Efficacy of elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil (E/C/F/TDF) in treatment-naïve and experienced patients with HIV infection was demonstrated in phase 3 trials. The primary objective of this study was to evaluate effectiveness and safety of E/C/F/TDF in real world settings.Retrospective, observational data collected by the Turkish ACTHIV-IST study group between May 2015 and December 2016 were analysed.A total of 387 patients were prescribed E/C/F/TDF; 210 patients with available data at 6th month were eligible; 91.5% were male, and mean age was 35.2 (SD: 10.8) years; 54.0% of males identified themselves as MSM. Sixty-three percent (133) of the study population were treatment-naïve patients, and 37% (77) were treatment experienced. HIV RNA level was below 100 copies/mL in 78.9% of treatment-naïve patients and 89.9% of treatment experienced patients at month 6. Median increase in CD4 T lymphocyte count was 218 copies/mL in treatment-naïve patients and remained stable or increased in treatment experienced patients. Adverse events were observed in 15% of the patients, and the regimen was discontinued in only six patients.Real world data on the effectiveness and safety of E/C/F/TDF is comparable with the phase 3 trial results Adverse events are uncommon and manageable. The World Health Organisation (WHO) estimates that 36.7 million people were living with human immunodeficiency virus (HIV) in 2016, and 1.8 million of them were newly infected in that year. Moreover, HIV infection caused approximately 1 million death globallyThe Turkish Public Health Authority reported that a total of 16644 people had been diagnosed with HIV infection from 1985 through June 2017, and 1537 people had been diagnosed with acquired immune deficiency syndrome (AIDS) during the same time periodSince life-long treatment with an ART regimen consisting of at least three drugs is recommended, multi-tablet regimens (MTRs) may compromise adherence to treatment and result in decrease in quality of life and treatment successThe first boosted integrase strand transfer inhibitor (INSTI)-based combination available was a fixed combination of elvitegravir (150 mg), cobicistat (150 mg), emtricitabine (200 mg) and tenofovir disoproxil fumarate (245 mg) Efficacy of E/C/F/TDF in ART naïve and treatment experienced patients compared to other combinations was shown in phase trials. The results of the phase 3 trials revealed that E/C/F/TDF was effective in more than 80% of treatment-naïve and 78–87% of treatment experienced patientsE/C/F/TDF has been well tolerated by both treatment-naïve and treatment-experienced patients. Most adverse events reported in these studies were mild or moderateE/C/F/TDF was approved in 2013 and has been widely used in Turkey. Real world data on the effectiveness and safety data of E/C/F/TDF has not been published in a detailed manner so far. We present real world data on the effectiveness and safety of E/C/F/TDF using one of the largest HIV databases in Turkey.ACTHIV-IST (ACTion against HIV in ISTanbul) is a study group founded in 2012. The aim of this local study group was to establish a database collecting data on PLWH from 5 major centres in Istanbul involving approximately 20% of HIV-infected patients in Turkey. The list of participating centres is included in the acknowledgement section.Due to the observational nature of the study, data collection procedures of ACTHIV-IST database do not require interventions in addition to the routine practice of the centres; however, the centres are expected to record all available data in the database. Collected data include demographics, medical characteristics, HIV infection and treatment history, including adherence, HIV RNA levels, CD4 T-lymphocyte results, and adverse events. The primary objective of this analysis was to evaluate effectiveness and safety of E/C/F/TDF in real world settings in HIV-infected patients. Data collected from all treatment-naïve and -experienced patients on E/C/F/TDF who provided written consent and had at least 6 months of follow-up were included. There were no exclusion criteria. The data analysed in the manuscript were collected between May 2015 and December 2016. Each centre used its local laboratory, and each laboratory followed its own standard operating procedures. HIV RNA measurement technique varied across laboratories, and with one hospital using a detection threshold of 100 copies/mL. Therefore, an undetectable HIV RNA viral load was defined as < 100 copies/mL in this study.Adverse events were classified and reported by using the medical dictionary for regulatory activities (Med-DRA).No formal sample size calculation was made since all eligible patients were included using a consecutive sampling technique. Participating centres were expected to show maximum effort to minimize loss to follow-up. No replacement or adjustment analysis for missing data was planned for the patients lost to follow-up. Due to the nature of the study, descriptive statistics  were used to summarize the data. Analyses were performed intent-to-treat population. Some analyses were repeated for all treatment-experienced patients, treatment-experienced patients with a history of prior treatment failure patients and treatment-naïve patients with baseline HIV RNA level ≥100,000 copies/mL.ACTHIV-IST database included data on 387 patients on E/C/F/TDF at the end of 2016 with 2736 patient-month follow-up data. Of those, 91.0% were male, mean age was 35.0 (SD: 10.7) years, and 53.4% of males described themselves as men who have sex with men (MSM). Of the 387 patients, 225 (58.1%) were ART naïve and 162 (41.9%) were antiretroviral treatment experienced .Most of the treatment-naïve patients (95.6%) were male, with mean age 33.7 (SD: 10.3) years; 54.0% of males described themselves as MSM. HBsAg (3.6%) and anti-HCV (1.3%) positivity were uncommon, and one-third of patients (31.1%) had at least one comorbidity. Treatment-naïve patients were diagnosed with HIV infection for a median duration of 16.0 months (IQR: 12.0–21.0) and were on E/C/F/TDF for a median of 11.0 months (IQR: 8.0–14.0). E/C/F/TDF was started within 3 months of HIV diagnosis in 55.1% of treatment-naïve patients .Of the 162 treatment-experienced patients, 84.6% were male and mean age was 36.8 (SD:11.0) years; 53.0% of males described themselves as MSM. HBsAg (5.6%) and anti-HCV (0.6%) positivity were uncommon, and one-third of patients (32.6%) had at least one comorbidity. Treatment-experienced patients were diagnosed with HIV infection for a median duration of 39.5 months (IQR:29.0–63.0), had been on ART for a median of 31.5 months (IQR:23.0–54.0), and on E/C/F/TDF for a median of 12.0 months (IOR:8.0–15.0). ART was started within 3 months of HIV diagnosis in 58.0% of treatment-experienced patients. The most commonly used previous NRTI backbone was F/TDF (96.3%); the three most commonly used regimens were F/TDF + lopinavir/ritonavir (46.9%), F/TDF + efavirenz (43.2%), and F/TDF + darunavir + ritonavir (4.3%). Of the treatment-experienced patients; 24.1% (39/162) were switched to E/C/F/TDF due to treatment failure, and 75.9% (123/162) changed treatment to simplify the treatment .To be able to determine the real world safety and effectiveness of E/C/F/TDF, only the patients with available data at 6 months were evaluated in this study. A total of 210 patients were eligible, of whom 91.5% were male and mean age was 35.2 years (SD 10.8), and 54.0% of males identified themselves as MSM. Sixty-three percent (133) of the study population were treatment-naïve patients, and 37% (77) were treatment experiencedMedian baseline HIV-RNA level in treatment-naive patients was 112,228 copies/mL . Six-month HIV RNA levels were available in 95% (127), and 38% (51) patients had available HIV RNA results at 12 months. HIV RNA level was below 100 copies/mL in 78.9% of treatment-naïve patients at month 6 . The patSix-month CD4 T lymphocyte results were available in all of the patients (133) and in in 37% (49) at 12 months. Median CD4 T lymphocyte level in all treatment-naïve patients was 384 (IQR: 281–516) copies/mL at baseline and increased to 602 (IQR: 444–745) copies/mL at month 6 (median increase 218 copies/mL).Patients were sub-grouped according to CD4 T lymphocyte level as group I: < 350 copies/mL (n=50), group II: 350 to <500 copies/mL (n=51), and group 3: ≥500 copies/mL (n=32).Median CD4 T lymphocyte level was 247, 405 and 601 copies/mL at baseline and increased to 432, 631 and 829 copies/mL at month 6 in group I, II and III, respectively. Median CD4 T lymphocyte level continued to increase to 539 copies/mL in group I and remained stable in groups II and III at month 12 .Median HIV-RNA level of all treatment experienced patients (n=77) was available at baseline and at 6th month. The patients were switched to E/C/F/TDF due to simplification in 60 patients and because of virologic failure in 17. Median HIV-RNA level of all treatment experienced patients was 20 (IQR: 0–72) and 0 (IQR: 0–20) copies/mL before E/C/F/TDF and at month 6, respectively, whereas HIV RNA level was below 100 copies/mL in 77.9 and 89.9% of all treatment experienced patients at before E/C/F/TDF and month 6, respectively . Median Median CD4 T lymphocyte level in treatment experienced patients before E/C/F/TDF and at month 6 was 611 (IQR:458–748) and 598 (IQR: 454–824) copies/mL, respectively. Since the number of available results was too small, 12-month data were not evaluated.Due to the greater number of the patients, the increase in CD4 T lymphocyte count was evaluated primarily in the simplification group. The patients were subgrouped according to CD4 T lymphocyte level as group I: < 350 copies/mL (n=7), group II: 350 to <500 copies/mL (n=11), and group 3: ≥500 copies/mL (n=35) .Median CD4 T lymphocyte level was 272, 446 and 696 copies/mL at baseline and increased to 319, 481 and 661 copies/mL at month 6 in group I, II and III respectively.Seventeen patients were included in virologic failure group. Median CD4 T lymphocyte levels were 143, 451 and 652 copies/mL at baseline and increased to 234, 560 and 839 copies/mL at month 6 in group I, II and III, respectively.Adverse events were analysed in 387 patients having withat least 6 months of follow-up. During E/C/F/TDF treatment, 14.9% (58) patients experienced adverse events. The adverse events (reported for >1.0% of patients) were nausea , diarrhea , pruritus , asthenia , headache , dizziness  and dyspepsia  .Two patients (0.5%) died during treatment; neither death was attributed to E/C/F/TDF. Of 387 patients, six 1.6%) drug discontinuations were reported: due to death 2, drug-drug interaction .6% drug INSTIs prevent or inhibit the binding of the preintegration complex to host cell DNA. INSTI-based regimens result in a rapid early-phase decay of plasma HIV-RNA. All currently available INSTIs are included now among the recommended regimens, and once-daily regimens are recommended for both treatment-naïve patients beginning ART and experienced patients receiving complex or poorly tolerated regimens and to use fixed-dose combinations (FDCs) and STRs when possible to decrease pill burdenE/C/F/TDF was approved by the U.S. Food and Drug Administration (FDA) in August 2012 in a once-a-day STR to treat ART-naïve patients or ART-experienced patients with no resistance to its componentsExcept for phase 3 studies and case reports, there is only one study evaluating the effectiveness and safety of E/C/F/TDF in the English literature so farThe results of the phase 3 trials revealed that E/C/F/TDF was effective in more than 80% of treatment-naïve and 78–87% of treatment experienced patientsIn a phase 2 study comparing E/C/F/TDF to efavirenz (EFV)/F/ TDF for initial therapy, 90% of patients using E/C/F/TDF had HIV-1 RNA < 50 copies/mL at week 24 and 48, and a median increase of CD4 T-lymphocyte of 205 copies/mL was observed at the end of 48 weeksA phase 3 study comparing E/C/F/TDF to EFV/FTDF revealed that 86.7% of treatment-naïve patients using E/C/F/TDF had HIV RNA < 50 copies/mL at week 48, and a median increase of CD4 T-lymphocyte of 239 copies/mL was observed after 48 weeks of treatmentIn a phase 3 ; E/C/F/TDF 89.5% of participants were suppressed at week 48, and 83.3% and 77.6% of patients maintained suppression at weeks 96 and 144, respectivelyIn the present study HIV RNA suppression to below 100 copies/mL was observed in 78.9% of the treatment-naïve patients at 24 weeks. When compared to the phase 2/3 studies, rates of virological suppression similar, but it should be emphasized that HIV RNA level was ≥ 1,00,000 copies/ml in 13% and ≥ 100.000 copies/ml in 40% in our study. Squillace and colleagues found that viral load was undetectable in 77% of their naive patients (n=78) at 24 weeks, similar to our resultsMedian 6-month increase in CD4 T lymphocyte count was 218 copies/mL in our study, which is similar to phase 2/3 studies results demonstrating median increase of 205 to 239 copies/mL at 12 months.Eighteen patients had HIV RNA >100 copies/mL at 24 weeks. When these patients were evaluated separately, 55% had baseline HIV RNA > 100,000 copies/ml, and 75% of the remaining patients were nonadherent to ART.Adverse events, treatment failure, greater pill burden, and more frequent dosing have been associated with early ART discontinuation or modificationSimilar to the phase 3 trials in our study HIV RNA level remained below 100 copies/mL in approximately 90% of the all treatment experienced patients at month 6, and CD4 T-lymphocyte levels remained stable or increased depending on the level of CD4 cells.E/C/F/TDF is well tolerated; the most common adverse events reported in registration trials were diarrhea, nausea, upper respiratory infection, and headacheIn our study approximately 15% patients experienced adverse events. The most commonly reported adverse effects were nausea (2.1%), diarrhea (1.8%), pruritus (1.6%), asthenia, headache, dizziness and dyspepsia. The discontinution rate was low (1.6%): two patients died due to reasons not attributed toE/C/F/TDF; four other discontinuations were reported due to drug-drug interactions, elevated creatinine, elevated liver enzyme, and physician decision.Tenofovir alafenamide (TAF) was first approved by FDA in the form of the STR E/C/F/TAF (Genvoya ®) November 2015Real world data on effectiveness and safety of E/C/F/TDF is comparable with the phase 3 trial results in both treatment-naïve and experienced patients. Adverse events are uncommon and manageable, and discontinuation rates are low. Since E/C/F/TAF and other newer regimens are not available worldwide, E/C/F/TDF may remain be recommended in the first-line regimens in treatment-naïve and experienced patients.


We had access to both components of a couple who became infected with human immunodeficiency virus (HIV)-1 through sexual behavior during the early initial phase of infection and before initiation of therapy. We analyzed blood samples obtained at the time of diagnosis and after six months of combined antiretroviral therapy. Next-generation sequencing (NGS) and phylogenetic analyses were used to investigate the transmission and evolution of HIV-1 quasispecies. Phylogenetic analyses were conducted using Bayesian inference methods. Both partners were infected with an HIV-1 B subtype. No evidence of viral recombination was observed. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (distances ranging from 0.102 to 0.148). One HIV-1 single variant was concluded to be dominant in all of the HIV-1 regions analyzed, although some minor variants could be observed. The same tree structure was observed both at baseline and after six months of therapy. These are the first extended phylogenetic analyses performed on both members of a therapy-naïve couple within a few weeks of infection, and in which the effect of antiretroviral therapy on viral evolution was analyzed. Understanding which HIV-1 variants are most likely to be transmitted would allow a better understanding of viral evolution, possibly playing a role in vaccine design and prevention strategies. During the course of the infection, human immunodeficiency virus (HIV)-1 typically accumulates a high level of genetic diversity . The HIVDuring the last decade it has been established that in the vast majority of primary infections the host is colonized by only one out of potentially thousands of HIV-1 variants . In factT/F variants are modeled by both stochastic and selective pressures, and the mucosal bottleneck plays a major role in this process . Thus, iSuccessful combined antiretroviral therapy (cART) leads to the suppression of viral replication, and HIV becomes undetectable in peripheral blood. However, HIV-1 hides in resting memory CD4+ T cells as a latent reservoir ,20. As sIn roughly 25% of HIV-1-positive patients undergoing cART, the virus reservoir increases over time despite treatments. Viral blips, which result in transient detectable viremia, are associated with a reservoir increase and/or a slower reservoir decay . Recent A better understanding of which variants are most likely to be found in a new infection could help vaccine design and prevention strategies. Moreover, since the initial phase of HIV-1 infection directly impacts the progression of the disease , future We characterized T/F variants in a young couple who became infected through heterosexual activity. As the phylogenetic analyses represent the gold standard in the study of HIV evolution and transmission, we adopted the Bayesian inference methods to study the variants transmitted between the two individuals in four viral genomic regions. This is the first study performing a phylogenetic analysis of the T/F variants in a therapy-naïve couple within a few weeks of infection and after the initiation of antiretroviral therapy.A fourteen-year-old female (M) was admitted on 16th June 2017 to the pediatric department at Luigi Sacco Hospital  for a 6-day persistent fever associated with pharyngodynia. During the hospitalization, blood was drawn in order to perform routine tests. As the HIV test result was positive, an immuno-virological evaluation was initiated. M reported having a sexual relationship with a fifteen-year-old male (G). G was visited at our pediatric infectious disease clinic and underwent immuno-virological tests on20th June 2017. Written informed consent was obtained from the parents or legal guardians of the adolescents and from the patients themselves, prior to study enrolment, following Helsinki declaration.Whole blood was collected from the two patients by venipuncture in vacutainer tubes containing EDTA  at the time of the diagnosis. After 6 months of treatment, blood was drawn again .® . The HIV-1 protease, RT, V1V2 and gp41 fragments were amplified from DNA using HIV genotype-specific primers (® (Promega). All amplicons were purified using the QIAquick PCR purification kit .Both patients, G and M, were analyzed at two different time points, before  and after the start of antiretroviral therapy . Total DNA was extracted from the whole blood by DNA purification using Maxwell primers . PCR was® XT DNA sample preparation and index kit  according to the manufacturer’s manual. Resulting libraries were normalized and pooled for subsequent sequencing on an Illumina MiSeq platform using the 2 × 150 cycle paired-end sequencing protocol.Library preparation for Illumina sequencing was done using a NexteraResults were mapped and aligned to the HXB2 reference. The consensus sequence for the reads obtained from the individual samples was generated by Geneious software .The BBMerge tool was used to merge the two overlapping paired reads into a single longer read, and reads shorter than 60 bp were deleted. An in-house pipeline was used to filter identical reads in the same sample. This approach allows limitation of PCR bias and recombination. However, it could affect the number of reads per template resulting in reduced coverage .http://hiv-web.lanl.gov; accessed on 27 February 2021) using the CLUSTAL algorithm implemented in BioEdit version 7.0.4 . Two to five strains representative of each of the nine pure subtypes  and all known circulating recombinant forms (CRFs) were chosen as references.Protease–RT and integrase sequences generated by resistance testing were trimmed to equivalent lengths  and aligned with representative sequences available in the Los Alamos database , the second including reads from samples during treatment , and the third was composed of reads of samples collected before and during treatment .For each amplified region three different datasets were built, the first only encompassed reads obtained from samples collected before treatment (n = 57).For dated phylogeny a distinct dataset was built including additional strains obtained from patients residing in the metropolitan area of Milan with the same subtype . Reads were deleted and