Home-based HIV counselling & testing (HBHCT) is highly acceptable and has the potential to increase HIV testing uptake in sub-Saharan Africa. However, data are lacking on strategies that can effectively link HIV-positive individuals identified through HBHCT to care. This trial was designed to assess the effectiveness of two brief home-based counselling sessions on linkage to care, provided subsequent to referral for care among HIV-positive patients identified through HBHCT in a rural community in Masaka district, Uganda.28 communities (clusters) were randomly allocated to control  and intervention  arms (n = 14 clusters/arm). Randomisation was stratified on distance from the district capital (≤10 km vs > 10 km) and cluster size , and restricted to ensure balance on selected cluster characteristics. A list of possible allocations was generated and one randomly selected at a public ceremony. HBHCT is being offered to all adults (≥18 years), and HIV-positive individuals not yet in care are eligible for enrolment. The intervention is provided at one and two months post-enrolment. Primary outcomes, assessed 6 months after enrolment, are: the proportion of individuals linking to HIV care within 6 months of HIV diagnosis and time to linkage. The primary analysis will be based on individual-level data.This study will provide evidence on the impact of a counselling intervention on linkage to care among adults identified with HIV infection through HBHCT. Interpretation of the trial outcomes will be aided by results from an on-going qualitative sub-study. Primary outcomes are: the proportion of individuals linking to care within 6 months of HIV diagnosis through HBHCT, and time between HIV diagnosis and linkage to care. Secondary outcomes include time between HIV diagnosis and receipt of CD4 cell count test results, time between HIV diagnosis and ART initiation, and the proportion of participants who report adherence to daily cotrimoxazole prophylaxis 6 months after HIV diagnosis. A further secondary outcome is the proportion of HIV-negative participants who agree to repeat HIV testing 6 months after HBHCT.22.12.1.1The study is an open-label cluster randomised controlled trial being conducted in three rural sub-counties in Masaka district, Uganda. It is based at the MRC/UVRI Unit station in Masaka. The study population consists of newly and previously diagnosed HIV-positive adults (≥18 years) identified through HBHCT who are not in care and are willing to provide informed consent and receive follow-up counselling at home. Exclusion criteria include: previous/current receipt of HIV care, on-going participation in other health-related research, planned change of residence in the next 6 months, and inability to provide informed consent.2.1.2HIV-negative adults (≥18 years) who are willing to provide informed consent, receive follow-up counselling at home, and have no intention of changing residence in the next 6 months are recruited from each randomised community primarily to reduce the possibility of revealing the HIV positive status of the main trial participants. Data from these participants will also be used to investigate whether follow-up counselling increases the uptake of repeat HIV testing.2.2Clusters comprised one or more villages i.e. the smallest administrative areas. All villages (n = 158) in the study area were mapped  and the 2.3Sample size was calculated to ensure adequate power to address the hypothesis that follow-up counselling would increase the proportion of individuals that link to care. Based on previous studies in the area k, of 0.25. This was based on past studies of linkage to HIV care k ranged from 0.12 to 0.33. We assumed linkage of 35% in the control arm based on findings from earlier HBHCT studies k of 0.25 and a harmonic mean of 17 participants completing the study in each cluster, we estimated that 11 clusters per arm would be required to have 90% power to detect an increase in linkage to care from 35% in the control arm to 60% in the intervention arm as statistically significant (p < 0.05). This sample size would also give us 95% power to detect a hazard ratio of 1.7 for the effect of the intervention on time to linkage, or 80% power to detect a hazard ratio of 1.5 We assumed a between cluster coefficient of variation, After completing enrolment in the first seven clusters, we observed that 12% (419/3546) of registered resident adults from the mapping exercise were not found at home and could not be contacted in spite of repeated attempts. Of those found at home, 11% (358/3127) declined to have HBHCT. Furthermore, among HIV positive persons, there was a higher than expected level of engagement in HIV care: only 26% (75/294) of those who tested positive in HBHCT were not in care. As a result, the number of participants enrolled per cluster was much lower (harmonic mean < 10) than anticipated. Based on these preliminary findings, the number of clusters was increased to 28 (14 per arm) with an expected harmonic mean of 7 participants completing the study in each cluster; this would allow for detection of the same difference in proportions with a power of 83%, or a hazard ratio of 1.7 with a power of 85%.2.4We used a combination of stratification and restriction in order to minimise between-cluster variation and achieve overall balance between study arms with regard to key variables. First, randomisation was stratified on distance (≤10 km or >10 km) from the Masaka district capital, as this may influence accessibility to HIV care, and on cluster make-up, i.e. whether the cluster was composed of a single large village or several small villages: 70,560 possible allocations (possible ways in which clusters could be randomised to intervention or control arms) were generated under the stratified design. Restricted randomisation was then applied to achieve balance on the following covariates: cluster size; presence of a trading centre; location along a major road; lakeshore location; and presence of an HIV clinic within 5 km. The tolerance thresholds for balance were defined through an iterative process in which different thresholds were tried and the number and the validity of the acceptable allocations examined. Of the possible allocations under the stratified design, 28,932 (41%) were found to satisfy the additional balance criteria after restriction. Of these, a list of 1000 acceptable allocations was randomly generated from which one was selected at public randomisation ceremony. Each allocation had a unique running number.2.4.1The six clusters added later were treated as a separate stratum. Possible allocations for these clusters were generated using the same procedure described above and one allocation selected at a second public randomisation ceremony on 30th June 2015.The ceremony was held on 13th March 2015 and attended by 1–2 leaders from each of the villages. Three sacks each containing 10 balls labelled 0 to 9 were prepared, representing allocations 001 to 999, and 000 representing 1000. The principle underlying the random selection procedure was carefully explained in simple words. Three community leaders, selected by their peers, were then invited and one after the other each asked to draw one ball from one sack  thus generating a 3-digit number, corresponding to the allocation running number on the list. 2.5Participants in the control and intervention clusters receive HBHCT and a written referral to an HIV care clinic of their choice if found to be HIV-positive. In addition, participants in the intervention clusters receive two follow-up counselling sessions at their home, delivered within 2 months after testing. Each session lasts about 45 min. The counselling intervention is designed to address common previously described psychosocial barriers of linkage to HIV care such as stigma 2.6A summary of the study procedures is provided in 2.6.1Mapping of the study area, community mobilisation meetings, staff recruitment and training, setting up of referral tracking and linkage verification procedures with HIV care providers were conducted in the three months prior to study initiation. In each cluster, additional community mobilisation is conducted one week prior to HBHCT.2.6.2Accompanied by a community guide, study counsellors visit all the households in the randomised clusters, enumerate all resident adults aged ≥18 years and offer them HBHCT. HCT is conducted in accordance with national guidelines Once 3–4 HIV-positive participants are enrolled, enrolment is also offered to the first consenting person that tests HIV negative in the next household. We expect to recruit at least 84 HIV-negative participants.2.6.3•Months 1 and 2 (intervention arm only): to provide follow-up counselling.•Month 6: to collect data on linkage to care and other outcomes, perform CD4 cell count testing for participants who have not had a CD4 count test at an HIV clinic or are not aware of the result, and repeat HCT for HIV-negative participants. CD4 cell count and repeat rapid HIV tests are conducted at the MRC/UVRI laboratory in Masaka town. Repeat rapid HIV tests are conducted at the laboratory instead of participants' homes in order to make the procedures for HIV-positive and HIV-negative participants as similar as possible and thus minimise the risk of revealing participants' HIV status.•Month 6 + 1 week: to provide CD4 cell count or repeat HIV test results.There are four post-enrolment home-based follow-up visits.Participants not found at home at any of the post-enrolment visits are revisited at least two more times.2.6.4Outcome data are collected using two methods: Firstly, a questionnaire is administered by the counsellor to collect information on whether and when the participant linked to care, provided a blood sample for CD4 cell count testing and received the results, was informed of his/her ART eligibility and initiated ART, initiated cotrimoxazole prophylaxis and whether he/she adhered to cotrimoxazole prophylaxis. Good adherence to cotrimoxazole prophylaxis is defined as self-report of having taken >80% (≥25) of 30 doses in the past month. Secondly, clinic records are reviewed, looking for a participant's referral letter and medical forms, and pre-ART and/or ART register books to verify self-reported linkage and other outcomes. Participants whose records are not found at the HIV clinic are re-contacted, informed of the verification outcome, and asked to clarify if they actually registered for care or not. Except for adherence to cotrimoxazole, which is not recorded in the clinic records, only verified data will be used for the analysis of primary and secondary outcomes.2.6.5Data are collected using paper-based questionnaires. Before data entry, questionnaires are checked for completeness and logical consistency. Data are double-entered and validated in Microsoft Access. Queries are run on the entered data every fortnight and reports sent to the study team for resolution. Data arising from the resolved queries are resubmitted for entry and the process is repeated until no more queries are generated.2.7Analyses will be by intention to treat and based on individual-level data, since there is a reasonable number of clusters per arm and the cluster size varies considerably ClinicalTrials.gov (NCT02497456).The initial study protocol and subsequent amendments were approved by Uganda