Average viral load increased 0.559 log10 copies per milliliter. CD4+ T cell count decreased as soon as ART was withdrawn, an average loss of 99.0 cells/mL. Forty-three percent of the mutations associated with antiretroviral resistance in PBMCs disappeared and fifty-seven percent of the mutations in plasma reverted to wild type, which was less than the 100% reversion expected. In PBMC, the PI mutations reverted more slowly than reverse transcriptase mutations. The patients were projected to need an average of 33.7 weeks for PI to revert compared with 20.9 weeks for NRTI and 19.8 weeks for NNRTI. Mutations in the pol gene can cause virological failure and difficulty in re-establishing effective virological suppression.HIV cure studies require patients to enter an analytical treatment interruption (ATI). Here, we describe previously unanalyzed data that sheds light on ATI dynamics in PLHIV (People Living with HIV). We present drug resistance mutation dynamics on the pol gene among individuals with antiretroviral virological failure who underwent ATI. The study involved a 12-week interruption in antiretroviral therapy (ART), monitoring of viral load, CD4+/CD8+ T cell counts, and sequencing of the pol gene from 38 individuals experiencing virological failure and harboring 3-class resistant HIV strains: nucleoside reverse transcriptase inhibitors (NRTI) non-nucleoside inhibitors (NNRTI), and protease inhibitors (PI). Protease and reverse transcriptase regions of the  In the late 1990s and early years of this century, researchers studied the clinical benefit that analytical treatment interruption (ATI) might offer HIV-1 patients experiencing antiretroviral virological failure given the limited options of salvage therapy available at that time. However, these studies led to the conclusion that complete ATI cannot provide sufficient clinical benefit to justify its use as an adjunct to antiretroviral therapy (ART) programs ,3,4,5.At the time the data were gathered for this study, researchers believed that ATI would hasten the reappearance of wild type virus, which in turn would facilitate the subsequent reintroduction of ART ,7,8,9. AFrom June 1999 to June 2000, the authors collected samples from 38 HIV-1 treatment-experienced patients experiencing antiretroviral virological failure who agreed to participate in a study of ATI. This paper analyzes the original data in a more consistent way than conducted previously. Although we no longer consider ATI a viable treatment strategy, there is still much to be learned from these data in terms of the evolution of resistance patterns in the reverse transcriptase and protease portions of the HIV-1 genome. The difference between the virus genotype with and without the selective pressure of ART offers important information about effective treatments for the future. Once infected, the patient’s HIV-1 begins to evolve and continues to do so even as it faces long-term replication suppression ,13. GiveSome anatomical sites such as the central nervous system and genital tract may be inaccessible to some classes of antiretroviral drugs. For example, the brain absorbs protease inhibitor (PI) medications poorly . These iViral fitness, the ability to create more adapted and highly replicating infectious progeny, is inversely related to the number of incorporated antiretroviral resistance related mutations ,26,27. IThe current study describes viral load and CD4+/CD8+ T cell count dynamics. We also characterize the drug-resistant mutation dynamics in the plasma and PBMC. We focus on mutations associated with HIV-1 resistance at the reverse transcriptase and protease regions of the pol gene in DNA and plasma RNA. The expectation of the study team was that, since the use of ART repressed the wild-type virus, it would proliferate on withdrawal of ART and that the resistant strains would disappear due to the greater replicative fitness of the wild type. Finally, we report on the presence of cell activation markers HLA-DR and CD38 on CD8+ T cells. These proteins are associated with activation of latent infected CD4+ T cells, making them available for attack by the patient’s immune system. Prior research has indicated that expression of these activation markers is associated with an increase in viral load and a decrease in CD4+ T cell counts ,34,35,36Previous use of antiretrovirals before the experimental period revealed a variety of antiretrovirals available then. This study included 36 patients with HIV-1 3-class antiretroviral resistance mutations and 2 patients with chronic HIV-1 infection but no detectable HIV-1 resistance related mutations. These latter patients served as controls. There were one male and one female control subjects. The mean age of the controls was 37.0 years (standard deviation of 1.41 years). The patients harboring resistance included 28 males (77.8%) and 8 females (22.2%). The mean age of the resistant group was 38.75 years (standard deviation of 7.77 years). All patients had a virus of subtype B except for patients 3 and 53, who had combination subtype B/F.For the reverse transcriptase and protease regions of the pol gene, the