distance, phylogenetic and phylodynamic analyses were repeated. The final dataset included 185, 559, 288 and 76 reads for protease, RT, V1V2 and gp41, respectively, from untreated samples; 137, 720 and 226 for the same regions from samples during treatment and 287, 599 and 513 reads from samples obtained before and after treatment.The Bayesian phylogenetic tree was reconstructed by means of both MrBayes and Beast using the general time-reversible (GTR)+ gamma distribution (G)+ proportion of the invariable sites (I) model, which was selected as the best-fitting evolutionary model using the JmodelTest . A MarkoTo evaluate if the sequence clustering was influenced by the application of a molecular clock, maximum-likelihood trees were also built using IQTree V.2 with 1000 bootstrap replicates and the previous selected evolutionary model .http://beast.bio.ed.ac.uk/Tracer; accessed on 27 February 2021). Convergence was assessed based on the effective sample size (ESS) value and only parameters with estimates with ESS > 300 were accepted. The maximum clade credibility (MCC) tree was then selected from the posterior tree distribution using TreeAnnotator v.1.8.4 available in the BEAST software package.The dated tree was built using Beast v.1.8.4  softwareThe MEGA X program was used to evaluate the genetic distance between and within sequences of each dataset . A root-Mycoplasma pneumonia. The serology for EBV showed the presence of a previous infection. The QuantiFERON test result was negative. The HIV test was performed both on anti-HIV specific antibodies and HIV antigens. The test for HIV1/2-Ab/Ag was positive. There were 9,404,000 copies/mL of HIV-RNA and the CD4+ T lymphocyte count was 438/mm3 (12.8%). Therefore, the diagnosis of HIV was confirmed and disclosed to M and her parents. A drug resistance test was then performed, and its result was negative. A four-drug antiretroviral therapy based on dolutegravir, abacavir and lamivudine (in a single-tablet regimen) in addition to a single tablet of darunavir 800 mg/ritonavir 100 mg was started. After three weeks of antiretroviral therapy, HIV-RNA in the blood was at 514 copies/mL and the CD4+ T lymphocyte count was 820/mm3 (41%). Darunavir/ritonavir was interrupted after three months, given the decrease in viral load. The therapy was successful and after six months HIV-RNA had < 37 copies/mL with a CD4+ T lymphocyte count of 888/mm3 (39%).The blood and urine culture returned a negative result for HSV1-2, CMV, toxoplasmosis, HCV, parvovirus, and 3 (16.8%). Upon drug resistance testing, which returned a negative result, the recommended therapeutic regimen was based on emtricitabine, tenofovir alafenamide and darunavir/c. The HCV antibody and HBs antigen tests were negative; HIV1/2-Ab/Ag were positive, as confirmed by Western blot analysis. After four weeks, HIV-RNA had 790 copies/mL and the CD4+ T lymphocyte count was 466/mm3 (28%). After six months, HIV-RNA had < 37 copies/mL and CD4+ T lymphocyte count was 838/mm3 (35%).HIV-RNA baseline viremia in G was 80,240 copies/mL with a CD4+ T lymphocyte count of 340/mmThe HIV-1 protease, RT, V1V2, and gp41 regions were amplified and sequenced. The phylogenetic trees confirmed that the viral subtype present in G and his partner was a B subtype. There was no evidence of viral recombination observed in any portion. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (M = 0.012 and G = 0.011) compared to samples during treatment at six months (distances ranging from 0.102 to 0.148). On the contrary, in the gp41 region a lower genetic distance was observed in samples during treatment at six months (M = 0.062 and G = 0.067) compared to the naïve samples (M = 0.113 and G = 0.129). Regarding the intra-patient genetic distances between the two time points, the lowest values were observed in the V1V2 fragment (M = 0.071 and G = 0.077) while a very high divergence was observed in the gp41 portion (M = 1.292 and G = 1.970). The tree obtained by analyzing all of the reads achieved from the two patients before the beginning of the therapy showed the presence of a single major variant in the protease A and RT Only one major common variant could be observed in the tree of the gp41 region D. When aComparison of the different coalescent models using the BF test on the entire dataset showed that the model best fitting the data was a coalescent prior BSP  under a log-normal relaxed clock (2lnBF strict vs. relaxed clock = 54.8). In the tree, all sequences clustered significantly together (pp = 1) with a G sequence obtained that arose externally. The dated tree indicated a TMRCA for the cluster encompassing all patient isolates at 22.25 months (95%HPD: 8–43.6 months) corresponding to February 2017. The node related to the M sequence dated 19.3 months (95%HPD: 6–20 months) corresponding to April 2017 (data not shown).During the last decade, the knowledge that HIV establishes new infections mostly through one single variant has been supported by a growing amount of evidence ,50,51. IWe performed phylogenetic analyses in a young couple who became infected through sexual activity using a next-generation sequencing (NGS) approach (Miseq platform); we analyzed the HIV-1 quasispecies present in samples and verified their evolution under treatment pressure. This event allowed us the unprecedented opportunity to study the T/F variants in a therapy-naïve couple and to verify the effect of antiretroviral therapy on viral evolution. The results showed the presence of a limited number of variants, ranging from one to two, which were transmitted from G to M.We chose to analyze HIV proviral DNA, which is the “archived” compartment, since we believe it is more informative than plasma. By sequencing different HIV-1 regions , we observed that the average genetic distance between the viruses present in the two partners increased over time, as already reported in different studies, with the exception of the gp41 region where treatment halved the viral genetic distance. In samples obtained before and six months after the initiation of therapy, the pol region appeared to be highly conserved, as expected, compared to other analyzed portions, and only one major T/F variant seemed to be present. The env region was found to be highly variable, as expected, in particular in the gp41 portion, highlighting the presence of three common variants. The evolution of HIV-1 quasispecies was evaluated considering only viral variants that were present after six months of therapy. Upon antiretroviral treatment, one HIV-1 single variant was clearly dominant, although some minor variants could be observed in all considered portions, with the exception of the gp41 region. These minor variants were not visible at baseline probably due to the overwhelming presence of the main variants. Apparently, the segregation of variants sampled before and after therapy involved the gp41 region, but not the V1V2 gp120 region. None of the employed therapeutic approaches target this region, but, as it is an immunogenic region, it is subdued to selective pressures, nonetheless. A possible explanation for this apparent discrepancy is that the dominating V1V2 observed variant was previously completely obscured by a different variant.In order to better understand the differences observed at the two evaluated time points, an additional analysis was conducted in which all the variants were included. When all the data from the two time points were pooled together, results showed that all the variants were already present at the time of transmission, with the exception of gp41. In fact, two variants were clearly identifiable among both the naïve and the post-therapy variants. Most likely, these two variants that were observed in all datasets can be considered the early T/F variants. This could be attributed to the sequestration of the virus in reservoir cells that were infected early in the course of infection, but were subsequently maintained at low levels in circulation as latently infected cells.In agreement with the literature, we observed that the viruses in the newly infected subject were closely related to viruses present in the donor; this is in sharp contrast with results obtained when transmission occurs in a late stage of the infection ,53.It has recently become clear that the transmitted/founder virus is rarely the dominant variant in the donor, suggesting that the transmission bottleneck is not entirely stochastic but also involves the selection of specific viral phenotypes . These fA strong point of this work is the short timeframe between virus transmission and sampling; however, further longitudinal analysis of couples in which the recipient is sampled close to the acute stage of disease will be needed to better determine the selection of variants, and the relation among ancestral genotypes and variants, found in newly infected subjects.The high homogeneity of quasispecies between the donor and the recipient in this study was due to the short interval between transmission and sampling, and to the high viral load in the transmitter, which negatively correlates with genetic distance between donor and recipient variants .Literature data reported, for whole blood, an increase in the average genetic distance with respect to the first sample. Usually overall, the average genetic distance between the viruses present in two partners increases over time; however, this remains less evident in the blood compared to the plasma compartment.Our phylogenetic analyses not only confirmed the probability of transmission, but also allowed us to date this event with good approximation. In addition to the existence of an epidemiological relationship between the two patients, the dated tree supported the time point and the probable direction of transmission. In detail, our analysis confirmed that G acquired the infection before in early 2017, while M acquired it a few months after (around April of the same year).This is the first time that phylogenetic analyses have been performed on an HIV-infected couple in both individuals at the therapy-naïve stage and upon cART. However, our study presented some limitations, the most relevant is intrinsic to the used sequencing methodology based on amplicons. The derived PCR bias could affect the interpretation of the viral variants. Understanding which HIV-1 variants are most likely to be transmitted is important for vaccine design and predicting virus evolution.