Virus Research Institute Research Ethics Committee (GC/127/14/12/491), the Uganda National Council for Science and Technology (HS 1732), and The London School of Hygiene and Tropical Medicine Ethics Committee (8833). Written informed consent  is obtained from each participant before study procedures are conducted. The trial is registered at 3This study is one of the few randomised trials from sub-Saharan Africa designed to evaluate the effect of an intervention to increase linkage to HIV care following HIV diagnosis through HBHCT. We are aware of five other such trials. The first is a recently completed individually randomised controlled trial from South Africa and Uganda that assessed whether community-based HIV testing (including HBHCT) with counsellor support  and POC CD4 cell count testing increases uptake of ART. In this trial, linkage to care was high (≥89%) across all linkage strategies used A major strength of our trial is that referrals for care are tracked and medical records examined to verify self-reported linkage. Hence it is unlikely that the proportion of persons linking to care will be overestimated. Despite concerns about inadequate routine record keeping at health care facilities in Uganda We randomised communities instead of individuals in order to minimise the risk of contamination between trial arms e.g. through sharing of follow-up counselling information, which would result in a dilution of intervention effects. Also, individual randomisation may not have been acceptable to participants especially if they perceive one of the interventions to be inferior to the other.The study has some limitations. First, some individuals in the study communities may have knowledge of their cluster allocation which may influence their decision to enrol in the study. Individuals may obtain prior knowledge of cluster allocation through community leaders who attended the pre-enrolment public randomisation ceremony or from those in the same community who have enrolled before them. To minimise the effect of this potentially differential enrolment, individuals are only informed of their cluster allocation after they have consented and enrolled in the study. Additionally, within each village, participants are enrolled over 2–4 days, to minimise the time available for individuals to find out their allocation from fellow village residents. Related to this is the risk of differential loss to follow-up after enrolment e.g. because different proportions of participants may withdraw from the trial after becoming aware of their allocation. To mitigate against this, part of the eligibility criteria is “availability for the entire duration of the study and willingness to be followed up at home”. Participants who indicate that they will not be available for follow-up are excluded from the study. Second, despite the creation of buffer zones between clusters, there might still be a risk of contamination resulting from possible interaction between study participants from different trial arms. Data on the occurrence of such interaction is collected at the month-6 visit; the results will be reported along with the main trial findings.We did not include a component to evaluate cost-effectiveness of the home-based follow-up intervention. Such evaluation may be necessary to inform policy if the intervention is found to be effective. However, given the fact that it is a relatively simple intervention, the costs of incorporating it into the routine HBHCT work are likely to be minimal.Whereas quantitative methods such as those used in our trial are appropriate for investigating the effect of a health care intervention, a qualitative exploration of experiences, attitudes, beliefs and understandings is needed to find out why the intervention works or does not work This study will contribute evidence on the impact of referral plus home-based follow-up counselling on linkage to care, as compared to only referral among adults identified with HIV infection through HBHCT in Uganda. Interpretation of trial outcomes will be aided by findings from the qualitative sub-study.UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 program supported by the European Union. The International AIDS Vaccine Initiative provided funds for HIV test kits. The Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical MedicineLondon School of Hygiene & Tropical Medicine provided PhD support funds to cover CD4 count tests.This study was jointly funded by the


HIV-1 infection cannot be cured because the virus persists as integrated proviral DNA in long-lived cells despite years of suppressive antiretroviral therapy (ART). In a previous paper  we documDOI:http://dx.doi.org/10.7554/eLife.18889.001 Combination antiretroviral therapy (ART) has had significant effects on the morbidity and mortality associated with human immunodeficiency virus type 1 (HIV-1) infection. ART very effectively suppresses active virus replication, but it cannot eradicate the infection because HIV-1 persists as integrated proviral DNA in long-lived cells that constitute a virus reservoir. Latently infected resting memory CD4+ T-lymphocytes (memory CD4 cells) represent the most solidly documented HIV-1 reservoir , 1995. FBecause of their importance to development of a cure for HIV-1 infection, many methods to quantify HIV-1 reservoirs have been developed. The quantitative virus outgrowth assay (QVOA) has been the ‘gold standard’ , but recThe HIV-1 reservoir is established early during primary infection and is remarkably quantitatively and qualitatively stable. Despite their significance for HIV-1 cure efforts, relatively little is known about the pre-ART establishment and turnover of the HIV-1 reservoir. In this study, we characterized the establishment and maintenance of the HIV-1 DNA reservoirs in 10 patients. We previously studied the evolution of HIV-1 in these patients before ART by whole genome deep-sequencing of HIV-1 RNA in longitudinal plasma samples . We now We found that the HIV-1 DNA populations remained genetically stable for up to 18 years after the start of suppressive ART. The absence of genetic changes indicates that viral evolution and replication are not important mechanisms for the maintenance of HIV-1 reservoirs during supressive ART. We also found that the variants that were replicating shortly before start of ART were overrepresented in the HIV-1 DNA reservoirs. This excess of late variants in the DNA reservoirs indicated that proviral HIV-1 variants continued to turn over with a half-life of approximately one year until the patients began therapy. ART effectively froze the composition of the HIV-1 DNA reservoir in the state it had at start of therapy.The study population consisted of 10 HIV-1 infected patients who were diagnosed in Sweden between 1990 and 2003. The following criteria were used to select the patients: (1) A relatively well-defined time of infection; (2) Being treatment-naive for ≥5 years; and (3) Receiving suppressive ART (plasma HIV-1 RNA levels continuously <50 copies/ml) for ≥2 years. We previously characterized HIV-1 RNA populations in longitudinal plasma samples (6–12 per patients over 5–8 years). Results for 9 out of the 10 patients were reported in For this study, we obtained sequence data from the HIV-1 DNA in viral reservoirs using deep sequencing of the p17gag region of the HIV-1 genome in DNA prepared from PBMCs. Longitudinal PBMC samples (1–3 samples per patient for up to a 2.6-year period) were obtained 3–18 years after the start of suppressive ART . We defiThe HIV-1 DNA sequences recapitulate the diversity observed in RNA sequences before treatment, often with exact sequence matches, The estimated numbers of HIV DNA templates, the numbers of distinct haplotypes observed, and the fractions of haplotypes present in multiple samples are presented in We found that substantial proportions  of the p17gag DNA sequences from the viral reservoirs were hypermutated and were therefore expected to be replication incompetent . This reWhether or not HIV-1 reservoirs are maintained by persistent replication remains controversial . We used−3/year. In contrast, DNA sequences showed no signal of statistically significant evolution in DNA reservoirs during suppressive ART.The results for the rates of evolution before and after the start of suppressive ART are presented in Taken together, our results did not indicate that persistent HIV-1 replication acts as a mechanism to maintain the HIV-1 reservoir during suppressive therapy.The phylogenetic analyses indicated that most of the HIV-1 DNA variants present in the PBMCs matched the HIV-1 RNA variants detected in the plasma samples obtained shortly before the start of therapy . HoweverTo investigate when the PBMC HIV-1 DNA variants were deposited in the viral reservoirs, we compared the on-treatment PBMC DNA sequences with the longitudinal pre-treatment plasma RNA sequences. For each p17gag DNA sequence, we determined the pre-treatment plasma sample and the RNA haplotype that was the most likely source. This method assigned most of the HIV-1 DNA sequences to the plasma samples closest to the start of treatment . A combiSequences that matched sequences at earlier plasma sampling time points were found as minor variants among the p17gag DNA sequences , panel BIn summary, viral variants that replicated shortly before the start of suppressive ART were overrepresented in the HIV-1 DNA reservoirs. This result indicated that the infected cells were turning over  for as long as the patients were untreated. Suppressive ART halted this turnover.We investigated the composition and turnover of HIV-1 DNA sequences in viral reservoirs in patients receiving long-term suppressive therapy. The reservoir HIV-1 DNA populations were remarkably stable and showed no evidence of active replication during ART. Since we had previously characterized the HIV-1 populations in these patients prior to ART, we could determined how sequences in the HIV-1 reservoir related to pre-treatment populations. In particular, we were able to show that sequences in HIV-1 DNA reservoirs mainly derived from the populations present during the last year before the start of suppressive therapy.−3 changes per site per year); these rates are approximately 3- to 5-fold greater than those typically observed in gag and pol of replicating RNA populations. Such rapid evolution is incompatible with the lack of observable changes in the reservoir sequences over the 20 times longer time intervals reported here . However, it is reasonable to assume that most of our HIV-1 DNA sequences were from memory CD4+ T-lymphocytes because others have found that these cells are the main HIV-1 reservoir , 1995. BIn summary, we found compelling evidence against persistent viral replication as a mechanism to maintain the latent HIV-1 DNA reservoir during suppressive therapy. We also found that most of the latently infected cells present during long-term suppressive ART were infected shortly before the start of ART, and that T-cell turnover slowed down dramatically when ART began.The study was performed according to Declaration of Helsinki requirements. Ethical approval was granted by the Regional Ethical Review Board, Stockholm, Sweden (Dnr 2012/505 and 2014/646). Written and oral informed consent was obtained from each patient that participated in the study.The study population consisted of 10 HIV-1-infected patients who were diagnosed in Sweden between 1990 and 2003. These patients had been included in a recent study of the population genomics of intrapatient HIV-1 evolution . The patWhole-genome deep-sequencing of the virus RNA populations present in the plasma samples obtained before the start of therapy was performed as previously described . BrieflyA total of approximately 70 ml fresh whole blood was split between seven Vacutainer (EDTA) tubes. The PMBCs were isolated using Ficoll-Paque PLUS  centrifugation according to the manufacturer’s instructions. Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit  or the QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions. The amount of DNA was measured using a Qubit dsDNA HS Assay Kit . Patient-specific nested primers (Integrated DNA Technologies) were used to amplify a 387 bp long portion of the p17gag gene corresponding to positions 787 to 1173 in the HxB2 reference sequence. The primers were designed based on the plasma RNA sequences from each patient . Outer pThe HIV-specific primers were flanked by NexteraXT adapters. To construct sequencing libraries, indices and sequencing primers were added in 12–15 cycles of additional PCR. The amplicons were sequenced using an Illumina MiSeq instrument and 2 × 250 cycle kits. A total of 6500 to 190,000  paired-end reads were generated per sample. The overlapping paired-end sequencing reads were merged to create synthetic reads spanning the entire p17gag amplicon. The nucleotide on the read with the higher quality score was used for the cases of disagreement between paired reads. We counted the numbers of times a particular p17gag sequence was observed, and performed subsequent analysis using read-abundance pairs. To reduce the effects of sequencing and PCR errors, we combined rare sequences (below frequency 0.002) with common sequences if they differed at no more than one position. Specifically, starting with the rarest sequences, we merged rare sequences with the most common sequence that was one base away. The cutoff value of 0.002 is the typical error frequency of the pipeline . Less thTo classify sequences into hypermutated and non-hypermutated sequences, we counted mutations at positions that did not vary in the RNA samples obtained before therapy. If greater than four mutations were observed, and at least one-half of them were G→A, the sequence was considered to be a hypermutant. The results for the distributions of the different transition mutations relative to the closest genome found in the RNA samples are presented in We reconstructed phylogenetic trees using the approximate maximum likelihood method implemented in the FastTree software . The tipThe root-to-tip distances were calculated as the mean distance between a sample and the founder sequence, approximated by the consensus sequence of the first RNA sample. This root-to-tip distance was regressed against time to determine the rate of evolution in the absence of treatment. The root-to-tip sequence of the last RNA sample and the DNA samples was regressed against time to determine the rate of evolution on treatment. To determine the most likely seeding time for a PBMC p17gag DNA sequence, we calculated the likelihood of sampling this sequence given the SNP frequencies in each plasma RNA sample and assigned the sequence to the sample that had the greatest likelihood value.https://github.com/neherlab/HIVEVO_reservoir.Scripts used for processing and analysis of the data as well as plotting the results are available at  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Arup Chakraborty as the Senior Editor, and he also served as the Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Establishment and stability of the latent HIV-1 DNA reservoir" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:You have carried out a nice study of HIV evolution in patients prior to and after initiation of antiretroviral therapy. You show that the virus continues to evolve until ART is started. During ART, there is no further evolution. This conclusion is consistent with many previous studies. Although most studies of HIV evolution during ART have concluded that ART stops viral replication and thus halts evolution, a recent Nature paper by Wolinsky and colleagues provided some evidence for continuing evolution. Thus, the current paper is timely. The issue is of particular importance with regard to finding a cure for HIV investigation. Most investigators believe that the real barrier to cure is a stable latent reservoir. However, the finding that evolution is still occurring during ART would suggest that an additional problem is inadequate suppression of viral replication. The demonstration that evolution is halted strongly favors the idea that reservoirs are the barrier to cure, not inadequate ART. For these reasons, we think that the paper is likely to be accepted, provided the following comments are addressed.1) You analyzed gag sequences in proviral DNA. However, it is not clear that most proviruses are profoundly defective. Even if the gag gene can be successfully amplified, there are likely to be defects elsewhere in the genome in many of the sequences. How does this affect the phylogenetic analysis? Is it appropriate to treat defective and replication-competent viral species the same way in such analyses? Some discussion of this issue would be helpful.2) Paragraph 2 in Introduction (on QVOA) is difficult to understand. First, none of the QVOA information is used later in the text. The study quantifies neither the number of defective viruses nor the size of the reservoir. Second, while this is a matter of scholarship, the statement about PHA partial stimulation is problematic. There is no evidence that PHA in culture results in partial T-cell activation. The cited paper, Ho et al. 2013 only shows that there is partial HIV reactivation in culture, not the mechanism of how it arises. Partial reactivation of HIV may be due to intrinsic properties of the virus and should also be mentioned .3) A very strong part of the study is the comparison between HIV DNA post-ART and the HIV RNA pre-ART. However, there was not discussion or analysis of this. In our opinion, 4) A more rigorous comparison to the data in Lorenzo-Redondo et al. (possibly in a new Discussion section) would be extremely helpful. The counter argument that will in all likelihood be used to rebut and limit this work as definitive is the classic "lack of data" argument  Related to the above point, why does the analysis not account for clonal expansion? The authors should have done this analysis  to estimate the extent of expansion rather than leave that to speculation (even for a single patient where they assumed this was the top contribution). This would have taken out a major source of possible confusion. What if the sample prep or methodology are somehow dominated by clonal expansion which is why they don't capture any variation?6)  […]1) You analyzed gag sequences in proviral DNA. However, it is not clear that most proviruses are profoundly defective. Even if the gag gene can be successfully amplified, there are likely to be defects elsewhere in the genome in many of the sequences. How does this affect the phylogenetic analysis? Is it appropriate to treat defective and replication-competent viral species the same way in such analyses? Some discussion of this issue would be helpful.It is true that most proviruses are likely to be defective. The dominating forms of inactivating genetic changes are large deletions and hypermutation. This has been fully explained in the Introduction (second paragraph) where we also have added a new reference . In our analyses we identified hypermutated sequences and performed analyses with and without these sequences, with very similar overall results (see 2) Paragraph 2 in Introduction (on QVOA) is difficult to understand. First, none of the QVOA information is used later in the text. The study quantifies neither the number of defective viruses nor the size of the reservoir. Second, while this is a matter of scholarship, the statement about PHA partial stimulation is problematic. There is no evidence that PHA in culture results in partial T-cell activation. The cited paper, Ho et al. 2013 only shows that there is partial HIV reactivation in culture, not the mechanism of how it arises. Partial reactivation of HIV may be due to intrinsic properties of the virus and should also be mentioned .We agree that it is not clear exactly why the QVOA underquantifies.Therefore, we have rephrased the text in the Introduction (second paragraph). We have also added the references that reviewer mentions together with the recent Bruner paper mentioned above.3) A very strong part of the study is the comparison between HIV DNA post-ART and the HIV RNA pre-ART. However, there was not discussion or analysis of this. In our opinion, We agree that this section of the Results (Time for deposition of reservoir HIV-1 DNA sequences) lacked clarity and important details. Therefore we have extensively rewritten this section. We have also completely revised 4) A more rigorous comparison to the data in Lorenzo-Redondo et al. (possibly in a new Discussion section) would be extremely helpful. The counter argument that will in all likelihood be used to rebut and limit this work as definitive is the classic "lack of data" argument . This paper estimated a rate between 0.5 and 0.8 × 10-3 per month. However, these estimates were for the C2-V5 region of gp120 using data by Shankarrappa et al., 1999. It is well-known that gag and pol evolve more slowly than gp120. Recent estimates suggest rates that are 3 to 10-fold lower . We have also added a supplementary figure  that ill5) Related to the above point, why does the analysis not account for clonal expansion? The authors should have done this analysis  to estimate the extent of expansion rather than leave that to speculation (even for a single patient where they assumed this was the top contribution). This would have taken out a major source of possible confusion. What if the sample prep or methodology are somehow dominated by clonal expansion which is why they don't capture any variation?With our data we cannot perform the type of analysis that Maldarelli et al. and Cohn et al. performed. They analyzed integration sites and thereby obtained molecular tags for each integrant/clone. We chose to analyze p17gag because it was part of our plasma HIV RNA dataset, in contrast to the extreme ends of the LTRs, and because it had sufficient genetic diversity to allow reliable comparison between plasma RNA and PBMC DNA HIV populations. We have expanded the discussion of the pros and cons of our sequence method and data .As pointed out in the revised Discussion, several lines of evidence suggest that clonal expansion has not affected our results in a qualitative manner. Clonal expansion would result in undue over-representation of a some sequences. To control against such expansion, we repeated the analysis in 6) We observed a total of 604 reads  that could not be assigned to a patient sample. Such aberrant reads were observed in 10 out of 30 samples and the majority of these reads mapped to the human genome. This has been clarified in the Methods section (subsection “Sequencing and read processing”).