patients were divided based on the degree to which they returned to the wild type. These categories are high reversion, low reversion, and non-reversion. We established the reversion group assignment on the return to wild-type pattern in the reverse transcriptase region of the plasma sequences. The decision rule for assignment to each group was based on a comparison of the number of mutations that the patient experienced between baseline (week 0) and week 12. The “None” group had an equal or greater number of mutations at the end than at the baseline. The “Low” group had fewer but not zero mutations. In other words, they had at least one mutation but less than they had at baseline. The “High” group experienced zero mutations at week 12 after showing one or more at baseline. In this scheme, the controls were those with zero mutations at baseline.The last line of 10 transformation of the raw data) over the twelve weeks of the study for all four groups (three reversion groups and the control group). The data underlying these results, the viral loads, CD4+ counts, and CD8+ counts for all patients at all sample collection times are included in the 10. However, the viral load of the no reversion group rose until week 8 and then declined. It only increased over the study period by 0.136 log10. Overall, the groups, including the controls, increased by an average of 0.568 log10. In addition, the high and low reversion groups’ viral load increase was statistically significant. A paired two-sample t-test of the difference between viral load in all resistant patients  at baseline and at week 12 had a value of t = 6.2919 with 35 degrees of freedom and p < 1.0 × 10−6. The linear increase in viral load in the current cohort contrasts with the rapid rise in viral load reported in a previous study [p-value < 1.0 × 10−6.Focusing on the three groups that showed initial resistance mutations, the decline in CD4 levels between baseline and week 12 was statically significant  did the number of mutations go to zero. In all other cases, there remained a small number of mutations per patient in week 12.That the resistant mutations would revert to the wild-type virus due to ATI among individuals with antiretroviral failure was only partially supported by the data from this study where the proviral DNA profile is concerned. We limited all of the analyses of reversion to wild type to the 36 patients who showed resistant mutations. However, we include in the The plasma sequences collected at baseline and study end (week 12) show an uneven reversion pattern despite the expectation that all mutated codons would revert to wild type after 12 weeks of ATI. Among the patients showing drug resistance, a mean of 14.95 codons evolved between the start and end of the treatment interruption . Of the 434 amino acids in the sequence studied, 111 different codons evolved during the study. Some of these 111 codons had variant amino acids at the end of the survey, so the number of different mutations during the treatment interruption totaled 164. For example, codon 20 showed an evolution of R10K in five patients and I10K in two other patients. We focused our analysis on those codons that evolved in more than one patient. Although our sample was drawn well before IAS began to report drug resistance mutations, we tested whether mutations in patients with drug resistance are consistent with the 2017 IAS list of drug-resistant mutations. In 35 of the 40 codons in the table (87.5%), the patients reverted to wild type as defined in the IAS 2017 interpretations.We conducted a parallel analysis of the PBMC samples from the 36 resistant patients for the evolution of both protease inhibitor and reverse transcriptase inhibitor resistance mutations. We hypothesized that the high reversion level that we would see at the plasma level would not be observed in this compartment. Of the PI codons on the IAS list, we initially had data on 22. These were codons 10, 20, 24, 30, 32, 36, 46, 47, 48, 50, 53, 54, 63, 71, 73, 77, 82, 84, 85, 88, 89, 90, and 93. We eliminated codons 32, 47, and 50 from our analysis as they either had 0 or 1 tests that showed that mutation and therefore did not allow comparison. For codon 89, only patient 3 exhibited a mutation. Codons 77 and 88 showed an increase in the number of patients exhibiting the mutation during the 12 weeks and thus had positive slopes in the regression. Accordingly, it was not possible to calculate a time to 0 patients using regression. However, the Kaplan–Meier survival analysis result showed a probability of the survival of the mutation at codon 77 of 0.318 (95% CI of 0.173–0.587) and at codon 88 of 0.176 (95% CI of 0.063–0.493). Given the overall sample size of 36 and the variable number of patients who showed the mutation , it is not possible to tell if this result is due to a sampling error or to some fundamental observation about these codons. p = 0.00013) using a Pearson’s Product Moment Correlation test. We included a figure with this result in the We evaluated the consistency of results between the time to 0 mutations for each codon and the survival analysis. We found a correlation between these values of 0.69. This correlation was significant  among the PI mutation codons. Graphs for the other codons are found in the data repository for the study .The mean number of weeks for the protease inhibitor mutations is 34.5 weeks with a standard deviation of 27.3 weeks. 