By increasing life expectancy of people living with HIV, the most clinical challenge is managing both drug-to-drug interactions and comorbidities . Doravirine (DOR), a new non-nucleoside reverse transcriptase inhibitor, recently approved for the treatment of HIV, could be a good companion of dolutegravir (DTG) in a dual regimen for experienced elderly patients with multimorbidity and polypharmacy.We herein report our preliminary experience in a small cohort of elderly patients (>50 years of age) with multimorbidity and on polypharmacy who were switched to DOR/DTG dual regimen and followed-up for 3 months. The study was conducted at the Infectious and Tropical Diseases Unit of Padua University Hospital, Italy.Eighteen patients were included, 72.2% males and 27.8% postmenopausal women, mean age was of 61.3 years (7.6), 50% experienced AIDS events. Switches to DOR and DTG were mainly due to high cardiovascular and metabolic risk (72.2%), and interactions among comedications (50%). Antiretrovirals that subjects were switched off were mostly boosted protease inhibitors 66.7%. We observed a viral suppression among all subjects. Interestingly, we observed a statistically significant reduction in body mass index, body weight and waist circumference, eGFR, and a significant increase in serum creatinine levels. No significant changes in CD4+ T cell count was observed from the baseline. Lipid and fasting glucose values did not change significantly.To the best of our knowledge this is the first experience reporting real-life outcome of switch to DTG + DOR in elderly with multimorbidity and on polypharmacy. From our very preliminary data the dual combination of DTG and DOR could be a good treatment strategy for these subjects. However, our findings need to be validated on a greater number of patients. It exists both as single agent and in combination with tenofovir diproxil emifumarate. European AIDS Clinical Society guidelines recommend the combination of DOR with tenofovir diproxil emifumarate as first line agent in naïve patients, while American guidelines prefer to limit its use in specific sub-groups of patients.[Results from clinical trials confirmed a good safety profile of DOR both on HIV virological control and on metabolic profile.[Prevalence of polypharmacy ranges from 37% to 94% in elderly people living with HIV (PLWH), and increases by age, as a direct consequence of an increasing proportion of multimorbidity in this population.2t test and Mann Whitney U test were used to compare continuous variables . Chi squared test was used to assess categorical variables, as appropriate. Level of significance was set up with a P value < .05.This study was conducted at Infectious and Tropical Diseases Unit of University Hospital of Padua and received approval form the Hospital Board. Patients’ consenting was waived as per Italian law for retrospective studies . Data of patients treated with DOR and DTG for 3 months (June–August 2021) were retrospectively collected from clinical health records. Multimorbidity and polypharmacy were defined as per literature.[3Clostridium difficile diarrhoea for 3 days, due to an ongoing antibiotic treatment. As for viro-immunological parameters, we observed a maintenance in viral suppression among all those who were undetectable at the baseline. Two people who were not undetectable at the baseline showed undetectable HIV RNA during the follow-up. Interestingly, we observed a statistically significant reduction (P < .05) in body mass index, body weight and waist circumference, eGFR, and a significant increase in serum creatinine levels  males and 5 (27.8%) postmenopausal women. Mean age was of 61.3 years (standard deviation: 7.6), with a mean length of HIV disease of 23 years (standard deviation: 8.5). Nine patients (50%) experienced AIDS events in their past medical history. Risk factors for HIV acquisition were mainly intravenous drug use  and unprotected sex with men . All patients had multimorbidity and polypharmacy, with severe polypharmacy in 5/18 (27.8%) cases. Comorbidities and class of comedications are depicted in Figure 4To the best of our knowledge, even if very limited by the low numbers, this is one of the first real-file experiences describing the impact of DTG + DOR dual regimen. Fear of possible worsening of comorbidities  in patients who were on a boosted PI was the main driver for switch in our small cohort, as well as the drug interactions perpetrated by the boosting agents. The major issue, in the era in which the burden of antiretroviral drugs decreased with the introduction of dual regimen, is the increasing number of non-cART medications. Psychiatric comedications are one of the most involved class in drug interactions, especially when administered with PIs.The effect on body weight and decrease of waist circumference, even if significant, need to be confirmed in greater cohort and now not conclusive. It could be more likely explained by the remotion of PIs from the antiretroviral regimen more than a “slimming effect” of DOR and we are not still able to declare if it will be maintained over time, and whether the presence in the regimen of DTG will contribute to weight gain. No significant effect was observed on lipids and glucose levels, probably because people with dyslipidaemia were on statin and people with diabetes on antidiabetic drugs daily, respectively, but also this finding will require future confirmation. The effect on kidney function could be explained by the decreased tubular secretion of creatinine induced by DTG (which was not present in the baseline regimen), rather than a real renal toxicity of the studied combination.From our very preliminary data the dual combination of DTG and DOR seems to be an option for elderly subjects, with multiple comorbidities and drug interaction/toxicity issues, and can be a patient's tailored treatment, even if not currently contemplated as association by guidelines. Further data on greater number of PLWH and with longer follow-up are necessary to confirm our results.MM and AMC were involved in designing the study. LS, DL and CP collected the data. MM analyzed the data and drafted the manuscript. All authors  were involved in revising the article.Conceptualization: Maria Mazzitelli, Anna Maria Cattelan.Data curation: Maria Mazzitelli, Lolita Sasset, Davide Leoni, Cristina Putaggio, Anna Maria Cattelan.Formal analysis: Maria Mazzitelli.Methodology: Maria Mazzitelli.Project administration: Maria Mazzitelli.Supervision: Maria Mazzitelli, Anna Maria Cattelan.Writing – original draft: Maria Mazzitelli, Anna Maria Cattelan.Writing – review & editing: Maria Mazzitelli, Lolita Sasset, Davide Leoni, Cristina Putaggio, Anna Maria Cattelan.


To describe the experience and resource requirements of implementing point-of-care testing for early infant diagnosis of HIV in rural Zambia.A demonstration project was conducted using a hub-and-spoke model in 2018–2019 at five clinics in rural Zambia. Two testing hubs were established, and all HIV-exposed infants were tested with the GeneXpert system. Data on costs, turnaround times and test results were collected.Seven hundred and eighty six tests were conducted. At the hubs, results were available a median of 2.4  hours after sample collection and most mothers (84%) received same-day results. At the spoke facilities, results were available a median of 9 days  after sample collection and provided to the mother a median of 16 days  after sample collection. Eleven children tested positive, and 9 (82%) started treatment a median of 13 days  after sample collection and on the day mothers received results. In contrast, results from matching samples sent for routine testing were available a median of 38 days  after sample collection and provided to the mother a median of 91 days  after sample collection.Implementing point-of-care testing in a network of rural health centres in Zambia required significant initial and ongoing investment in infrastructure, training and supervision. However, point-of-care testing can rapidly diagnose HIV-infected infants, so they can benefit from early treatment. In 2019, an estimated 150 000 children were newly infected with HIV globally, including 126 000 in sub-Saharan Africa and 6000 in Zambia ). Hardcopy results were transported back to the clinics using the same transport system. As the time for results to return to the clinics was variable, mothers typically received the result at their next clinic visit. In the event of a positive result, mothers were contacted by clinic staff to return as soon as possible.The NSEBA Demonstration Project was conducted in Choma District, Southern Province, Zambia, in 2018–2019 and was nested within the Novel Screening for Exposed Babies (NSEBA) Study. In Southern Province, the HIV prevalence was estimated to be 13.3% among adults 15–59 years of age in 2016 [The NSEBA Study was previously described . BrieflyThe NSEBA Demonstration Project was conducted using the GeneXpert platform  see . Two cli® K3 EDTA, Sarstedt AG & Co, Germany) and transported to the laboratory where it was processed and tested the same day. EID testing was prioritised, such that EID samples were tested immediately upon arrival at the laboratory. Results were delivered to the clinic to be given to the mother, with the goal of same-day delivery.At the hub facilities, the GeneXpert test was used as a point-of-care test. A liquid whole blood sample was collected from the infant in an EDTA blood collection tube  or they were contacted by phone or through community health workers when results were available and requested to return to the clinic to receive them.Results from GeneXpert testing were documented as well as the dates and times for each step in the testing process and any resulting linkage to care and ART initiation. Consistent with the standard of care, when results from the central laboratory were returned, they were provided to the mother at their next clinic visit if concordant with the GeneXpert result. If the result from the central laboratory was discordant, the mother was contacted to return to the clinic. Results from the central laboratory were documented as well as the dates for each step in the testing process. At the end of the study period, an attempt was made to retrieve results that were not returned from the laboratory by examining the lab database.All costs associated with implementing GeneXpert testing, including