To determine the postnatal course of neurosteroid levels in relation to gender, mode of delivery and the extent of skin-to-skin (STS) contact during the first days of life in healthy term newborns.Prospective observational study of 39 neonates in which parents recorded total duration of STS in the first 2 days and nine neurosteroids  were assayed from blood samples at birth and at 1–2 days of age.All nine neurosteroid levels declined significantly during the first 2 days of life. Gender did not significantly affect the change in neurosteroid levels. The decline in neurosteroid levels was generally more pronounced in vaginal deliveries, and there was a trend toward a larger decline with more exposure to STS.Ongoing studies may better characterize the role of neurosteroids and the influence of STS in more critically ill and premature neonates. We studied nine of the neuroactive steroid compounds most commonly reported in the literature to act on the human brain.This was a prospective observational study of healthy newborns in the delivery room and postpartum unit at Lucile Packard Children's Hospital Stanford during 2015 and 2016. Institutional Review Board approval was granted by Stanford University. Prospective participants were screened through the electronic medical record, and written informed consent was obtained from infants' parents prior to delivery, on admission or at preoperative appointments in the Labor and Delivery unit.−1) or other high-risk medical/surgical co-morbidities (as determined by the clinician).Inclusion criteria for study eligibility were inborn infants with gestational age ⩾37 weeks, birth weight ⩾2000 g, cardio-respiratory stability (as determined by clinician) and prior written informed consent. Exclusion criteria were congenital anomalies, cardio-respiratory instability, invasive ventilation, hypotension requiring pressor support, acidosis (pH<7.2), blood culture-positive sepsis, hypernatremia  and infant gender were recorded. Parents were given logs to record the duration in minutes of all episodes of STS contact  in the first 2 days of life. Parents were taught the definition of STS contact and instructed how to fill out the log by study personnel at the time of consent. The completed logs were then reviewed with families for any discrepancies or unclear entries at the time of collection by study personnel.One milliliter of neonatal blood was collected in lithium heparin vacutainer and microtainer tubes  from the placental side of the cut umbilical cord at birth and from a routine heel stick concurrent with standard-of-care laboratory tests drawn between 24 and 48 h of age. Whole blood was centrifuged within 15 min of collection, and 300 μl of plasma was aliquoted, immediately frozen with liquid nitrogen and stored at −80 °C. Plasma samples were packaged with dry ice and shipped overnight to the Gaikwad Laboratory at the University of California Davis, Davis, CA, USA where samples were assayed for nine neuroactive steroids.13 using a Waters Acquity UPLC system connected with the high performance Xevo-TQ mass spectrometer. Resulting data was processed by using the TargetLynx 4.1 software , and all neurosteroid concentrations are reported in pg ml−1.Plasma samples were extracted with little modification and subjected to ultra-performance liquid chromatography tandem mass spectrometry analysis as described previouslyP=0.05. The Wilcoxon signed-rank test was used to compare steroid levels at birth with those at 1–2 days of age. The two-sample Wilcoxon rank-sum (Mann–Whitney) test was used to compare the changes in steroid levels in the first 2 days based on infant gender, mode of delivery and duration of STS. Statistical analyses were performed using Stata 13 .Owing to the large range and non-normal distribution of the steroid levels, statistical analyses were carried out with non-parametric tests at a significance level of We approached 90 expecting mothers and obtained 49 consents. Two infants were withdrawn owing to admission to the neonatal intensive care unit for cardio-respiratory instability. Forty-seven infants were enrolled in the study and 87 samples were assayed. We analyzed 39 infants with gestational age ⩾37 weeks  who had complete data, which included a cord blood sample from birth, a blood sample from 1–2 days of age and a completed STS log. Of the eight remaining infants who were excluded, one had gestational age <37 weeks, three were missing a cord blood sample and four were missing a blood sample from 1 to 2 days of age.P=0.03, using the two-sample Wilcoxon rank-sum test).For the 39 infants, the mean birth weight was 3383 g (s.d. 507), and the mean gestational age was 38.8 weeks (s.d. 0.8). Twenty-four (62%) of the infants were male, and 30 (77%) were delivered by c-section. Twenty-eight (72%) of the deliveries were scheduled c-sections without preceding labor. Steroid levels after birth were collected concurrently with routine hospital blood draws, which were performed at a mean age of 26 h (range 24–45 h). Total duration of STS episodes in the first 2 days of life ranged from 0 to 1530 min with median 346 min (interquartile range (IQR) 450). For c-section versus vaginal deliveries, the median STS duration in the first 2 days was 462 (IQR 495) versus 240 (IQR 146) minutes  and more premature infants (28–33 weeks' gestation) to determine the changes in neurosteroid levels based on the degree of prematurity and its resulting clinical treatments, which may preclude STS provision. Recent literature20 has also indicated that peripheral concentrations of neurosteroids may not correlate with central nervous system concentrations. Thus future studies, first in neonatal animal models, and then potentially in human neonates, would ideally include neurosteroid levels in cerebrospinal fluid when it is already being obtained for other diagnostic or therapeutic purposes.A limited number of neuroactive steroids have been studied in human neonates. For example, pregnenolone concentrations in healthy human neonates display a rapid and significant fall in the first days of life in both full-term and late preterm infants.per se. Further investigations into the reasons for c-section, the duration of labor in both vaginal and c-section deliveries preceded by labor and preexisting maternal and fetal morbidities will be necessary.Given the predominance in our study of c-section deliveries not preceded by labor, we had the ability to compare newborns who experienced the ‘squeeze' of a vaginal delivery to those who did not. We saw that, similar to foals, neonates born by c-section had less of a decline in three neurosteroids . Thus the effect of delivery mode on the decline in neurosteroid levels in the first 2 days may be due to the lack of labor rather than the mode of delivery n=30) versus vaginal deliveries (n=9) and overall small sample size are potential limitations. However, differential in sample size increases the chances of a type II  error but not a type I  error. The unequal group sizes would have therefore favored not finding a difference in the decline in neurosteroid level based on delivery mode. Instead, we did find significant differences for progesterone, epipregnanolone and pregnanolone-sulfate. Sample size calculations  indicate that increasing the total sample size to 80–100 infants  would give power to detect significant differences in the change in steroid level based on delivery mode for two additional neurosteroids (DHEAS and pregnenolone-sulfate).The large differential in c-section deliveries (Parental report of STS contact in the first 2 days may have been a contributing factor to the small number of neurosteroids that showed a significant relationship between duration of STS and decline in steroid levels. Despite training of parents on the STS logs at study consent and review of the logs with families after completion, both under-reporting and over-reporting of STS duration are possible. However, parents in a postpartum unit where the mothers and babies are cared for in the same room are the best positioned observers and recorders of STS that we currently have. In the future, as STS becomes standard of care, documentation by medical personnel of STS duration in the electronic medical record will become more standardized or, potentially, wearable devices that track proximity and body temperature may assist in more objectively tracking STS.Although there was a trend toward a larger decline in neurosteroid levels over the first 2 days associated with more exposure to STS, this relationship was only significant for pregnanolone in all deliveries and for progesterone in vaginal deliveries. Given the findings in foals, we would have expected that exposure to STS would have a more significant impact on newborns born by c-section, where they potentially did not receive as much ‘squeeze' or ‘conditioning' during the birthing process. We did not, however, see this association in our small sample, which may be explained by the fact that these were healthy newborns. Healthy newborns may not be the correct analog to the neurologically abnormal foals with NEMS who respond to the ‘squeeze technique', and it may be that STS has a much larger effect on newborns where the normal prenatal, perinatal and postnatal processes are altered. This includes premature newborns, newborns with birth hypoxia resulting in neurological impairment and other critically ill newborns in the neonatal intensive care unit.In addition to expanding the study population to more critically ill and more premature infants, future studies should also extend the study period beyond the first days of life. In particular, tracking the levels of neurosteroids along with the ‘dose' of STS contact in the first days to weeks to months of life could lead to the discovery of a biomarker for STS contact, which optimizes the neurodevelopment of these vulnerable patients.Neurosteroid levels decline during the first days of life in healthy term newborns. This decline is generally more pronounced in vaginal deliveries, and there is a trend toward a larger decline with more exposure to STS. Ongoing studies may better characterize the role of neurosteroids and the influence of STS in more critically ill and premature neonates.


Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brain tumors, whereby the standard of care is focal radiation, a treatment that provides temporary relief for most patients. Surprisingly, decades of clinical trials have failed to identify additional therapies that can prolong survival in this disease. In this conference manuscript, we discuss how genetic engineered mouse modeling techniques with the use of a retroviral gene delivery system can help dissect the complex pathophysiology of this disease. With this approach, autochthonous murine DIPG models can be readily induced to (1) help interrogate the function of novel genetic alterations in tumorigenesis, (2) identify candidate cells of origin for this disease, (3) address how region-specific differences in the central nervous system influence the process of gliomagenesis, and (4) evaluate novel therapeutics in an immunocompetent model. Pediatric brain tumors are now the leading cause of cancer-related death in children . Of the Cancer Cell , due to its high prevalence in DIPGs at approximately 80%  and it being the first time that a cancer-associated SNV was identified in a histone gene itself . Other Scer Cell . Besidescer Cell , 18. TheIn this review, we are focusing on the use of the RCAS/tumor virus A (TVA) system to induce murine DIPGs Figure  but it iDiffuse intrinsic pontine gliomas arises in a unique part of the brain, the pons, a part of the brainstem that harbors critical neurons regulating basic functions such as respiration, heart rate, and blood pressure. This is why surgical resection is not an option for DIPG. One application of genetic mouse models using retroviruses is to interrogate region-specific differences within the central nervous system by generating age- and strain-matched murine gliomas induced by the same genetic events and comparing their differences and similarities using expression profiling. We performed one such experiment with our initial DIPG model driven by PDGF-B and Ink4a-ARF loss. By comparing the expression profile of brainstem gliomas and gliomas induced in the cerebral cortex that were initiated with the same two genetic alterations, we identified a small number of significantly differentially expressed genes. We focused on one such gene, paired box protein 3 or Pax3, a transcription factor that we observed to be upregulated in brainstem gliomas relative to gliomas arising in the cerebral cortex and relative to normal brainstem. Additional experiments demonstrated that pax3 is expressed in tumor cells, that it cooperates with PDGF-B in promoting gliomagenesis, and that a distinct subset of human DIPGs, approximately 40%, also have high expression of pax3 .Accurate genetic mouse modeling of DIPG requires introduction of the genetic alterations to the correct cell of origin during the development of the pons. One challenge is that it is difficult to be certain of the correct cell of origin of the human disease as DIPG is often diagnosed once patients develop sign and symptoms, a relatively late time-point in the natural history of the disease, where the tumor occupies the majority of the pons. With this caveat in mind, there have been a few studies using human postmortem pontine tissue to investigate candidate cell of origin for DIPG. In one study, candidate cells of origin for DIPG were identified as a Nestin+/Vimentin+/Olig2+ cell population located in the ventral pons during middle childhood as this cell population mirrors the age-specific incidence of DIPG . In a seOne important aspect of DIPG from a therapeutic perspective is its relatively intact blood–brain barrier which is most evident on MRI imaging as DIPGs rarely enhance with gadolinium on T1 sequences. A detailed discussion of the composition of the blood–brain barrier is outside the scope of this communication, but in brief, two main components of the blood–brain barrier are the tight junctions between endothelial cells and efflux pumps such as ABCB1 and ABCG2 that are expressed by endothelial cells and efflux drugs back to the bloodstream. As it is unclear whether it is the tumor location, or its genetic alterations that most contribute to the relatively intact blood–brain barrier of DIPG, we investigated whether the brainstem location or the histone mutation or both that contributes to the relatively intact blood–brain barrier in DIPG. Using the RCAS/TVA genetic mouse modeling approach, we generated murine DIPGs with and without H3.3K27M in both the cerebral cortex and the brainstem, and measured the relative openness of the blood–brain barrier with dynamic contrast-enhanced MRI. Interestingly, we observed that blood–brain barrier permeability was 67% significantly higher in PDGF-B driven gliomas that were induced in the cerebral cortex vs. those that were induced in the brainstem while H3.3K27M did not significantly impact the openness of the blood–brain barrier . These rWhen the K27M H3 mutations in DIPG were first discovered in 2012, we investigated whether their expression in nestin progenitors of the neonatal brainstem would be sufficient to drive gliomagenesis in a similar manner to PDGF-B. As 20% of human DIPGs do not harbor the H3 mutations but are clinically indistinguishable, it is likely that H3 mutations are not necessary for DIPG formation. Perhaps not surprisingly, expressing the mutant histone alone (H3.3K27M) in nestin progenitors of the neonatal brainstem did not induce tumors by 12 weeks of age. Interestingly, expressing H3.3K27M with p53 loss in nestin progenitors of the neonatal brainstem resulted in the development of ectopic proliferating cell clusters in approximately 70% of the mice by 12 weeks of age but no overt tumors were observed . As PDGFin utero electroporation using piggyBac transposon-based vectors of H3.3K27M and p53 deletion using CRISPR did succeed in inducing gliomas with these two genetic events suggesting that these two genetic events may be sufficient to drive gliomagenesis in certain settings  of PDGF-B; H3.3K27M; p53-deficient tumors and PDGF-B; p53-deficient tumors and compared their transcriptome. It is worth noting that we did observe significantly reduced global H3K27me3 in the PDGF-B; H3.3K27M; p53-deficient tumors relative to PDGF-B; p53-deficient tumors in a similar fashion to the reduced global H3K27me3 observed in human DIPGs with K27M H3 mutations , 35, 36.hibition .in vivo in the PDGF-B; p53-deficient DIPG model and observed a modest prolongation of survival. Interestingly, the survival benefit with dasatinib was more substantial when we treated PDGF-B; p53-deficient DIPG-bearing mice that were deficient for ABCG2 and ABCB1  significantly prolonged survival , 40. It nd ABCB1 . Evaluatpatients .Genetic mouse modeling of DIPG with the RCAS/TVA system provides a complementary approach to studying human DIPGs to better understand its complex biology with the ultimate goal of developing improved therapies. Increased research efforts focusing on DIPG in the past decade have ushered in new insights regarding DIPG pathogenesis as well as the development of improved DIPG models, both patient-derived and genetic models. Of course, there are many, many questions that remain to be addressed. Is the relatively intact blood–brain barrier the reason for the failure thus far to identify a single drug that can significantly prolong survival in this disease? Is the K27M histone mutation required for tumor maintenance and is it an important therapeutic target? Are some of the epigenetic changes it induces irreversible as has recently been shown for the IDH mutation ? Can we OB wrote the manuscript. CH and CS prepared figures for the review. All the authors read and approved the submission of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We describe a case of pemetrexed toxicities related to reabsorption by an ileal neobladder, which caused prolonged hematotoxicity and nephrotoxicity.A 59-year-old white man was diagnosed with metastatic wild-type adenocarcinoma of the upper lobe of his right lung. After a first cycle of cisplatin and pemetrexed, he had unusually prolonged aplasia and acute kidney injury.The prolonged aplasia was caused by pemetrexed reabsorption by the ileal mucosa of the neobladder as pemetrexed was eliminated renally in an active form and is partly lipophilic.Pemetrexed may be reabsorbed by the ileal mucosa of the neobladder because of its hydrophobic structure and renal excretion in its active form. Acute urinary retention may maintain this phenomenon. Published data excluded a potential role for cisplatin in this toxicity; furthermore, we could not assess pemetrexed concentrations in the blood or urine as these assay