2 indicates the degree to which the line explains the points.This figure shows the number of patients with a mutation at each test and a regression of these points. The regression equation shows the parameters determining the line, and the RWe had data on nine of the IAS’s list of 15 NRTI codons . We eliminated codon 65 from further analysis as it had only one patient with a mutation at this location at week 0. Codon 75 just had two data points: one in week 0 and one at week 12. 2 measure indicates the relative variability within the group.Codon 184 (M184V) comes first in the Stanford University database of NRTI mutations because of its 100-fold reduction in patient susceptibility to Lamivudine/Emtricitabine but its enhancement of the susceptibility to Zidovudine and Tenofovir Disoproxil Fumarate . The othp = 1.0). However, the TAMs considered together take a significantly longer time to revert to wild type than codon 184 according to a one-sided Wilcoxon Signed Rank test (p = 0.016). This started with 28 patients exhibiting the Valine mutation, the loss of one carbon atom that distinguishes it from the Isoleucine wild type. By the end of the 12 weeks, the number showing this mutation had dropped to 8, and our linear regression model for this codon projected it would be eliminated by week 16. The TAM groups showed fewer patients with the mutations at the start of the interruption (20.7 for TAM 1 and 17.7 for TAM 2), declining to 8.3 and 7.7, respectively, at the end of the study. The higher rate of reversion for I184V than the TAMS can be seen in greater detail in the panels of Both TAM groups have mean values very close to each other. According to a sample of 3 per group, there is no difference between the two TAM types with a Wilcoxon Rank Sum test (Our data included 7 of the 16 non-nucleoside reverse transcriptase inhibitors (NNRTI) on the IAS list . We eliminated codons 106 and 108 from the analysis as they had insufficient data to permit a regression to be performed. We also removed codons 179 and 188 from the analysis as the counts of patients with the mutation only varied between 1 and 3, which strongly suggested that our sample was not typical of the population. The last line of The average number of weeks for NNRTI mutations to revert to wild type was 19.8 weeks with a standard deviation of 2.2 weeks. Although the graphs and tables we display here show the range of rates of reversion to wild type among the codons studied, the average for each of the three classes of antiretroviral medications indicates that this is a prolonged process that may last as long as 8 months (in the case of protease inhibitor mutations). The study sample showed a greater presence of CD38+ and HLA-DR+ proteins on CD8+ T cells in patients who returned to wild type. The no reversion group, whose mutations remained constant despite the treatment interruption, expressed a significantly lower level of these cell activation biomarkers (measured by the percentage of CD8+ T-cells with both CD38 and HLA-DR present) than did the high reversion group. This result parallels the observation that cell activation markers would be expressed in greater quantity in untreated HIV-1 patients than in patients receiving treatment . 10 viral load by dividing the cell activation markers’ expression by the log10 viral load. As To create an index of cell activity absent viral load, we corrected the cell activation marker levels for the logp-value < 1.0 × 10−6). However, the correlation between cell activation and CD4+ T-cell levels is stronger and statistically significant  showing that the higher the T cell activation, the lower the CD4+ T-cell counts. We compared the T cell activation level with the viral load and CD4+ T-cell levels for all the patients. Overall, the correlation with viral load was low but statistically significant  without changing their HIV genetic profile, which argues in favor of the activity of antiretrovirals in use, although probably without a good adherence to current treatment. As was expected, the viral load increased for patients subject to ATI, and CD4+ T-cell counts decreased. The viral load increase results from an increase in active viral replication after ATI. This is an important factor in replenishing the proviral reservoir even for this short period. This has been shown previously in this same patient population . One facThe picture for reversion to wild type is not clear. This study shows that reversion is not as direct a process as previously thought for resistance-related mutations selected by all three classes of antiretrovirals studied ,30. WhilWhat enables such a result? First, the period chosen for the treatment interruption—twelve weeks—may not be sufficiently long for complete reversion to the wild-type profile at the DNA level, although it was the standard for