upgrading the laboratories and purchasing and maintaining equipment and supplies, were documented during the study period.Descriptive statistics, including proportions for categorical variables and medians, interquartile ranges and ranges for continuous variables, were used to summarise the data overall and by hub. Implementation data, including costs and supervisory visits, were summarised. The study population was described in terms of maternal and child characteristics. Testing data, including the number of tests, proportion of tests returned within the target time frame (same day for hub and within two weeks for spoke facilities), turnaround times from sample collection to return of results to the clinic and mother, and test results, were summarised. The proportion of mothers receiving the results on the same day at the hub facilities were compared by year and location of testing using a chi-square test. Turnaround times were compared between the hub and spoke facilities using a Wilcoxon rank-sum test. The analysis was performed using Stata Statistical Software, Version 16 .The NSEBA Study was approved by the Institutional Review Boards at Macha Research Trust, the Johns Hopkins Bloomberg School of Public Health and the National Health Research Authority in Zambia. The NSEBA Demonstration Project was conducted after consultation with the Ministry of Health of Zambia. All parents or guardians provided written informed consent for participation in the study. As GeneXpert is approved as a diagnostic test for HIV by the WHO, results were provided to healthcare providers and caregivers and used for clinical decision-making.The hospital hub was already set up to perform the assay, so testing was initiated immediately and all spoke facilities were initially designated to send samples to the hospital hub. The health centre hub required upgrades to the laboratory before testing could begin, including installation of ceiling boards and an air conditioner, and upgrades to plumbing and electrical fittings and door closures . In addiDuring the evaluation, repairs were required for both the laboratory and the GeneXpert equipment at the health centre hub , includiAs testing was new to the lab technicians at the health centre hub, a plan to provide support and supervision was created and carried out by the lab technicians at the hospital hub. This included weekly visits to the health centre laboratory in the first month of testing and then monthly visits thereafter or as needed. Twenty-four scheduled visits were conducted during the study period, and five issues were identified and resolved , and for children tested after birth in the maternal and child health clinics than at birth in the maternity ward  after sample collection . Most moA total of 830 cartridges were used to obtain a final result for the 786 tests . The final result was positive for 17 tests, negative for 767 tests and invalid for two tests . The 17 Results from the central laboratory were received and available for comparison for 729 tests (92.7%). Fifty-two samples (6.6%) were lost at the central laboratory, one sample was not sent as the child was found to have been recently tested, and four results (0.5%) were not available by completion of the study. Discrepant results were found for four tests (0.5%) from four children who were negative by GeneXpert and positive by the central laboratory. All four children were later determined to be uninfected on repeat testing , consistDuring the study period, the GeneXpert machines at the hubs were also used for tuberculosis testing for all patients  and HIV viral load testing (both hubs) for paediatric patients seen at those facilities. In addition to EID, on average the hospital hub performed 37 tests for tuberculosis and 38 HIV viral load tests per month, and the health centre hub performed 2 HIV viral load tests per month . The labThis project demonstrated the public health importance of point-of-care platforms to improve the timeliness of EID in a rural area as well as the resources required to implement the programme using a hub-and-spoke model. In addition to investing in the point-of-care technology and assays, implementing the programme required that lab facilities be upgraded and maintained, and the health centre hub required ongoing support and supervision for the duration of the project. With this investment, implementing point-of-care testing improved EID testing, resulting in shorter turnaround times for results for all children and ART initiation for children testing positive.Consistent with other studies , 11, 13,The decrease in turnaround times for results led to rapid ART initiation for children testing positive, the ultimate goal of point-of-care testing given the clinical and survival benefits of early treatment . Prior tThis study highlighted the challenges of centralised testing, not only in lengthy turnaround times for results to return to the clinic, but also for results to get to the mother and to be used for clinical decisions. With point-of-care testing, almost all mothers received their child’s test results during the study period. In comparison, 6% of samples were lost on their way to or at the central laboratory and thus would not have generated results, and prior studies in this area suggest that 13–30% of mothers did not receive the results from testing at the central laboratory , 23. In Implementing point-of-care testing and achieving the clinical and survival benefits required significant investment in personnel, infrastructure and financial resources. Use of the GeneXpert platform in this project necessitated that the new health centre hub facility be upgraded and maintained to satisfy the requirements for testing, as has been seen in other studies , 25. WhiSeveral studies conducted in sub-Saharan Africa have found point-of-care testing to be cost-effective in comparison with centralised testing, in terms of cost per test result returned within 30 days , additioSeveral additional limitations to this demonstration project should be noted. First, a relatively small number of tests were conducted and only a small number of HIV-infected children were identified. This is a challenge for all studies of EID given the widespread availability of ART and high coverage of PMTCT in this region that has resulted in a dramatic decline in mother-to-child transmission . Second,In summary, implementing point-of-care testing in a network of rural health centres in Zambia required significant initial and ongoing investment in infrastructure, training and supervision. However, point-of-care testing can rapidly diagnose HIV-infected infants so they can receive the benefits of early care and treatment.tS1-S2Table S1. Supervisory visits to new hub at the Zonal Health Centre.Table S2. Testing and treatment histories for children with discrepant results between GeneXpert and the central laboratory.fS1-S4Figure S1. Map of study area.Figure S2. Algorithms for testing with GeneXpert with (A) liquid whole blood and (B) dried blood spots.Figure S3. Time from sample collection to return ofresults to mothers for (A) all samples; (B) samples tested at the hospital hub; and C) samples tested at the health center hub.Figure S4. Proportion of mothers receiving results on the same day at the hub facilities by (A) year and month; and (B) location of testing.


Combined antiretroviral treatment (cART) traditionally consists of three antiretroviral medications, while two-drug regimens (2DR), historically used infrequently, recently been suggested to be non-inferior to three-drug regimens, is emerging as a potential treatment option and is currently a recommended option for treatment initiation in many guidelines.Characterize the indications and clinical efficacy of 2DR use at a real-life setting in a nation-wide survey.A cross-sectional survey of Israeli patients treated by 2DR until July 2019, included demographic, immunologic, virologic, genotypic and biochemical/metabolic parameters at diagnosis, ART initiation, 2DR initiation and following 24, 48, 96 and 144 weeks of 2DR treatment.176 patients were included in the study. In contrast to historical data implicating ART resistance and adverse effects as the major reasons leading to 2DR switching, treatment simplification was the main reason leading to 2DR treatment in 2019. 2DR that included INSTI and PI were more commonly used in cases of drug resistance, while a combination of INSTI and NNRTI was used in all other 2DR indications. A switch to 2DR induced a mean CD4 T cell increase from 599 cells/μl at treatment initiation to 680 cells/μl at 96 weeks of treatment p<0.001 and viral suppression improvement from 73.9% at initiation to 87.0% at 48 weeks of treatment (p = 0.004). PI and INSTI 2DR was inferior in suppressing viral levels compared to other 2DRs but used for subset of more complex patients.2DR in a large-scale real-life nation-wide survey proved to be safe and effective. Most 2DRs, other than PI and INSTI, were similarly effective in suppressing HIV viremia and in elevating CD4 T cell counts. Combined antiretroviral treatment (cART) evolved from a single drug therapy  to a triA total of 7970 people living with HIV in Israel, are currently followed and treated in eight HIV centers. The aim of this study was to evaluate the real-life usage of two drug regimens (2DR) in Israel, to characterize the different regimens used in different HIV centers, the rationale leading to switching to 2DR, and the clinical outcomes of this practice.Patient information was retrieved from the local databases of seven Israeli HIV centers between July 2019 to August 2019. Included were all the HIV patients who were treated by two drug regimens (2DR) at the time point of data collection in these centers . No exclusion criteria were implemented to reflect the real-life implications of dual therapy. The data collected included demographic, immunologic, virologic, resistance profile and biochemical/metabolic parameters at diagnosis, ART initiation, 2DR initiation and after 24, 48, 96 and 144 weeks of 2DR treatment±8 weeks. Immunologic and virologic data included CD4 count (cells/μl), CD8 count (cells/μl), HIV viral load , Hepatitis B virus (HBV) and Hepatitis C virus (HCV) serology. The biochemical/metabolic parameters included renal function , liver aminotransferase activity , lipid profile, including triglycerides , total cholesterol (mg/dl), low density