile plaques containing amyloid β (Aβ), intracellular aggregates of tau protein in neurofibrillary tangles, and progressive neuronal loss. The amyloid cascade hypothesis41 posits that overproduction of Aβ, or failure to clear this peptide, causes AD because of the aggregation of monomeric Aβ species into higher-molecular-weight Aβ oligomers that results in neuronal cell loss42.AD is a neurodegenerative disorder characterized by memory loss, cognitive decline, and neuropsychiatric symptoms that interfere with normal daily activitiesCurrent drug therapies for AD treat only the symptoms, such as memory loss, while having no effect on the progression of the disease. Drugs included in this group are cholinesterase inhibitors , which are approved for treating mild to moderate AD, and memantine, an NMDA receptor antagonist, which is used to treat patients with moderate to severe AD.42. To date, this effort has failed to yield any clinically effective drugs. One of the difficulties in testing such compounds has been the lack of reliable biomarkers identifying patients in the early stage of the disease. For this reason, most clinical trials were conducted in patients in advanced stages of the disease after irreversible damage to the brain had already occurred. However, the criteria for the diagnosis of AD, revised in 2011 by the National Institute on Aging and the Alzheimer’s Association workgroup43, now include biomarkers for identifying AD patients earlier. Such individuals are much better candidates for treatment with disease-modifying drugs44. The development of these biomarkers increases the likelihood of identifying that cohort of patients that is most likely to respond to disease-modifying drugs.Much effort has been expended in developing disease-modifying drugs for the treatment of this condition. Such agents would be able to slow the progression of the pathological changes and their effects would persist even after terminating treatment45. Given problems with vaccines, Aβ passive immunotherapy is currently the most popular approach for developing disease-modifying drugs for the treatment of AD46.Immunotherapy directed towards Aβ has been considered a promising approach because it would, in theory, decrease the aggregation and brain deposition of Aβ. Tau immunotherapy has also been explored as a possible means for inhibiting disease progression in AD48. Unfortunately, encouraging preliminary results obtained with solanezumab have not been confirmed in recent phase III clinical trials48. This drug is, however, still in clinical development in prodromal AD patients (NCT02008357 and NCT02760602).As antibody-based immunotherapy against Aβ has so far failed to yield positive clinical results, questions are being raised about the validity of the amyloid hypothesis of AD. However, it remains unknown whether the clinical failures reported for bapineuzumab, gantenerumab, and solanezumab are due to their inability to reduce the formation of Aβ oligomers or because they were tested in inappropriate populations of AD patients50 suggest that the amyloid hypothesis is still consistent with the development of new disease-modifying drugs, based on this hypothesis, in the near future. Aducanumab is the first example of an antibody developed by selecting human B-cell clones triggered by neo-epitopes present in soluble oligomers and insoluble fibrils. That is, only the pathogenic forms of Aβ49 are affected without interfering with Aβ monomers that exert a critical role in maintaining neuronal survival, learning, and memory51. It has been established that aducanumab enters the brain and that 1 year of monthly intravenous infusion reduces brain Aβ in a dose- and time-dependent manner in patients with prodromal or mild AD. The greatest effects are observed at doses of 3 and 10 mg/kg. This effect was accompanied by a slowing of the clinical decline as measured by the Clinical Dementia Rating—Sum of Boxes and Mini Mental State Examination scores. As already observed with solanezumab, amyloid-related imaging abnormalities (ARIA), such as vasogenic edema, indicate a dose-dependent response in AD patients49. The clinical efficacy of aducanumab must be confirmed in the long-term extension phase of this study as well as in the ongoing phase III clinical trials, which may finally validate the amyloid hypothesis in the field of AD.The recent positive results with aducanumab in prodromal and mild AD in a phase Ib trialAs indicated in this report, the pathology-to-drug discovery approach is now being applied for the identification of new drugs for the treatment of psychiatric disorders . This ha5-HT, serotonin; Aβ, amyloid beta; AD, Alzheimer’s disease; CBD, cannabidiol; CB1 receptor, cannabinoid 1 receptor; D2R, dopamine 2 receptor; D3R, dopamine 3 receptor; D4R, dopamine D4 receptor; eF2, elongation factor; MDD, major depressive disorder; mGluR, metabotropic glutamate receptor; mTOR, mammalian target of rapamycin; NMDA, N-methyl-D-aspartate; SNRI, serotonin and noradrenaline reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; THC, delta-9-tetrahydrocannabinol; TRD, treatment-resistant depression.