techniques are not validated. Thus, care is needed when giving chemotherapy to patients with a neobladder. We present a case of pemetrexed toxicities due to reabsorption by an ileal neobladder that occurred in a 59-year-old white man with wild-type metastatic lung adenocarcinoma. He developed unusually extended aplasia and acute kidney injury after a cycle of cisplatin and pemetrexed. We considered that ileal reabsorption of pemetrexed by the neobladder was responsible for the prolonged hematotoxicity. In fact, ileal mucosa absorption is possible because pemetrexed is partly lipophilic and eliminated in an active form.EGFR , ALK (anaplastic lymphoma kinase), KRAS , ERBB2 (erb-b2 receptor tyrosine kinase 2), and B-Raf  wild-types. He had a previous history of radical cystectomy, prostatectomy, and vesiculectomy and received a Hautmann neobladder in 2001. The neobladder was constructed from ~ 70 cm of distal ileum, which was anastomosed to his ureters and urethra. He initiated cisplatin and pemetrexed chemotherapy, and a vitamin B12 and folic acid supplementation had been prescribed prior to the initiation of treatment.We report a case of a 59-year-old white man with a metastatic adenocarcinoma of the lung diagnosed in April 2013. This tumor was After 10 days of chemotherapy he was hospitalized because of fever above 39.9 °C. A blood sample showed neutropenia > 0.3 G/L and thrombocytopenia > 21 G/L. Thus, amoxicillin-clavulanate plus ciprofloxacin treatment was started.On the 4th day after hospitalization he had acute renal failure, metabolic acidosis, hyponatremia, and hyperchloremia.An abdominopelvic computed tomography (CT) scan showed major distension of the neobladder and bilateral pyelocaliceal dilatation. Urethral catheterization collected 2 L of a gelatinous liquid.The electrolyte disorders were corrected by 23 days after hospitalization and his renal function was restored at 16 days of hospitalization (Fig. Our patient presented unusual hematological adverse effects after a first cycle of chemotherapy. In patients with an advanced stage of non-small-cell lung cancer, pemetrexed is known to cause grade 3 and 4 neutropenia in 15% of cases, grade 3 and 4 thrombocytopenia in 4% of cases . In contIt is to be noted that in patients with locally advanced or metastatic bladder cancer treated with pemetrexed, the hematotoxicity rate is more significant so that cases of grade 3 to 4 neutropenia are up to 75% . The hemBecause of the hydrophilicity and the low oral bioavailability of cisplatin, it can be assumed that cisplatin was not absorbed by the ileal mucosa. In a phase I study of pemetrexed, 78% of the administrated dose was found in its active form within the urine. Its plasma half-life is ~ 3.1 hours when renal function is normal .During our patient’s urine-retention episode, we supposed that pemetrexed was stored in an active form by the ileal neobladder due to its lipophilic nature, which maintains toxicities. Cisplatin could not have caused this prolonged toxicity because of its hydrophilicity and so could not be reabsorbed by the ileal mucosa.Pemetrexed was the main cause of the adverse effects observed because of its absorption by the ileal neobladder, with this being exacerbated by our patient’s urinary retention.It was not possible to determine the plasma and urine concentrations of pemetrexed as these techniques are not routinely validated.


South Africa has the largest HIV epidemic in the world, with 19% of the global number of people living with HIV, 15% of new infections and 11% of AIDS-related deaths. Even though HIV testing is mandated in all hospital-based facilities in South Africa (SA), it is rarely implemented in the Emergency Department (ED). The ED provides episodic care to large volumes of undifferentiated who present with unplanned injury or illness. Thus, the ED may provide an opportunity to capture patients with undiagnosed HIV infection missed by clinic-based screening programs.In this prospective exploratory study, we implemented the National South African HIV testing guidelines  for 24-hours a day at Frere Hospital in the Eastern Cape from September 1st to November 30th, 2016. The purpose of our study was to quantify the burden of undiagnosed HIV infection in a South African ED setting. Furthermore, we sought to evaluate the effectiveness of the nationally recommended HIV testing strategy in the ED. All patients who presented for care in the ED during the study period, and who were clinically stable and fully conscious, were eligible to be approached by HIV counseling and testing (HCT) staff to receive a rapid point-of-care HIV test. A total of 2355 of the 9583 (24.6%) patients who presented to the ED for care during the study period were approached by the HCT staff, of whom 1714 (72.8%) accepted HIV testing. There was a high uptake of HIV testing (78.6%) among a predominantly male (58%) patient group who mostly presented with traumatic injuries (70.8%). Four hundred (21.6%) patients were HIV positive, including 115 (6.2%) with newly diagnosed HIV infection. The overall prevalence of HIV infection was twice as high in females (29.8%) compared to males (15.4%). Both sexes had a similar prevalence of newly diagnosed HIV infection  in the ED.Overall there was high HIV testing acceptance by ED patients. A non-targeted testing approached revealed a high HIV prevalence with a significant burden of undiagnosed HIV infection in the ED. Unfortunately, a counselor-driven HIV testing approach fell short of meeting the testing needs in this setting, with over 75% of ED patients not approached by HCT staff. HIV testing is the critical first step to meeting the WHO global strategy goal of having 90% of HIV-infected individuals tested, 90% of those on anti-retroviral treatment (ART), and 90% of those virally suppressed (90-90-90). Early deIn 2016, South Africa had 270,000 new HIV infections, 110,000 AIDS-related deaths and over 7.1 million people living with HIV., 4 The bRecent innovations in HIV testing often focus on difficult to access and high-risk populations such as prisoners, commercial sex workers, and injecting drug users. –11 One sDespite the 2015 National South African HIV testing guidelines mandating universal testing in all healthcare facilities, ED-based HIV testing is not routinely implemented in SA., 30 Freqst to November 30th, 2016. Frere Hospital is an urban tertiary care center that provides 24-hour emergency medical and trauma care to the greater East London area, as well as for referrals from regional and district hospitals up to 180km away. The ED has approximately 35 beds and two doctors on staff at all times. There is no Electronic Health Record (EHR) or patient tracking system. Patients wait in line and are seen in order of presentation unless they are critically ill.This prospective study was conducted at Frere Hospital in East London, SA from September 1During the study period, HIV testing was implemented and conducted per the recommendations and protocols in the 2015 National HCT guidelines, as was required by the local ethics review board.  The coreth-grade education. HCT staff received remuneration in line with current salaries for lay HCT providers (approximately 150 ZAR ($10)/day). Before starting the study, all staff were required to complete Good Clinical Practice (GCP) and data collection training, as well as an HCT refresher course provided by Department of Health certified trainers from the antenatal clinic at Frere Hospital. Staff worked in 12-hour shifts to cover the ED 24 hours a day. At all times two HCT staff were available to conduct HIV testing.HIV testing in SA is often conducted by lay counselors . All HCT staff had been previously trained in rapid POC HCT and had been previously employed as lay HCT providers in the neighboring area. Staff were required to be fluent in English, Xhosa and/or Afrikaans and have a minimum 12TM Rapid Anti-HIV 1& 2 test . Non-reactive specimens were considered HIV-negative and reported as such. All reactive specimens were confirmed using an ABON™ HIV 1/2/O Tri-line HIV Rapid Test . The Advanced testing kit provided results within 10 minutes and the ABON kit within 15 minutes. The total time to complete HCT for an HIV negative patient was between 30–40 minutes, and for an HIV positive patient was up to 60 minutes.The HIV testing algorithm implemented used rapid POC blood tests approved by the South African Department of Health. Blood samples were obtained using a finger-prick. All patients were first tested with the Advanced QualityAs per the 2015 National Testing Guidelines, patients with two reactive rapid tests were considered to be HIV infected, and no further