lipoprotein , high density lipoprotein levels , and glucose levels (mg/dl). We documented the indication/s for 2DR initiation, the type of ART used before and after the switch to 2DR, the number of regimens that the patient has been treated with until 2DR. Resistance mutations documented at diagnosis, during follow up to 2DR switching and after 2DR initiation. Drug-resistance mutations were identified using the Stanford University HIV Drug-Resistance Database . ChanginTo evaluate the clinical efficacy of 2DR therapy over time, repeated measures ANOVA model was applied for assessment of quantitative variables. Assessing trend over time for quantitative variables which were not normally distributed, was performed using the non-parametric Friedman test. Testing the change between two time points for quantitative variables, was performed using a paired t-test. Testing the change between two time points for categorical variables, was performed by applying the McNemar or the McNemar-Bowker test. Pearson’s Chi-square test and Fisher’s exact test were used for testing the association between two categorical variables. All statistical tests were two-tailed and a p-value of less than 0.05 was considered statistically significant. Statistical analysis was carried out using IBM SPSS Statistics version 24.0.The study was approved by IRB committees of all participating HIV Centers.Approval number of Hadassah Medical center IRB 0574-18-HMO, approval number of Rambam Medical Center IRB 0428-19-RMC, approval number of Kaplan Medical Center IRB 0036-10-KMC, approval number of Meir Medical Center 001-19-MMC, approval number of Sourasky Medical Center IRB 0741-18-TLV, approval number of Sheba Medical Center IRB 5838-19-SMC. No approval was required from Hillel Yaffe Medical Center as no patients were treated with 2DR at the study data collection. All committees granted a formal waiver of informed consents as data was retrospective and analyzed anonymously.In reviewing all the medical records of patients living with HIV and followed by the participating 7 HIV centers, we identified 173 patients who were switched to dual therapy, and 3 patients who initiated 2 antiretroviral drug regimens as their primary ART regimen until 31.7.2019. In total, we were able to survey the data of 94% of 7970 people living with HIV in Israel, as one HIV center did not participate in the study. The rate of patients treated with dual therapy was comparable between the centers and ranged from 1.6% to 4%. Seventy-one precents of the patients were males and 40% were born in Israel, while Ethiopia was the main country of origin for immigrants. Patients were previously heavily treated for a mean of 8.57 years and a mean number of treatment switches totaled 2.08 as indicated in The most common reasons for switching to 2DR were renal toxicity (26.1%), therapy simplification (25.0%) and resistance to prior ART (22.7%). Other, less common reasons were cardiovascular toxicity, osteoporosis, and gastrointestinal adverse effects. Forty patients featured more than one reason for 2DR initiation, all of which were documented . The indTwenty-four patients (13.6%) experienced a second switch to a different 2DR, performed primarily for treatment simplification, hyperlipidemia, drug interactions, and renal toxicity. The majority of patients that underwent a second switch to 2DR (75%) were treated with a PI combined with INSTI as the initial 2DR and most of them were switched DTG+RPV as second 2DR. Nine patients (5.1%) were switched back to triple drug therapy, due to DDI, simplification, cardiotoxicity and other non-specified adverse events or unknown reasons .Repeated CD4 count (cells/μl) measurements conducted in 92 patients with a complete follow up at 24, 48 and 96 weeks after 2DR initiation demonstrated a significant gradual increase in CD4 counts upon 2DR treatment from an average of 599 cells/μl at treatment initiation, to 614 cells/μl, 645 cells/μl and 680 cells/μl at 24, 48 and 96 weeks, respectively ”, “low VL” (UD<VL<200), and “high VL” (VL = >200). Comparing the undetectable VL rate at 2DR initiation and 48 weeks demonstrated a significant increase in undetectability rate from 74.6% to 87.3% and a decrease in the low and high VL groups (P = 0.004). Significant results were also observed when comparing VL at 2DR initiation, 24 and 144 weeks of follow-up .To differentiate the efficacy of different 2DR regimens, we compared the changes in viral suppression between patients who were switched to the newer and approved 2DR regimens dolutegravir+rilpivirine (DTG+RPV) or dolutegravir+lamivudine (DTG+3TC) and those treated by other 2DR regimens. As treatment simplification was the main indication to change to DTG+RPV or DTG+3TC, this patient subset featured a higher rate of undetectable VL at 2DR initiation . We further compared the rate of patients who were undetectable at the 2DR initiation but became detectable at week 24, 48, 96 or 144 in each of the 2DR subsets. In the DTG+RPV and DTG+3TC group, the rate was significantly lower than in the other regimens . Additionally, 72.2% of the DTG+RPV or DTG+3TC patients who initiated the 2DR with detectable viral load featured a completely suppressed viral load at all subsequent follow up time points, while the conversion rate to undetectability was only 52.2% in a group that included patients treated with other 2DR regimens. This difference did not reach statistically significance (p = 0.091), but the total effect indicated favorably to DTG+RPV and DTG+3TC treatment (p = 0.004). Application of the same analysis to other 2DR regimens indicated that 45% of patients treated with INSTI & PI, became completely suppressed, compared with an 73.9% in patients treated by other regimens (p = 0.038), . This anDrug resistance associated mutations that were documented as naïve patients or due to virological failure, were documented in 48 (27.2%) of the patients and 24.3%, 14.7%, 10.2% and 2.3% of the patients had a least 1 mutation for NRTI, NNRTI, PI and INSTI respectively. The Mutation M184V in RT was documented in 18.6% of the patients and K103N in 4.5%. The rate of resistance was 40% in patients that were detectable at 2DR initiation with 32.5%, 22.2%, 7.5% and 5% of the patients harboring at least one mutation to NRTI, NNRTI, PI and INSTI respectively. As described above, resistance prevalence was 40% in patients that were detectable at 2DR initiation and 32% of the patients that were not completely virologically suppressed after 2DR switch had previously documented resistance- 27% for NRTI, 13.5% for NNRTI and 16.2% PI. No resistance mutations were documented to evolve in any of the patients that had detectable VL after 2DR.One hundred and thirty eight patients had virological data at week 48, 18 of whom (13%) had a detectable viral load. Nine of these non-virologically suppressed patients, had a viral load of 0–200 copies/ml, while 9 featured a viral load of above 200 copies/ml. Eleven (61.1%) who featured a detectable viral load at week 48 of 2DR treatment also had a detectable viral load at treatment initiation. In comparison, only 25% of virally undetected patients at 48W featured a detectible viral load at treatment initiation (p<0.01). Surprisingly, the rate of documented resistance was lower (but non significantly) in the 48W detectable patients in comparison to the undetectable patients (16.6% vs 30.5% p = 0.414). Analysis of the patients who were detectable vs. undetectable in 96W yielded similar results. These results possibly stemming from adherence issues or resistance that was not documented due to low viremia.A non-significant increase in triglycerides (p = 0.591), glucose (p = 0.562) creatinine (p = 0.426) and HDL levels (p = 0.355), and a decrease in AST (p = 0.41), ALT (p = 0.157) and total cholesterol levels (p = 0.834) were documented following the switch. Analyzing LDL levels in 46 patients with available results at 2DR initiation, 24W, 48W and 96W, of whom 23.9% were pretreated with PI, and in 26.1% PI was one of the 2DR components, indicated a significantly decreased from 112.98±30 mg/dl to 103.4± 29.8 mg/dl (p = 0.021). This effect also trended by comparing by paired T test of LDL at dual initiation and 96W of 79 patients (113.6±32 mg/dl vs 106.99±31.2 p = 0.067) but was not reproduced in 24W and 48W (p = 0.29 and P = 0.665 respectively).Dual therapy for HIV-infected patients has been proven to be non-inferior to standard triple anti-retroviral therapy in randomized, double-blind controlled trials both in experienced  and naivSecond, our study demonstrates that in many cases, the rationale for 2DR switch is motivated by a need to reduce previous treatment toxicity (58% of switches). In our cohort, we noted a trend towards 2DR improving hyperlipidemia, which reached statistical significance by 96W. This trend merits further larger-scale follow-up studies.An observational study evaluating 121 patients treated with RAL combined with PI, reported that approximately 70% of them remained on this regimen with a median treatment duration of 60 months, with treatment simplification being the main reason for 2DR discontinuation in half the 2DR-failing patients . MoreoveOur study features several limitations stemming mainly from its retrospective design, including a lack of a control group treated with 3DR, and heterogenous ART regimens used before and after the switch to 2DR. Additionally, since 2DR was not prevalent before 2017, patients were excluded from our long-term metabolic and efficacy analysis due to a short follow up. Furthermore, due to the cross-sectional design of the study, we may have not identified patients that were switched to 2DR and switched back to 3DR before July 2019 and might have missed an excessive discontinuation rate of certain combinations.In summary, our study indicates that two drug regimens are safe and effective in a real-life context, taking into account the impact of such treatment on virological suppression, immunological response and metabolic effects. The combination of PI and INSTI provided to a complex subset of patients was documented to be inferior to other 2DR combinations in this setting. Other long-term effects, such as enhanced cognitive impairment, the effect of viral transmission by sexual contact or from mother to child should be further explored by prospective, long term follow-up studies.S1 Dataset(XLSX)Click here for additional data file.