Use of oral anticoagulation therapy in patients with atrial fibrillation (AF) involves a trade-off between a reduced risk of ischemic stroke and an increased risk of bleeding events. Different anticoagulation therapies have different safety profiles and data on the societal costs of both ischemic stroke and bleeding events are necessary for assessing the cost-effectiveness and budgetary impact of different treatment options. To our knowledge, no previous studies have estimated the societal costs of bleeding events in patients with AF.The objective of this study was to estimate the 3-years societal costs of first-incident intracranial, gastrointestinal and other major bleeding events in Danish patients with AF.The study was an incidence-based cost-of-illness study carried out from a societal perspective and based on data from national Danish registries covering the period 2002-2012. Costs were estimated using a propensity score matching and multivariable regression analysis (first difference OLS) in a cohort design.Average 3-years societal costs attributable to intracranial, gastrointestinal and other major bleeding events were 27,627, 17,868, and 12,384 EUR per patient, respectively (2015 prices). Existing evidence shows that the corresponding costs of ischemic stroke were 24,084 EUR per patient (2012 prices). The average costs of bleeding events did not differ between patients with AF who were on oral anticoagulation therapy prior to the event and patients who were not.The societal costs attributable to major bleeding events in patients with AF are significant. Intracranial haemorrhages are most costly to society with average costs of similar magnitude as the costs of ischemic stroke. The average costs of gastrointestinal and other major bleeding events are lower than the costs of intracranial haemorrhages, but still substantial. Knowledge about the relative size of the costs of bleeding events compared to ischemic stroke in patients with AF constitutes valuable evidence for decisions-makers in Denmark as well as in other countries.The online version of this article (doi:10.1186/s12913-017-2331-z) contains supplementary material, which is available to authorized users. Over 6 million Europeans currently suffer from atrial fibrillation (AF), and projections suggest that the prevalence of AF will at least double by 2050 due to the aging population –3. The pEstimates of the societal costs of both ischemic stroke and bleeding events in patients with AF are necessary for assessing the cost-effectiveness and budgetary impact of different treatment options, including oral anticoagulation therapy versus no therapy and various oral anticoagulation therapies with different safety profiles. A recent Danish registry study found that the average 3-years societal costs of first-incident ischemic stroke in patients with AF are 24,084 EUR per patient 2012 prices), including direct costs related to healthcare and social care services and indirect costs due to production lost to society  prices, –16. HoweThe aim of this study was to estimate the 3-years societal costs of first-incident intracranial, gastrointestinal and other major bleeding events in Danish patients with AF, including costs of healthcare, social care services and production lost to society. A subgroup analysis was performed for patients with AF who were on oral anticoagulation therapy prior to the bleeding event.This study was an incidence-based cost-of-illness study carried out from a societal perspective and based on data from national registries covering the entire Danish population. Every Danish citizen has a permanent personal registration number that enables linkage between registries at the individual level. The costs attributable to intracranial, gastrointestinal and other bleeding events in patients with AF were estimated in a cohort design using propensity score matching and multivariable regression analysis (first difference OLS (Ordinary Least Squares)) .The source population was identified from the National Patient Registry as all patients who were hospitalised in the period 1994-2012 with AF as the primary or secondary diagnosis (ICD 10: I48) .From the source population, the bleeding groups (exposed) were identified for each year during the period from January 1st 2002 till December 31st 2012 as patients who were hospitalised in the year in question with an ICD 10 bleeding diagnosis as the primary or secondary diagnosis following or at the same time as the AF diagnosis . Patients in the control group assigned to patients with intracranial haemorrhages were not censored from this control group if they were hospitalised with gastrointestinal or other bleeding events. Similarly, patients in the control group assigned to patients with gastrointestinal and other bleeding events were not censored from these control groups if they were hospitalised with intracranial/other bleeding events or intracranial/gastrointestinal bleeding events, respectively.Patients with AF on oral anticoagulant therapy were identified from the Registry of Medical Products Statistics (Prescription Registry) as patients who, in the year before the bleeding event, had redeemed at least two prescriptions with the ATC codes B01AA (vitamin k antagonists (VKA)) or B01AE07 (dabigatran) which were the only oral anticoagulation therapies on the Danish market before 2012 . The stuThe average annual costs attributable to bleeding events were estimated over a 3-years period from the year in which the bleeding event occurred (the incidence year) and onwards using the matched control/regression approach which is considered the gold standard method of cost-of-illness studies .t = 0, 1, 2) after the incidence year minus the costs incurred in the year before the