ATI studies at the time. A 1992 study from Sweden indicates that patients taking zidovudine (AZT) who developed resistance to the medication and subsequently abandoned treatment still carried some of the mutations as long as nine months after stopping treatment . As noteWe also need to consider why each mutation disappears with its dynamics. There appears to be little or no uniformity in the rate of reversion. Theoretically, once ART’s selective pressure that gives an advantage to the resistant mutations is withdrawn, the wild-type strain should reemerge rapidly. Interestingly, once ART is stopped and its selective pressure withdrawn, viruses with distinct resistant-related mutation profiles may reemerge as latent cells resume replication and/or based on the differential replicative capacity of distinct strains in each HIV quasispecies. When single genome amplification of antiretroviral resistant strains is performed, mutations that are detected in the population sequence analyses may not be present in all HIV strains but dispersed among distinct viruses of the HIV quasispecies at a given moment . We mustAfter antiretroviral treatment interruption, HIV recombination at the pol gene may dynamically occur in distinct HIV strains with resistant and wild-type profiles, which may in turn seed new proviral populations. As mentioned before, studies exploring partial treatment interruption show that, once NRTIs are withdrawn and PIs are maintained, wild-type HIV emerges at the reverse transcriptase region of the pol gene while the protease region remains resistant . In contNonetheless, we have previously demonstrated for this same patient population during the ATI period that the disturbance caused by interrupting the selective pressure imposed by antiretrovirals at the pol gene resulted in ancestral viral progeny activation in a very distinct (and distant) HIV genomic region such as the gp120V3 HIV-1 env region, which repopulated the cell reservoir . In contrast with what has been previously demonstrated in vitro, PI-related mutations’ persistence was longer than the persistence of NNRTI-related mutations. As NNRTI mutations do not impair the HIV-1 fitness as with NRTIs and PIs mutations ,45, it wAnother feature that deserves further attention is the fact that M184V waned more rapidly than other studied mutations. Although transmitted drug resistance mutations tend to persist in general, M184V tends to revert to wild type in this specific population . It is aWe recognize that a more detailed analysis of HIV quasispecies in this study would greatly benefit from single genome amplification or next-generation sequencing (NGS) tools. It has been recently demonstrated using NGS that transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion . In thisWe previously described that there are two pathways for NNRTI resistance depending upon the drug used. The use of efavirenz primarily selects for the 103 mutation, which is accompanied by the mutations at positions 100 and 225, whereas nevirapine predominantly selects for the 181 mutation, which is generally accompanied by the 101 and 190 mutations  and 25–3We also recognize that more intensive cell population evaluations, additional immunologic markers, or direct HIV fitness evaluation could provide additional insights into the mechanisms of viral dynamics after ATI among patients experiencing virological antiretroviral failure. However, the number of patients evaluated here is larger than what has been described in other similar studies, and this is the first study analyzing the impact of such a strategy at a DNA level at multiple time points compared with the plasma compartment, where we sequenced only the baseline and study completion (12-week) time points. We, therefore, have been able to show how the reversion of the virus to a wild type after ATI among individuals failing antiretroviral therapy is not a clear and consistent process across the full range of pol codons studied. We have also provided insight into the persistence of distinct resistant-related mutations selected by different antiretroviral classes. We studied specific areas of the pol gene related to their known patterns of resistance to the three classes of ART medications in common use at the experiment time. However, our results suggest that ART and subsequent treatment interruption can induce other viral genome changes in which the impact on resistance profiles is not yet well understood. The region between codons 293 and 560 of the reverse transcriptase region is one area of interest. For example, a small number of studies have indicated that Tyr-318 is subject to a range of mutations that show some history of resistance to NNRTIs . AnotherThirty-six HIV-1 treatment experienced patients who were treated at the Federal University of Sao Paulo, Brazil in 1999, agreed to participate in the study on ATI. Two other patients under the same conditions, who suffered from toxic side effects but had no detected HIV-1 resistance mutations, submitted to ATI during the same period and were included in the evaluation. The samples were collected from June 1999 to June 