Mycobacterium malmoense pulmonary infection and HIV-1 chronic co-infection in a 60-year-old man while participating in an HIV-1 therapeutic vaccine clinical trial and during the analytical treatment interruption. We present clinical and therapeutic features of a complicated M. malmoense pulmonary infection along with discussion of the possible relation to the HIV-1 cure-related interventions.We report a case of  Mycobacterium malmoense is a slow-growing non-tuberculous mycobacterium (NTM) that was first isolated in 1954 in Malmö, Sweden. Clinical cases have been extensively described in north and north western Europe [M. malmoense infection include pediatric isolated lymphadenitis and adult tuberculous-like pulmonary infection. To date, very few cases of M. malmoense have been reported in Spain and only one case in an individual that was co-infected with HIV-1 [n Europe  but infeth HIV-1 .HIV-1 antiretroviral treatment (ART) has improved life expectancy and quality of life in people living with HIV-1, but it is a lifetime treatment unable to cure or eradicate the infection. Therefore, finding new therapeutic strategies has become a priority, and the most promising ones are vaccines .M. malmoense pulmonary infection in an HIV-1 co-infected man living in Barcelona and participating in the referred trial.In a tertiary center in Barcelona, we conducted a clinical trial evaluating a dendritic cell-based (DC) vaccine in combination with interferon alpha 2a (IFNα2a) followed by a 12-week analytical treatment interruption (ATI) (NCT02767193). We describe a case of 3 CD4 T-cell count and 4.53log10 viral load (VL). The chest X-ray was normal and mantoux test negative. A year later antiretroviral treatment was started. Since then, plasma VL has been suppressed and CD4 T-cell count > 500 cells/mm3.The patient is a 60-year-old Swedish male and chronic smoker. He was diagnosed with an HIV-1 infection in February 2008 with 752 cells/mm10, the CD4 T-cell count maintained >500 cells/mm3 and the patient remained asymptomatic. Ten months after restarting ART he started vaccination and received two doses of DC vaccine bi-weekly for 6 weeks. Vaccine was well tolerated and no complications were reported. The same day he received the last dose of DC vaccine, ART was interrupted and continued study treatment with pegylated INFα2a in a subcutaneous 180 mcg dose every week for 3 weeks. The patient referred to having chills, asthenia, myalgias and fever after each of the first two doses of INFα2a. Since symptoms had been described as related to interferon-based treatments, no further assessment was conducted.In May 2016 he was enrolled in an HIV therapeutic vaccine clinical trial. For vaccine production we needed autologous virus, therefore the patient had to stop ART after enrollment and during 9 weeks then ART was restarted. Although plasma VL increased up to 4.2logMycobacterium tuberculosis so a NTM infection was confirmed. Antimicrobial therapy was started with levofloxacin 750 mg, azithromycin 500 mg, rifampin 600 mg and ethambutol 1200 mg daily. At this moment the CD4 T-cell count was 787 cells/mm3 and VL 3.9log10 copies/mL. Due to the patient’s preference, ART was restarted until 2 weeks later. A Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to characterize the Mycobacterium as malmoense and whole genome sequencing confirmed the results. The culture was positive and isolates were identified as Mycobacterium malmoense. Although the patient was originally from Sweden, he had been living in Spain for more than 15 years, so detailed epidemiologic and potential environmental exposure was assessed and revealed him travelling and working at least each spring in Sweden. After 16 weeks since starting antimicrobial therapy, we decided to continue with a three-drug regimen and stopped ETB. Treatment prescriptions and modifications are presented in 3 and undetectable VL.After 1 week since the last dose of INFα2a and 3 weeks after ATI, the patient’s symptoms worsened and he also had productive cough and dyspnea. A chest X-ray was performed  and showM. malmoense isolation, the culture was negative and it was decided to continue this treatment for 12 more months.The antibiogram was obtained after approximately 18 weeks and the initial treatment was adjusted . Due to In November 2018, patient came to the clinic referring non-life-threatening hemoptysis. We suspected therapy failure and adjusted treatment . A chestDuring subsequent follow-up the patient referred to intermittent mild hemoptysis and, considering the chest CT findings, we proposed surgical intervention. In January 2020 he underwent a left lung upper successful lobectomy. Pathology described acid-fast bacilli microorganisms within multiple necrosating granulomas. Unluckily, no culture was performed. With these results, a persistent mycobacterial infection could not be discarded. We decided to continue the assessments with positron emission tomography (PET) and restarted the antimicrobial regimen. Initially, PET reported pathological uptake of fluor-deoxi-glucose (FDG) in various nodular lesions on the left lung parenchyma and several thoracic lymphadenopathy that was interpreted as related to mycobacterial infection. Later images showed a gradual and significant improvement, so after 9 months treatment was interrupted.To date patient is in good health, asymptomatic and continues his follow-up at an HIV-clinic.M. malmoense infection during supervised antiretroviral treatment interruption in a chronic HIV-1 co-infected patient participating in an HIV-1 therapeutic vaccine clinical trial. The most frequent disease presentation is pulmonary infection that resembles mycobacterium tuberculosis (TBC) [M. malmoense [To the best of our knowledge, we describe the first reported case of is (TBC)  and pre-almoense .Our patiM. malmoense pulmonary disease with rifampin, ethambutol and a macrolide and if severe, to add an injectable aminoglycoside [M. malmoense reactivation and outcome.Treatment for NTM is long-lasting and it should be maintained for a minimum of 12 months after culture conversion. The British Thoracic Society (BTS) guidelines recommend treating lycoside . These rlycoside ,9 but bolycoside . During lycoside ,12. Intelycoside , and thulycoside . This salycoside . In contlycoside ,17. No clycoside . There iExcept for a few cases describing non-supervised ART interruption and retroviral rebound syndrome ,20 with M. malmoense pulmonary infection in an HIV co-infected individual with a torpid outcome, and although this mycobacterium is highly pathogenic and difficult to treat, we cannot discard the effect of our interventions in the reactivation and evolution of this infection.We described an Particular attention should be paid to latent infections, beyond TBC, when using immunotherapy. Routine chest X-ray has not been implemented in HIV therapeutic vaccine clinical trials; our case report emphasizes the need to implement this evaluation, particularly before ATI.


Key populations (KP) are disproportionately infected with HIV and experience barriers to HIV care. KP include men who have sex with men (MSM), female sex workers (FSW), persons who inject drugs (PWID) and transgender people (TG). We implemented three different approaches to the delivery of community-based antiretroviral therapy for KP (KP-CBART) in Benue State Nigeria, including One Stop Shop clinics (OSS), community drop-in-centres (DIC), and outreach venues. OSS are community-based health facilities serving KP only. DIC are small facilities led by lay healthcare providers and supported by an outreach team. Outreach venues are places in the community served by the outreach team. We studied long-term attrition of KP and virological non-suppression.This is a retrospective cohort study of KP living with HIV (KPLHIV) starting ART between 2016 and 2019 in 3 0SS, 2 DIC and 8 outreach venues. Attrition included lost to follow-up (LTFU) and death. A viral load >1000 copies/mL showed viral non-suppression. Survival analysis was used to assess retention on ART. Cox regression and Firth logistic regression were used to assess risk factors for attrition and virological non-suppression respectively.Of 3495 KPLHIV initiated on ART in KP-CBART, 51.8% (n = 1812) were enrolled in OSS, 28.1% (n = 982) in DIC, and 20.1% (n = 701) through outreach venues. The majority of participants were FSW—54.2% (n = 1896), while 29.8% (n = 1040), 15.8% (n = 551) and 0.2% (n = 8) were MSM, PWID, and TG respectively. The overall retention in the programme was 63.5%, 55.4%, 51.2%, and 46.7% at 1 year, 2 years, 3 years, and 4 years on ART. Of 1650 with attrition, 2.5% (n = 41) died and others were LTFU. Once adjusted for other factors , KP-CBART approach did not predict attrition. MSM were at a higher risk of attrition  1.27; 95%CI: 1.14–1.42). Of 3495 patients, 48.4% (n = 1691) had a viral load test. Of those, 97.8% (n = 1654) were virally suppressed.Although long-term retention in care is low, the virological suppression was optimal for KP on ART and retained in community-based ART care. However, viral load testing coverage was sub-optimal. Future research should explore the perspectives of clients on reasons for LTFU and how to adapt approach to CBART to meet individual client needs. The World Health Organization (WHO) guidelines on HIV prevention, diagnosis, care, and treatment for key populations (KP) identified female sex workers (FSW), men who have sex with men (MSM), prisoners, persons who inject drugs (PWID), and transgender people (TG) as key populations, at substantial risk for becoming infected with HIV and also potentially driving HIV transmission . The burThe HIV epidemic in Nigeria is mixed, meaning that HIV prevalence is high in both the KP and general population. Findings from the various surveys showed that KP have a greater HIV burden compared to the general population –6. In 20One of the barriers to effective treatment for KP living with HIV (KPLHIV) is poor access to care. Moreover, retention on ART is a major challenge for many ART programmes, across different settings and populations . SeveralTo overcome barriers to access and engagement in care, the WHO recommended community-based approaches to ART care , 7. MoreA trial and a prospective study in Tanzania showed high retention rates in ART care among FSW receiving ART through a community-based ART programme for Key populations (KP-CBART) , 14 HoweIn Benue State, KP-CBART includes provision of ART through three different approaches. Hereafter referred to as KP-CBART approach. There are three community-based health centres, also known as “One Stop Shop clinic (OSS)”, two drop-in-centres (DIC), and eight community outreach venues. The OSS is a community-based health centre that provides comprehensive HIV care to KP. The Drop-in-Centre is a mini-OSS led by lay healthcare providers. As the term shows, outreach venues do not rely on a physical health infrastructure but deliver services in agreed locations within the community. In community venues medical health care workers (HCW) liaise with a network of KP-led and KP-competent civil society organizations. Both DIC and outreach venues are served by a mobile multidisciplinary health team that ensures medical care.The aim of this study is to evaluate the performance of KP-CBART in Benue State, in Nigeria using routine programme data. We described the characteristics of KP stratified by KP-CBART approach, explored the HIV virological suppression and attrition rates and the predictors of these outcomes.This is a retrospective cohort study of KP-CBART using routine programmatic data and the report is based on the reporting guidelines of studies conducted using observational routinely-collected health Data (RECORD) .Benue State has a KP-CBART programme that is part of the national HIV programme since 2016. This model of care was designed for HIV-positive MSM, FSW, PWID, and TG to increase their access to HIV prevention, care, and treatment in the state and to improve their treatment outcomes. This programme is implemented by the Agency for the Control of AIDS, Ministry of Health, and implementing partners with support from PEPFAR through the Centre for Disease Control.The three OSS are community-based health