bleeding event (the baseline year). Costs incurred in the baseline year were deducted as it was not possible to isolate costs according to diagnosis when including costs outside hospitals. Furthermore, patients were assumed to consume healthcare and social care resources even if they had not experienced the bleeding event as the majority of the study population was elderly with other comorbidities.Firstly, standardised costs were calculated for patients in the bleeding groups and control groups as costs in year t  and other healthcare professionals in the primary care setting, and costs of prescription medicine. The Danish healthcare system is public and mainly tax-financed. Hospital services and services provided by GPs and specialist physicians in the primary care setting are free of charge. Services provided by other healthcare professionals outside hospitals and prescription medicine are reimbursed by the National Health Insurance with a patient co-payment added. Data on hospital costs were collected from the National Patient Registry . Data onCosts of social care services included costs of home help and nursing homes that are financed by the municipalities in Denmark. Data on home help were obtained from Statistics Denmark (the Elderly Care Documentation) and data on use of nursing homes were obtained from the Building Regulations Register linked to the Civil Registration System . The cosEstimation of the production lost to society was based on the human capital approach which is a commonly used method for valuation of indirect costs assuming that the production lost to society is equal to the value of lost earnings , 29. Thet = 0, 1, 2) after the incidence year were estimated for each bleeding group. Costs of social care services and production lost to society were estimated for the bleeding groups 2002-2012 and healthcare costs were estimated for the bleeding groups 2002-2011 as data on hospital tariffs were only available until 2011. The average annual attributable costs reported here are average costs across bleeding groups, i.e. the estimated costs in the incidence year cover the bleeding groups 2002-2012 whereas the estimated costs in year 1 and 2 after the bleeding event cover the bleeding groups 2002-2011 and 2002-2010, respectively.The average annual attributable costs in year t (All costs were inflated to 2015 prices and converted to euros (EUR) based on the average exchange rate in 2015 (100 EUR = 746 DKK). The present value of the 3-years costs was calculated using a discount rate of 4% as currently recommended by the Danish Ministry of Finance .The Charlson index and HAS-BLED score were included as covariates to adjust for differences between the bleeding and control groups with regard to comorbidity and risk of bleeding events, respectively , 32. TheFurthermore, age, sex, region of residence, and socioeconomic status measured by education, employment, and income were included as covariates. Data on these variables were obtained from The Civil Registration System as well as the Education Register, the Integrated Database for Labour Market Research, and the Income Register of Statistics Denmark –35.Nearest neighbour propensity score matching with replacement was used to select four controls for each patient in the bleeding groups . MatchinAn absolute standardised difference below 10% and a variance ratio between 0.8 and 1.25 were considered to support the assumption of balance between groups .1 (first difference OLS):Average costs attributable to intracranial, gastrointestinal and other major bleeding events were estimated in a differences-in-differences regression model as βi = dummy variable indicating whether the patient belongs to the bleeding or the control group, Xi = covariates (see Table where mentclass2pt{minim0: μ = 0 against the hypothesis H1: μ ≠ 0). This was equivalent to testing whether average standardised costs per patient in the bleeding group were significantly different from average standardised costs per patient in the control group. The statistical significance level was set at P < 0.05. A possible skewed distribution of costs was not considered a problem in this study due to the large sample size [The statistical significance of cost estimates were evaluated using a two-sided t-test .Analyses were performed using the statistical package SAS 9.3 for Windows  and Stata 13.1 .In total, 21,046 patients with AF were hospitalised with a first-incident bleeding event during the period 2002-2012. Of these, 4529 patients experienced intracranial haemorrhages while 8507 and 8010 patients experienced gastrointestinal and other major bleeding events, respectively.Table The estimated average 3-years societal costs attributable to intracranial haemorrhages were 27,627 EUR per patient  corresponding to 18,049 EUR in the incidence year, 6922 EUR in the year after, and 4356 EUR 2 years after the event when not discounted to present value . Existing evidence shows that the corresponding costs attributable of ischemic stroke are 24,084 EUR per patient  [This study has a strong methodological design using an incidence-based case-control approach and propensity score matching to minimise the risk of confounding by age, sex, co-morbidity, bleeding risk, and socio-economic characteristics. Furthermore, the use of national registry data reduces the risk of selection and information bias as the registries cover the entire Danish population, and as data are prospectively