confirmatory testing was done. Patients were provided with their HIV results during the ED stay . HIV positive patients were provided with a letter stating their results, as is standard of care, to facilitate referral to a local anti-retroviral (ARV) clinic. Specimens that were reactive on the first assay but non-reactive on the second were subject to an ELISA laboratory test and recorded as discordant (no discordant results were generated during this study). Testing kits were stored in a cool room, with daily room temperature logs. Each kit was first quality control-checked using known reactive and non-reactive controls.All patients who presented for care in the ED during the study period and were over the age of 18 years, fully conscious, and clinically stable were eligible to be approached by HCT staff. As implemented, the HIV counselors were instructed to approach all patients who were eligible as soon as they completed the triage process. There was a private waiting area after the triage station where HCT providers could approach patients. Staff were instructed to try and capture all patients who presented for care to the ED.The primary outcome measure was the proportion of patients with newly diagnosed HIV infection among those who were tested by the HCT staff in the ED. Secondary outcome measures included patient characteristics such as age, sex, presenting complaint, South African Triage Scale (SATS) score, disposition and previous diagnosis of HIV infection. Presenting complaint was recorded as free text and coded using Medical Dictionary for Regulatory Activities (MedDRA). The severity of illness was quantified using SATS , as is standard of care. Data werBased on the recent survey data from the 2012 South African National HIV prevalence study, HIV prevalence is estimated at 12.2%. Our studThis study was approved by the Johns Hopkins University School of Medicine Institutional Review Board, the University of Cape Town Human Research Ethics Committee, and the Walter Sisulu University Human Research Ethics Committee. Written consent was obtained from all participants who enrolled in the study and was required for both the collection of demographic data and POC HIV testing results. Approval was not given to collect de-identified data on patients that presented to the ED but did not participate in the study, i.e., did not consent to an HIV test.A total of 2355 of the 9583 (24.6%) patients that presented to the ED for care during the study period were approached by the HCT staff .Of the 2355 patients approached by the HCT staff, 1714 patients (72.8%) accepted testing, and 641 patients (27.2%) declined to test. Of those who declined HIV testing, 138 patients (27.4%) stated that they had a known diagnosis of HIV. In this study, we could not collect any further data on the reasons for refusing to test. Of the 1714 rapid POC HIV tests performed, 262 tests were positive and 1452 tests negative. Of the 262 patients who had a positive HIV test in the ED 115 patients stated that this was a new HIV diagnosis . One hunBaseline demographics were collected on all patients approached by HCT staff . CompareHIV status was determined in 1852 patients  and those who declined testing but were known positive (n = 138)) . Of thesThe overall burden of newly diagnosed HIV infection in the ED was similar in females (6.0%) and males (6.4%). However, the prevalence of known HIV infection in females (23.8%) was more than twice that in males (9.0%). This led to an overall prevalence of HIV infection for females of 29.8% and 15.4% for males . Fig 2 pA significantly higher proportion of patients presented with traumatic injuries in all three groups  . There wThe critical focus of an HIV testing intervention is to capture those patients with a new diagnosis of HIV infection (n = 115) among those whose status is unknown (n = 1567). Overall the relative risk of a new diagnosis of HIV infection was not significantly impacted by gender, time of testing or symptomatology . FurtherIn This exploratory study demonstrates a high burden of HIV infection in a South African ED setting. The prevalence of HIV infection in our ED population (21.6%) is considerably higher than the reported estimates for the Eastern Cape province (12.2%), and the local district 13.6%).[.6%.[3] TThe overall HIV prevalence in females was almost twice that of males in our study. This rate is higher when compared to the national HIV figures where females outnumber males by a factor of 1.45 (14.4% compared to 9.9%) and thus may reflect higher population burden of HIV among females in the area compared to the national average.  We also Over 78% of patients accepted an HIV test when offered, demonstrating that patient refusal was not a barrier to the implementing an ED-based testing program. Acceptance of testing in our study was considerably higher than the testing uptake reported in a South African out-patient setting (49%), and the National household survey (67.5%).  Although men were more likely to refuse to test than women, our study still managed to capture a majority of male participants for HCT (56.9%). One of the challenges and arguments against providing routine HCT in the ED is the high stress, sometimes chaotic environment, with critically ill patients requiring urgent care. Documenting an HIV diagnosis in this setting is not thought of as a priority., 40, 41 Unfortunately, despite having two HCT counselors providing testing services 24 hours a day, we were only able to approach a quarter of the patients who presented for care. In part, we believe that this poor capture was due to time-consuming nature of the HIV testing process that was implemented. The current testing guideline, require extensive pre- and post-test counseling and can take 30 to 40 minutes to complete . Some studies have also cited that long wait times may contribute to test refusal.The number needed to test for each new positive case was 16:1 yielding on average one new HIV case per day. Some may suggest targeted testing in a setting where HCT resources are limited. However, our study also demonstrated that a targeted testing approach by gender, presenting complaint, or the presence of TB symptoms, would miss many of the patients we captured with undiagnosed HIV infection and underlines the importance of a universal HIV screening approach in the ED. , 42–46The implemented strategy required an eight-person HCT team to provide 24-hour testing services, costing on average 600 ZAR ($40)/day. This human resource burden may be difficult to maintain in a real-world setting where human resources are limited. Also, because a significant proportion of HIV testing (55.3%) was conducted outside of regular business hours (defined as 9 am to 5 pm), a traditional facility-based HCT strategy would have missed this population. Studies have identified that lay counselor programs lack standardized training and supervision and that competing administrative/auxiliary duties may lead to reduced service delivery. A more iA significant limitation of this study is that only a quarter of the ED population was approached by HCT staff. Due to the lack of an EHR/patient-tracking system, we cannot capture information on those who were missed. Thus, we were unable to perform a sensitivity analysis on the patients that were missed. It is possible that there is a bias in the patients included in this study, such as they were easier to approach, more likely to speak the same language as the HCT staff, and presented at times when the patient volume in the ED was lower. This may limit the generalizability of the study to the overall population attending the ED and in the community. Nevertheless, our study did reveal a relatively high prevalence of HIV infection among those who were tested. Future studies will need to capture the whole of the ED population to present a more accurate assessment of the true burden of HIV in this setting. Furthermore, information on linkage to care for these patients is not available, which would have been necessary to assess the downstream impact of the testing program on the 90-90-90 targets.This evaluation of the nationally recommended HCT strategy in a South African ED demonstrated a high acceptance of HIV testing in the ED, with 6.2% of those being tested having a new diagnosis of HIV infection. There were no significant demographic or clinical risks for HIV infection identified, which argues for routine universal HIV testing in the ED. Further innovation and implementation research is required to develop a sustainable testing strategy integrated into the clinical environment so that service delivery in the ED can be improved.