centres that provide comprehensive HIV services strictly to KP in an environment that aims at being free of stigma and discrimination. The OSS is located in the urban setting. relies on a team of HCW, including medical staff providing HIV testing and clinical care, and lay staff or peer educators for adherence counselling, client tracing, support group meetings, and escort services to the Tuberculosis (TB) treatment centres. HCW participated in a comprehensive ART training including adherence to build their capacity to provide HIV care and treatment services to patients. Besides clinical activities, the OSS serves as a meeting place for KP to engage in recreational activities. OSS also have outreach teams, which consists of an ART clinician, a peer educator, a triage nurse, and/or pharmacist. These outreach teams ensure a comprehensive package of outreach activities at DIC and community outreach venues.The two DIC are mini-OSS and are safe places where KP can meet to socialize, make friends and also access clinical activities when the outreach team is around. To ensure easy access, DIC are located close to “hotspots”  and are located in semi-urban areas. The main difference between OSS and DIC is that OSS are led by medical HCW while DIC are led by both a Nurse Case Manager and lay healthcare providers (community facilitators). Community facilitators are actively involved in the planning and conduct of outreach, support group meetings, and drug refill by proxy. The DIC do not have their own ART clinician but rely on the outreach team for clinical care. Similar to the OSS, DIC offer a comprehensive package of services, such as HIV-testing services, adherence counselling, condom distribution, ART initiation, ART refill and referral if needed.Community outreach venues are locations within the community where the outreach teams and community facilitators from the OSS provide ART care to KPLHIV. These venues are located in communities without operational DIC or OSS. OSS and DIC are located in urban and semi-urban areas while outreach venues are mostly found in rural communities. The community facilitators mobilise their peers for HIV diagnosis, ART initiation, ART refill, and other services while treatment officers plan HIV outreach services. In addition, within communities outreach teams visit hotspots to sensitize and mobilise clients for HIV testing and ART initiation. Community facilitators provide voluntary HIV testing and counselling. Community facilitators are engaged by civil society organizations and they work within the community, pay home visits, and render services at outreach venues. Some community facilitators and treatment officers are KP (eg peer educators) and rely on their own experiences of living with HIV. Community facilitators were trained by KP-led and KP-friendly civil society organizations to provide HIV services. They receive HIV counselling and testing (HTC) training for a minimum of 10 days.Stable patients on ART, adherent to medication/clinic appointments, can benefit from drug pick-up by proxy after 6–12 months of clinical stability. Drug pick-up by proxy is available in OSS, DIC and outreach venues. It involves ART drug dispensing through a diverse group of lay providers (community facilitators and treatment partners) to patients on ART who in turn provide ART to the client.Patients that are lost to follow-up (LTFU) are tracked through phone calls and home visits. LTFU refers to no clinical contact or drug refill from any of the KP-CBART approaches for more than 28 days since the last expected contact. Lists of patients LTFU are generated using appointment registers at the end of each scheduled outreach/clinic for immediate tracking by the community facilitators.Patients’ viral load status is assessed in line with the 2020 National Guidelines for HIV Prevention, Care, and Treatment in Nigeria. Viral load testing is conducted for HIV infected persons at 6 and 12 months on antiretroviral treatment, and thereafter annually. In the KP-CBART, clients’ clinic and ART refill appointment are structured to align with viral load testing schedule to ensure that clients do not miss their viral load test.Adult (18 years or older) KPLHIV who initiated ART in the KP-CBART programme between January 1, 2016 and December 31, 2019 and who were followed up until January 31, 2021 in Benue State Nigeria. Clients were initiated on ART based on the Test and Treat criteria , coherent with the Nigeria Guidelines for HIV Prevention and Treatment.Individual-level data of patients enrolled into the KP-CBART programme between January 2016 and December 2019 were extracted from the electronic medical record (EMR) into a standardized excel-based data extraction template. EMR data were triangulated with patient record in the folders and registers for missing data. Variables that were extracted included demographic data , clinical variables  and treatment outcomes .Attrition referred to those who died (either by direct observation or by family or close contact report) or were LTFU. LTFU was defined as no clinical contact or drug refill from any of the above described approaches for more than 28 days since the last expected contact. Retention in care on ART was defined as the proportion of patients that are active in care and on ART, among those who commenced ART and those transferred out to another HIV programme or facility. Virological non-suppression was defined as having a last viral load higher than 1000 copies/mL.Baseline characteristics were summarised stratified by KP-CBART approach. Proportions were calculated for categorical variables while medians and interquartile ranges were calculated for continuous variables. The Chi-squared test was used to identify associations between categorical variables.We employed bivariable and multivariable Cox regression to estimate the association between explanatory variables and attrition on ART. Patients transferred out were censored on the date they were transferred out. Patients retained in care at the end of the study period were censored on the last contact with the KP-CBART programme. We considered patients who were LTFU and those that died as having experienced the event and were censored on the date of their last ART refill appointment or on the date of their death. In patients with a viral load result, Firth’s logistic regression for rare events was used to determine the association between explanatory variables and virological non-suppression. Also, we estimated factors associated with not having a viral load test done among patients retained in care at the end of the study period. For both the Cox and logistic regression first saturated multivariable models were constructed. These were then stepwise simplified until only the variables of interest (KP-CBART approach and KP type) and variables associated with the event remained. Kaplan–Meier techniques  were empThe study was approved by the Institutional Research Board of APIN Public Health Initiatives (IRB022-FR), Benue State Ministry of Health and Human Services, and the Institute of Tropical Medicine Antwerp (1503/21), providing a waiver for the obligation to seek informed consent. Study participants were protected as all data were anonymized before accessing them. The database did not include personal identifiers such as names or addresses.In this study, we extracted data for 3495 KPLHIV initiated on ART in the CBART programme between 2016 and 2019. An estimated 51.8% (n = 1812) of participants were enrolled in OSS while 28.1% (n = 982) were enrolled in the community DIC and 20.1% (n = 701) through community outreach .At baseline, the median age of participants was 34 years (IQR: 29–40) and more than half  of all participants were female. About 45.6% (n = 1595) of the participants were living in the urban area while 13.6% (n = 475) and 37.8% (n = 1321) lived in semi-urban and rural areas respectively. The vast majority of participants were in the early stage of HIV disease at the time of diagnosis and enrolment: 91% (n = 3179) of them were diagnosed with WHO Stage 1 HIV disease.The majority of participants enrolled in KP-CBART were FSW , while 29.8% (n = 1040), 15.8% (n = 551) and 0.2% (n = 8) were MSM, PWID, and TG, respectively. In OSS and community outreach the proportion of KP that were FSW were nearly the same (57.5% vs 56.6%). The proportion of KP that were MSM (26.2%) in OSS was lower than in DIC (35.6%). Overall, only 0.2% (n = 8) TG were enrolled in the programme: 7 in DIC and 1 in community outreach. In community outreach the proportion (12.6%) of KP that were PWID was the lowest.Overall, retention in care was 63.5%, 55.4%, 51.2%, and 46.7% at 1 year, 2 years, 3 years, and 4 years on ART . Of 1650In the bivariable Cox regression analysis, age, sex, residence, year of ART initiation, KP type, and KP-CBART approach were significantly associated with attrition . The hazard of attrition was higher among participants aged 25–39 years  1.27; 95%CI: 1.13–1.43), among MSM , among participants started on ART in the 2017  and 2018 cohort  as compared to those who started ART 2019, and among KP living in rural  and urban areas  as compared to those living in the semi-urban areas.Of 3495 patients 48.4% (n = 1691) had a VL . Of 1691In bivariable logistic regression, sex, place of residence, and WHO Stage at ART enrolment were significantly associated with virological non-suppression.In the multivariable logistic regression model, virological non-suppression was associated with WHO Stage 2 (adjusted odds ratio (aOR) 3.76, 95%CI: 1.45–9.75) compared to WHO stage 1 at baseline. The type of KP population group and the KP-CBART approach were not associated with virological non-suppression .We studied long-term retention and predictors of attrition among KP receiving ART through KP-CBART in Benue State Nigeria. Attrition rates increased with increasing duration on ART, with less than half retained after 4 years since the start of treatment. Attrition mainly consisted of patients LTFU. Once adjusted for other predictors, type of KP or the approach to KP-CBART did not predict attrition. The high LTFU rate among KP enrolled into the CBART models explains the sub-optimal viral load coverage in the programme. However, among clients with recent viral test result, viral load suppression was optimal. The rate of viral suppression in the Benue KP-CBART model is higher compared to previous CBART studies among KP and the general population , 20, 21.Our study showed data for all KP subgroups and showed data on long-term retention. Most previous CBART studies were designed for a specific KP subgroup, without data for retention after 12 months on ART , 17. OneThe definition of LTFU in our study is more stringent compared to most studies that defined LTFU as being 2–6 months late for their last clinic appointment , 16, 21,The three approaches to KP-CBART described in this study are examples of differentiated service delivery (DSD). DSD is defined as ‘‘a person-centred approach that simplifies and adapts HIV services across the cascade in ways that both serve the needs of people living with and vulnerable to HIV and optimize available resources in health systems” , 25. In The strengths of this study include a large study cohort and long duration of follow-up in a non-research setting. The outcomes of this study reflect the real challenges influencing treatment outcomes of KP in the CBART. A major limitation of this study was that we could not verify the true outcomes of those reported as LTFU. Previous studies showed that some patients LTFU are underreported deaths, while a substantial proportion is active in care elsewhere, thus erroneously reported as LTFU . We repoIn conclusion, long-term attrition on ART is high among KP in CBART in Benue state, while virological suppression was high among those retained in care. How to reach best vulnerable groups remains unresolved. Further differentiation may be required, offering a true choice to individuals. Qualitative research should explore how to adapt KP-CBART for each of the KP subgroups. Moreover, the risks and benefits of innovative monitoring systems that correct for silent transfers should be studied.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.