registered and the quality is considered to be high. Another strength involves the use of a large data set covering a 10-years period, making the cost estimates more robust. The inclusion of social care costs related to home help and nursing homes, additional to healthcare costs, is another important strength of this study. Social care costs related to rehabilitation, assistive devices, home modifications, and informal care were not included as data were not available from the national registries. The cost estimates therefore remain conservative.The study also has a number of limitations. Firstly, the cost estimates concern major bleeding events as only patients who had been hospitalised with bleeding events as the primary or secondary diagnosis were included in the study. Patients with minor bleeding events who were not hospitalised were probably less costly.Secondly, only patients who had been hospitalised in the period 1994-2012 with AF as the primary or secondary diagnosis were included in the source population of the study from which both cases and controls were drawn. Thus, the study estimates the costs of bleeding events among patients with severe AF/comorbidity. The estimated costs would probably have been higher if all patients with AF had been included in the source population due to lower costs in the control group. However, the number of patients with AF not registered in hospital records is low in Denmark as national clinical guidelines recommend evaluation in hospital.Thirdly, there is a risk of confounding due to the observational nature of the study. The risk of confounding related to observable background characteristics was minimized by a well-balanced propensity score-matched and multivariable regression design. However, the risk of confounding increased over time after the bleeding event as the comparability between the bleeding and control groups diminished due to differences in mortality , 4% had had a previous stroke, and 6% had a stroke in the years after the bleeding events (the follow-up period). In the control groups, 12% had a stroke during the follow-up period. The fraction of patients in the control groups who had a stroke in the incidence year (t = 0) or earlier was below 0.5%. Thus, the incidence of ischemic stroke was 12% in both the bleeding groups and the control groups from the incidence year (t = 0) and onwards.It is possible that some of the differences in costs between the bleeding and control groups in this study were attributable to ischemic stroke. However, as a differences-in-differences approach was used and as the incidence of ischemic stroke in the bleeding and control groups was similar, this problem is not regarded a major concern. Overall, 16% of the patients in the bleeding groups had an ischemic stroke during the observation period 1994-2012 compared to 12% in the matched control groups. In the bleeding group, 6% had a stroke the same year as the bleeding event  is assumed to be equal to the present value of all lost future earnings until retirement from the labour market. The human capital approach has been criticised for not taking into account the existence of involuntary unemployment . AnotherFinally, the lack of observable differences between cost estimates of patients with AF in general and cost estimates of patients with AF who were on oral anticoagulation therapy prior to the bleeding event could be due to inaccuracy related to the identification of the latter group. The cost estimates of patients with AF on oral anticoagulation therapy were expected to be higher as evidence suggests that bleeding events associated with oral anticoagulant therapy are generally more severe . In the Only few studies on the costs of bleeding events in patients with AF have been published so far. The cost estimates of the present study are higher than the cost estimates of existing studies as these studies have focused on hospital costs only. Cotté et al. (2014) estimated the 2-years hospital costs of bleeding events in 61,582 patients with AF in France who were eligible for anticoagulation treatment based on data from the French national hospital claims system covering the period 2006-2008 . Mean 2-In conclusion, this study is the first study to estimate the societal costs attributable to intracranial, gastrointestinal and other major bleeding events in patients with AF, including both direct healthcare and social care costs and indirect costs due to production lost to society. This study documents that these costs are significant. Even though the cost estimates of this study may not be directly transferable to another country, the study does provide an indication of the relative size of the costs of bleeding events compared to the costs of ischemic stroke in patients with AF which is valuable evidence for decision-makers in every country. Intracranial haemorrhages are most costly to society with average costs of similar magnitude as the costs of ischemic stroke . The aveAdditional file 1: Figure S1.Extent of replacement. The figure shows number of times each control is used as a match in the study. (TIFF 72 kb)Additional file 2: Figure S2.Intracranial haemorrhages – standardised differences and variance ratios in covariates. The figure illustrates the quality of the matching between patients with intracranial haemorrhages and their matched controls. (PNG 895 kb)Additional file 3: Figure S3.Gastrointestinal bleeding events – standardised differences and variance ratios in covariates. The figure illustrates the quality of the matching between patients with gastrointestinal bleeding events and their matched controls. (PNG 896 kb)Additional file 4: Figure S4.Other bleeding events – standardised differences and variance ratios in covariates. The figure illustrates the quality of the matching between patients with other bleeding events and their matched controls. (PNG 895 kb)Additional file 5: Figure S5.Kaplan Meier survival curves. The figure shows the percentage of patients in the bleeding and control groups who are alive at the beginning of each year. (TIFF 187 kb)