Peer support is considered to be an important framework of support for mothers experiencing postpartum depression (PPD); however, some barriers exist that may limit its use including peer availability and mothers’ lack of time due to child care.This non-randomized study was designed to examine the feasibility, acceptance, and preliminary clinical outcomes of using 7 Cups of Tea (7Cups), a digital platform that delivers self-help tools and 24/7 emotional support delivered by trained volunteers, as an adjunct treatment for mothers diagnosed with PPD.Mothers with PPD were referred during intake to the study coach who provided guidance about 7Cups. 7Cups features included self-help tools and chats with trained volunteers who had experienced a perinatal mood disorder in their past. Acceptability was measured by examining self-reports and user engagement with the program. The primary outcome was the Edinburgh Postnatal Depression Scale (EPDS) change score between pre- and postintervention at 2 months, as collected in usual care by clinicians blinded to the study questions. Using a propensity score matching to control for potential confounders, we compared women receiving 7Cups to women receiving treatment as usual (TAU).P<.001, Cohen d=1.17). No significant difference in EPDS decrease over time was found between 7Cups and TAU, yet the effect size was medium favoring 7Cups .Participants (n=19) proactively logged into 7Cups for a median of 12 times and 175 minutes. Program use was mostly through the mobile app (median of mobile use 94%) and between 18:00 and 08:00 when clinicians are unavailable . Participants chatted with volunteers for a total of 3064 minutes and have indicated in their responses 0 instances in which they felt unsafe. Intent-to-treat analysis revealed that 7Cups recipients experienced significant decreases in EPDS scores (d=0.58) favoring the 7Cups group relative to TAU suggests that 7Cups might enhance treatment outcomes. A fully powered trial has to be conducted to examine this effect.This study supports using a computerized method to train lay people, without any in-person guidance or screening, and engage them with patients diagnosed with mental illness as part of usual care. The medium effect size ( Postpartum depression (PPD) affects 10% to 15% of mothers within the first year after giving birth , PPD carPeer support is considered to be another important framework of support for mothers experiencing PPD -14. For 7 Cups of Tea (7Cups) was chosen for this project because its solution enables, in a scalable way, training users from the community and engaging them with those who seek their support. Another reason to choose 7Cups was the high volume of available volunteers on the platform , openingWorking in collaboration between researchers, clinicians, and patients, we adapted 7Cups to supplement treatment for women with perinatal mood disorders . The firBased on the previous study results, modifications were made, including (1) recruitment of women listeners who had a personal experience of perinatal mood disorders, (2) providing group support tools, (3) integrating relevant evidenced-based self-help tools within the platform, and (4) appropriately presenting relevant information about the listener. Once we were satisfied with the modification process we conducted this pilot study. The primary objectives were to examine (1) whether using 7Cups as an adjunct to treatment is feasible in terms of patient recruitment and timeline (ability to offer the intervention as planned shortly after diagnosis and to assess study outcomes), (2) program acceptability by obtaining participant attitudes toward the use of 7Cups as an adjunct to treatment and examining user behavior on the platform , and (3) preliminary efficacy using standardized outcome measures. Our secondary objectives were to explore use patterns in order to understand user preferences and examine whether the experience of receiving lay people’s support felt safe to participants of the study. Patients were referred to use 7Cups in their own environment. We set ourselves the aim of keeping the referral process as close as possible to real-world settings such that patients learned about 7Cups enhancement option as part of their regular intake process.Participants were recruited from December 2015 to May 2017 from the perinatal program at the Adult Outpatient Department in the Zucker Hillside Hospital. We aimed to recruit more than 15 participants to enable identify a medium (0.5) pre to post effect size with 90% power and 2-sided 5% significance . OutpatiThe Feinstein Institute for Medical Research Institutional Review Board approved the study. Patients meeting eligibility criteria who consented to participate came for an in-person meeting with a health technology coach who guided them through the use of 7Cups. Participants completed assessments at baseline and 30 days after. They received up to US $110 compensation for completing assessments, regardless of their use of either 7Cups or usual care.The adaptation of 7Cups as part of the program to supplement treatment for perinatal mood disorders was described in a previous paper . 7Cups pStudy participants had the option to browse through the list of general listeners allowing them to filter listeners based on different categories . ParticiThe second component is a personalized growth path . Growth paths provide users with a tailored map to help them go from their current state to feeling better . They prThe third component is audio-based mindfulness exercises covering topics taken from the mindfulness and relaxation world such as calming meditation and acceptance of thoughts. Enrolled participants could access 7Cups whenever and wherever they chose, through the 7Cups app installed on their device or by computer log-in. The use of 7Cups was completely on demand and patients were not automatically prompted to use it in certain times or situations.Coaching had several aims: installing the app and guiding participants on the program features, tackling any technical difficulties, and encouraging beneficial use through constructive feedback. To enable proper feedback, the coach had access to a platform that presented all user activity in 7Cups. Coaching included 30 minutes in-person guidance (mostly conducted before or after a meeting scheduled for the patient with her clinicians) where users signed in to 7Cups on a computer and then installed it on their mobile device. The health technology coach also proactively contacted each participant 2 to 4 days after enrollment to address any technical difficulties that may have emerged and encourage users to find the features that are most beneficial for them. If, based on program use pattern, patients were not engaged, the coach tried to reach them 1 additional time. Thereafter, participants received 1 to 2 texts within the first 30 days to encourage use and check in on any difficulties that may occur. The study coach had a master’s degree in clinical psychology and did not have any prior clinical experience.In this pilot study, we aimed to collect relevant data while keeping the use of the program as close as possible to real-world conditions. Therefore, assessments and analysis were based on 2 paths. First, all participants enrolled into this study received 7Cups as an adjunct to treatment and were measured at preintervention and 30 days after. Second, in order to examine whether 7Cups enhances treatment outcomes, we compared 7Cups users to a different group of patients based on one outcome measure that was available to us. These assessments and analysis are further described in details below.At baseline, participants completed a demographic questionnaire . At baseline and 30 days after enrollment, participants completed online versions of the Beck Depression Inventory II (BDI-II), Edinburgh Postnatal Depression Scale (EPDS), and Beck Anxiety Inventory (BAI). Participants were asked to complete the BAI since symptoms of anxiety might be more common in the perinatal period than in other depressions . EPDS scFollowing recommendations of assessing feasibility in applied intervention research ,26, we et tests for paired samples.Data analysis included descriptive statistics of the multiple-choice questions and program use patterns. Pre-post analysis of self-reported symptoms in BDI-II, EPDS, and BAI were conducted using As described above, the 7Cups enhancement group also received usual care; therefore, it would be difficult to determine whether recorded pre-post effect was only the natural result of usual care. To account for that effect, we created a retrospective comparison group of patients receiving treatment as usual without 7Cups (TAU). As an outcome measure we used the difference in EPDS scores between baseline and 2 months as recorded by the treatment team on a usual basis for all patients in the perinatal program. To control for potential confounders between the groups, several steps were carried out. First, only patients who were not offered 7Cups and met eligibility criteria could be included in TAU group. This condition was possible because different staff members and hospital residents conducted intakes but only some of these “intakers” received guidance and therefore offered patients the use of 7Cups. Second, only patients who had an intake during the study time period could be included in the TAU.Of these remaining patients, a propensity score matching paradigm with the nearest neighbor matching was used to balance the groups across potential covariates ,33. Potet tests for paired samples. Due to the pilot nature of the study and limitations in statistical power, between-group effect size  was also used to compare 7Cups and TAU ) and TAU (mean difference 3.47 [SD 4.42]) groups, yet the effect size was medium: t32=1.67, P=.05, Cohen d=0.58. The percentage of participants who experienced a drop in at least 1 level of symptom severity during the 2-month examination was 71% (12/17) for 7Cups and 47% (8/17) for TAU.This comparison eventually included 17 participants from each group since 2 participants who used 7Cups  did not have postintervention clinician-based assessment documented at 2 months after enrollment. These 2 participants did not differ from other participants in their use patterns and self-reports. Intent-to-treat analysis of EPDS scores administered by clinicians revealed that patients in the 7Cups  schedule. It is also worth noting that while mothers received equivalent guidelines about the use of 7Cups via computer and mobile device, most program use was through mobile device (median of mobile use 93.7%), probably since mobile phones are more accessible when needed. Finally, about a third of mothers who chose not to participate in the study explained they did not have time to attend the enrollment meeting. Overall, these findings are congruent with a previous study showing that for mothers with PPD, one of the main perceived advantages of an online program revolves around its flexibility and accessibility, due to the mothers’ need to manage themselves around the children’s schedules .This study also demonstrated that women with PPD find online peers who have been recruited, trained, and screened using only a computerized program to be helpful and find the experience safe. Participants chatted for a total of 3064 minutes with not one noted incident of feeling unsafe. While a previous study demonstrated that people in emotional distress may find online peer support to be helpful, that study sample was biased as it was composed only of 7Cups native users . Our stud=1.17), the results did not indicate a significant difference between 7Cups enhancement and TAU groups. Since the perinatal program at Zucker Hillside Hospital provides evidence-based care including pharmacologic and psychosocial interventions , it was expected that TAU would yield significant improvement in symptoms of depression as well. However, the medium effect size (d=0.58) favoring 7Cups relative to TAU suggests that 7Cups might improve treatment outcomes. A fully powered trial must be conducted to examine this effect. It is also worth noting that participants did not report a significant drop in symptoms of anxiety, although this may be attributed to lower rates of experienced anxiety (BAI mean 20.47) in comparison to depression (BDI-II mean 26.11) at preintervention.While 7Cups had a very large pre to post positive effect on symptoms of depression . Listeners can also receive a verified listener badge, which means that they were inspected for their listening skills by an experienced listener and passed this inspection. In terms of safety and therapeutic effectiveness, our study was focused on the use of 7Cups as an adjunct to treatment and not as a standalone, which largely affects the safeguards in place. More has to be discussed and examined when it comes to whether there should be other methods for supervision and/or evaluation of actual conversations that occur based on the settings in which such programs are being used.d=0.58), it was not significant (P=.05). This is not surprising given that the nature of this study was more focused toward evaluation of potential effects, which could be helpful for researchers prior to the initiation of a fully powered examination. Second, the difference in outcomes between the groups is not based on a randomized controlled trial. While the propensity matching paradigm did account for several confounders, there might be other confounders affecting the results. For example, it is possible that there are other factors related to patients enrolling in the study, such as readiness to change, that affected outcomes but could not be balanced . We believe, however, that the large effect size found for the study group as well as the self-reported effects provide sufficient evidence that could be further investigated using a randomized controlled trial. Third, it might be that study compensation contributed to engagement with the program; however, this is unlikely given that participants were clearly informed that they would receive their compensation regardless of their use of 7Cups and that they would be compensated approximately 4 months after enrollment.This study has several limitations. First, while the difference in EPDS scores between 7Cups and TAU was not small (This study provides data regarding the potential value of 7Cups within the therapeutic process by retrieving user-based empirical data in real-world conditions . Given t