The high rate of attrition along the care cascade of infection with human immunodeficiency virus (HIV) results in lost opportunities to provide timely antiretroviral therapy (ART) and to prevent unnecessarily high mortality. This study aims to assess the effectiveness of a structural intervention, the one-stop (“One4All”) strategy that streamlines China’s HIV care cascade with the intent to improve testing completeness, ART initiation, viral suppression, and mortality.A two-arm, cluster-randomized controlled trial was implemented in twelve county hospitals in Guangxi China to test the effectiveness of the One4All strategy (intervention arm) compared to the current standard of care . The twelve study hospitals were selected for homogeneity and allocated one-to-one to the intervention and control arms. All patients screening HIV positive in study hospitals were enrolled. Target study enrollment was 180 participants per arm, 30 participants per hospital. Basic demographic information was collected as well as HIV risk behavior and route of infection. In intervention hospitals, patients then went on to receive point-of-care CD4 testing and in-parallel viral load (VL) testing whereas patients in control hospitals progressed through the usual SOC cascade. The primary outcome measure was testing completeness within 30 days of positive initial HIV screening result. Testing completeness was defined as receipt of all tests, test results, and post-test counseling. The secondary outcome measure was ART initiation (receipt of first ART prescriptions) within 90 days of positive initial HIV screening result. Tertiary outcome measures were viral suppression (≤200 copies/mL) and all-cause mortality at 12 months.We expect that this first-ever, cluster-randomized controlled trial of a bundle of interventions intended to streamline the HIV care cascade in China (the One4All strategy) will provide strong evidence for the benefit of accelerating diagnosis, thorough clinical assessment, and ART initiation via an optimized HIV care cascade. We furthermore anticipate that this evidence will be valuable to policymakers looking to elevate China’s overall HIV/AIDS response to meet the UNAIDS 90-90-90 targets and the broader, global goal of eradication of the HIV/AIDS epidemic.NCT02084316. ClinicalTrials.gov # In both resource-rich and resource-limited settings alike, patients are commonly lost at each step along the care cascade of infection with human immunodeficiency virus (HIV) that causes acquired immunodeficiency syndromes (AIDS) . In ChinAlthough it seems obvious, it is critically important that people living with HIV (PLHIV) step through all the milestones on the HIV care cascade in order for to fully realize the benefits of ART—they must become aware of their HIV-positive status, receive a diagnosis and clinical assessment, attend counseling, enroll in care and initiate ART, and adhere to their treatment regimens and follow-up schedules , 6. HoweThe strong evidence for the clinical benefit of immediate ART for PLHIV , 8, takeTesting completeness within 30 days of HIV-positive screening (primary)ART initiation within 90 days of HIV-positive screening (secondary)3) at 12 months after HIV-positive screening (tertiary)Viral suppression The objective of the present clinical trial was to evaluate a streamlined pathway for patients from screening HIV-positive to initiating ART, testing the effectiveness of a structural intervention—called the one-stop (“One4All”) strategy. This experimental strategy consisted of a new algorithm incorporating rapid, point-of-care (POC) HIV screening and CD4 testing, and in-parallel plasma viral load (VL) testing to promote fast and complete HIV diagnosis and staging, followed by immediate counseling and ART initiation for eligible patients. The One4All intervention was compared to the current SOC (control arm), and four outcomes were assessed:A cluster-randomized trial was designed as diagrammed in Fig. The setting for this study was county hospitals in Guangxi Zhuang Autonomous Region (hereafter referred to as Guangxi). In 2011, Guangxi reported the second highest cumulative number of HIV/AIDS cases in China, the highest number of newly-reported HIV/AIDS cases, the highest number of newly-reported AIDS cases, and the highest number of AIDS-related deaths . As showThe twelve selected study hospitals were stratified by historical rates of testing completeness followed by post-test counseling during the first six months of 2013 prior to being allocated to the intervention or control study arms. Eight hospitals were within the low historical testing completion stratum of <20%, and these eight hospitals were randomized, four into the intervention arm and four into the control arm. Four hospitals were within the high historical testing completion stratum of ≥20%, and these four hospitals were randomized, two into the intervention arm and two into the control arm. All patients who screened HIV-positive in the six intervention hospitals received the One4All intervention and all patients who screened HIV-positive in the six control hospitals during the same study period received SOC services. There was no blinding required since the unit of randomization was the study hospital and each hospital only offered the HIV care cascade it was assigned during randomization—SOC or One4All.Study eligibility criteria were: [a] being 18 years of age or older, [b] having a positive result on an HIV enzyme immunoassay (EIA) screening test, [c] seeking care in a study hospital, either inpatient or outpatient, and [d] residing or intending to reside within the study catchment area. Study exclusion criteria were: [a] having previously received confirmation of HIV infection in any setting, [b] being a prisoner or detainee at time of screening, or [c] being a pregnant woman.No special strategies to ensure adequate participant enrollment were employed. All patients who were found to be eligible were enrolled in the study. After enrollment, basic demographic information was collected. Information on self-reported risk behavior and route of HIV acquisition were also collected.The protocol and consent process were reviewed and approved by the US National Institutes of Health (NIH), National Institute on Drug Abuse (NIDA) Protocol Review Board and Data and Safety Monitoring Board, as well as respective institutional review boards (IRBs) of the University of California, Los Angeles (UCLA) and the National Center for AIDS/STD Control and Prevention (NCAIDS), China Center for Disease Control and Prevention (CDC).Individual informed consent was not obtained. Study information was shared with participants and participants were given opportunities ask questions. While participants could refuse to participate, or to share their data, their enrollment still counted toward the total number of study participants. No study-related reimbursement for participants was provided.All patient data were protected and kept confidential according to standard procedures present within China’s healthcare system. Participant data extracted from hospital records, and from China’s National HIV/AIDS Comprehensive Response Information Management System  , were asA minimum sample size of 180 participants per arm, across 12 clusters (or 30 participants per cluster) was selected. This sample size was expected to achieve 93% power based on a one-sided test  to detect a difference between the group proportions of 0.28, where under the alternative hypothesis the One4All arm proportion was assumed to be 0.50 and the control arm proportion was assumed to be 0.22. This calculation assumed an intra-class correlation (ICC) within hospitals of 0.082 based on preliminary data. Although 30 participants per cluster  was the target, it was expected that some hospitals would enroll participants more quickly than others. Therefore, a final study population of greater than 180 participants per arm was anticipated as enrollment at all hospitals remained open until the last hospital met its 30-participant goal.3. Each of the steps in the HIV care cascade up to assessment of ART eligibility are described in detail below for both the SOC condition and the One4All condition. In both care cascades, all participants received post-test counselling each and every time they received test results.A high-level overview of the HIV care cascade in SOC (control arm) and One4All (intervention arm) hospitals is depicted in Fig. In SOC study hospitals, the Wantai Screening HIV (1 + 2) Ag&Ab EIA (Beijing Wantai Manufacturer of Infectious Diseases Diagnostics) was used as the initial screening test followed by two different EIAs that varied between sites.In One4All study hospitals, the first HIV screening test was the also the Wantai Screening HIV (1 + 2) Ag&Ab EIA. The second and the third EIAs were the Determine HIV-1/2® rapid test (Abbott Laboratories) and the InTec HIV rapid test (Xiameng InTec Products).In SOC study hospitals, an additional blood sample was collected immediately following screening or on a subsequent study hospital visit. The sample was sent to the local city CDC laboratory for Western blot (WB) confirmatory testing according to usual practice. As confirmatory testing required approximately 10 to 15 days to be completed, participants typically had to be contacted and asked to return to the study hospital to receive their results.In One4All study hospitals, WB was not used for confirmatory testing. Rather, VL testing served as the confirmatory test (see below).In SOC study hospitals, participants were asked to return to the study hospital for another blood draw for CD4 testing after they were notified that the result of their confirmatory testing was positive. Blood specimens were again sent to the city CDC laboratory, this time for CD4 testing. CD4 testing also required approximately 10 to 15 days to complete. Once CD4 test results were available, participants were again asked to return to be notified.In One4All study hospitals, POC CD4 testing was performed in the study hospital with a POC Pima™ CD4 Analyzer  using whole blood samples. Results were available within 30 min. Participants were notified of their results as soon as possible, but no later than the next day if they had left the study hospital during the test.In SOC study hospitals, VL testing was conducted approximately one year after ART initiation. Participants were asked to return to the study hospital for blood sample collection. Blood samples were sent to the provincial CDC laboratory for plasma VL testing, which required 10 to 15 days to complete. Participants were contacted and asked to return to the study hospital to receive VL testing results once they were available.In One4All study hospitals, blood samples for VL testing were collected immediately following receipt of positive screening results, at the same time as the blood draw for CD4 testing (see above). VL testing was conducted again after one year. Plasma VL testing itself was conducted in the same manner as in SOC study hospitals—samples were sent to the provincial CDC laboratory, plasma VL testing required 10 to 15 days, and participants asked to return to the study hospital to receive VL test results.This intervention condition posed no specific safety risk to participants. In comparison to SOC study hospitals, the HIV care cascade in One4All study hospitals involved fewer blood draws and there was no increase in the volume of blood drawn or the typical risk associated with blood draws.3) and mortality at 12 months. Mortality was assessed as the number of reported deaths divided by the number of patients enrolled.The primary outcome measure was the proportion of participants who achieved testing completeness and received test results and post-test counseling within 30 days of positive screening. Testing completeness was defined as completion of three required components: 1) initial HIV screening (one to two tests in One4All and two to four in SOC), 2) CD4 testing, and 3) confirmatory HIV testing—WB in the SOC arm, or VL in the One4All arm. “Success” was defined as completion of all three required tests and notification of test result along with receipt of counseling after each test. Success was compared at 30 days after initial HIV-positive screening. The secondary outcome measure was the proportion of participants who initiated ART within 90 days from the date of HIV-positive screening. ART-initiation was defined as the receipt of the first ART prescription. Tertiary outcomes included the proportion of participants who achieved viral suppression . CRF data were stored in a web-based database constructed specifically for the study. This database served as the centralized location for electronic storage of all study-related data. Study-related data from each hospital system was retrieved weekly and uploaded to this study database.To supplement the data contained in the study-specific database, records were extracted from CRIMS . Each coA centralized Data and Statistics Team (DST) was responsible for the validation of the web-based study database, retrieval of study hospital data, data extraction from CRIMS, merger of the two datasets into one, assurance of data integrity and security, and development and delivery of training for participating hospital and CDC staff members on applicable data management procedures.To adjust for both the clustering effect of hospitals  and baseline participant-level and hospital-level confounding factors, analysis of the primary outcome measure was performed using G-side GLIMMIX modeling. Participant-level factors included in the model were age, gender, ethnicity, education, occupation, marital status, transmission route, and treatment setting (inpatient/outpatient), and hospital-level factors included baseline test completion rate prior to randomization. To assess the consistency of the primary outcome results, a Chi-square test adjusted for clustering was also used to measure the association between treatment arm and the primary outcome , althougp-value was calculated by referring the observed (unadjusted) Chi-square value to the distribution of Chi-square values obtained by randomly permuting observed treatments over hospitals, with the adjusted p-value defined as the proportion of 'permuted' Chi-square values at least as large as observed. To test between-arm differences in continuous data, a t-test adjusted for clustering was performed [In comparing differences in baseline characteristics between the two arms, a Chi-square test adjusted for clustering was performed for binary variables. When baseline categorical variables had more than 2 responses, an adjusted erformed .p-values and confidence intervals for multiple testing and multiple comparisons were not performed. All data analyses were performed using SAS software .Analytical methods and the adjustment for clustering and covariates effects used in the analysis of the primary outcome were also applied to the ART initiation secondary outcome and viral suppression tertiary outcome. As with the secondary analysis of the primary outcome, Kaplan-Meier analyses were also performed for time to initiation of ART, outside the 90-day window, and time to death. In addition, for the mortality outcome, a random effects Cox (shared frailty) model accounting for the hospital clustering effect was used to calculate the intervention effect on the hazard ratio (HR) of death while also controlling for other covariates. Adjustments to The trial is registered with ClinicalTrials.gov (#NCT02084316) and the original protocol has been published on CTNDisseminationLibrary.org. The dataset is available upon request to the corresponding author.To our knowledge this is the first controlled clinical trial to examine a bundle of interventions intended to improve multiple points in China’s HIV care cascade. A notable strength of this study lies in the rigor with which the effectiveness of the One4All strategy was tested. We expected that it would provide strong evidence in support of a streamlined HIV care cascade that accelerates diagnosis, complete clinical assessment, and treatment initiation having a significant clinical benefit over the current SOC.With growing global evidence of the clear benefits of immediate ART for all PLHIV, both for the purposes of treatment of HIV infection and of prevention of HIV transmission , 8, 10, 3 ART eligibility criterion in place, with exceptions allowed only for PLHIV who were pregnant or in serodiscordant relationships [3 in 2013 [3 threshold. One prospectively examined immediate ART for all participants regardless of CD4 count [3 on an exception basis or as a part of the NCAIDS-sponsored “Prevention and Treatment of Major Infectious Diseases” project launched in 2012, which consisted of a series of cluster-randomized controlled trials among key populations. Both studies found significant improvements in mortality. We envision the combination of this evidence with the outcome of the present study supporting the complete replacement of China’s current SOC HIV care cascade with a new, streamlined cascade that accelerates diagnosis, complete clinical assessment, and ART initiation for all PLHIV.At the time the present study was conducted, China still had a CD4 count ≤350 cells/mmionships  Thus, we in 2013 , and the in 2013 . Two stuSeveral important challenges were anticipated prior to trial commencement. For example, there was known variability in HIV SOC offered between different counties in Guangxi. A major cause of poor testing completeness and treatment initiation in rural Guangxi is disjointed HIV services as a result of structural and cultural constraints. County hospitals and CDCs struggle to follow national guidelines, mainly because of limited laboratory capacity and complex referral systems between hospitals and CDCs. The study team debated whether to provide strict guidelines and training to control arm study hospitals in an effort to standardize their current practice and improve homogeneity. However, we ultimately decided against tampering with the control condition, as we determined that a control comparator that represented the reality of the current standard HIV care cascade was more valuable than an artificial ideal control environment we would have created only for the benefit of the study. Therefore, during our baseline hospital investigations, the investigators engaged with hospital leaders and key personnel involved in HIV care and collected the key characteristics of the routine practice adopted at each county. A detailed testing to treatment flowchart was developed for each of the hospitals, documenting the key deviations from national guidelines. Then, CRFs were adjusted in order to accurately reflect the reality of testing and linkage to care procedures in each hospital.Another key challenge was in the size of the study. The study was originally designed to include 24 hospitals (or clusters), 12 each in the control and intervention arms. This would have provided adequate power to detect differences between arms in all four outcomes. However, due to both the known high degree of variability observed in SOC services across counties in Guangxi and historically low numbers of patients screening HIV positive in many hospitals, not to mention the inherent difficulties with implementing such a large study, we decided to scale back the number of hospitals to 12. Although we suspected that we would still find statistically significant differences in several of the outcomes, the study was truly only adequately powered to detect between-arm differences in the primary outcome of testing completeness by 30 days.As the study included no intervention related to ART regimen adherence, we anticipated the possibility that we may find no between-arm differences in achievement of viral suppression. We chose to define viral suppression as ≤200 copies/mL based on the equipment used for the study in local laboratories, recognizing that this target was aggressive. Although above the <50 copies/mL definition of viral suppression commonly recognized in high-income settings, it is well below the <1,000 copies/mL threshold recognized by the WHO as achievement of viral suppression in low- and middle-income countries such as China. It is also below the 300 to 500 copies per/mL target commonly used by research teams in studies in low- and middle-income settings . Future This first-ever, cluster-randomized controlled trial of a bundle of interventions intended to streamline the HIV care cascade in China (the One4All strategy) was evaluated on four outcomes: testing completeness within 30 days, ART initiation within 90 days, viral suppression at 12 months, and all-cause mortality at 12 months. We expect that the outcome of this trial will provide strong evidence for the benefit of accelerating diagnosis, thorough clinical assessment, and ART initiation via an optimized HIV care cascade such as the One4All strategy. Furthermore, we anticipate that this evidence taken together with the strong, new evidence of both treatment and prevention benefits of immediate ART for all PLHIV regardless of CD4 count , 8, 10,