2000. All patients were followed for 12 weeks. The total sample size of 38 was large for studies of this type at the time it was carried out. A non-systematic review of 13 studies on treatment interruption that parallel this study in intent and method published between 1999 and 2004 had a median number of participants of 24 and an interquartile range of 28 (10–38). Of the studies reviewed, only four had more or the same number of participants as the present study. See Eligible male and female patients met the following criteria: 18 years or older; experienced multiple ART failures while using nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside analog reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs); had detectable viral loads above 5000 copies/mL; and showed no signs of AIDS. At the time the experiment took place, the design was an interventional study based on the hypothesis that, if the ART drugs were withdrawn, the wild-type virus would replicate and eliminate the presence of resistant strains as the wild type had greater replicative fitness. All patients enrolled on ATI at day zero started a prophylactic regimen against opportunistic agents (data on file). CD4+ T cell counts were taken at 15-day intervals; viral loads were performed monthly; the protease and reverse transcriptase regions of the pol gene were sequenced every four weeks from PBMC; and two plasma genotyping tests were conducted at baseline and week 12 from protease: reverse transcriptase and the p17 region of gag gene. No patient presented AIDS-related symptoms during the study period.HIV-1 RNA in plasma was measured using the NASBA methodology , according to the manufacturer’s instructions. This assay has a limit of detection of 80 copies/mL.At each lymphocyte count, we determined sub-populations of CD4+ and CD8+ T cells by three-color flow cytometry. We used fresh samples for direct staining with appropriate monoclonal antibodies (mAb): CD4-PerCP, CD8-PerCP. Mouse immunoglobulin isotypes conjugated with phycoerythrin (PE), or fluorescein isothiocyanate (FITC) were used as negative controls for non-specific binding. The stained cells were analyzed on a FacScan flow cytometer. For the analysis of genotypic resistance of PBMC, proviral DNA was extracted from cells using QIAamp DNA Blood Mini kit  and plasma RNA was purified using QIAamp RNA kit  and reverse-transcribed using as previously described . The proThe nested-PCR fragments were purified by PCR purification kit  and sequenced using Thermo Sequenase fluorescent-labeled primer cycle sequencing kit—RPN 2436 . The primers were labeled by CY5.5. The products of the sequence reaction were analyzed using a DNA—Long Reader Tower (Visible Genetics Inc.). Base-calling was performed by GeneObjects software , and the bases were aligned and assembled using GeneLibrarian software . The resulting sequences for each sample were interpreted using the 2017 edition of the IAS–USA drug resistance mutations list . TM HIV-1 Genotyping System v2.0  per the manufacturer’s instructions. All codons of the HIV-1 protease region were sequenced. The reverse transcriptase region was sequenced up to codon 320. The HIV-1 gag gene amplification products were sequenced in an ABI 3100 Genetic Analyzer .Nucleic acid isolation, reverse transcription-PCR amplification, and sequencing of the HIV-1 protease and reverse transcriptase regions of the pol gene were performed using ViroSeqThe analysis compared the viral load and CD4+ T cell count dynamics considering patients’ reversion categories. The dynamics of the protease and reverse transcriptase mutations were analyzed by linear regression and Kaplan–Meier survival analysis ,59,60,61The data analysis was conducted using the R Statistical Computing System on a Macintosh (version 3.5.0) . AlgoritWe calculated the number of patients who reverted to wild type for each codon in the protease and reverse transcriptase regions on the IAS resistance mutation list for which we had PBMC data. We used the PBMC data for this analysis rather than the plasma data as the former permitted us to examine the evolution over four tests rather than just looking at the beginning and endpoints of the study. We had data on 9 of the IAS list of 15 nucleoside reverse transcriptase codons (NRTI) and on 7 of the 16 NNRTI codons on the IAS list. Twenty-two of the thirty-five IAS mutations affecting PI appeared in our sample. We aligned the data that had been gathered in 2000 with the codons on the IAS list. We removed those codons with no patients showing the mutation or had missing values for any of the four visits. We then performed a simple linear regression of the number of patients showing the mutation during the study period against the time since ATI initiation and projected from the parameters of the regression line how many weeks would be needed to reach 0 patients, i.e., how many weeks would be required for the mutation to be eliminated from the cohort. In five cases , the regression slope was positive rather than negative, indicating that patients were exhibiting new mutations after the ATI. Additionally, we conducted a Kaplan–Meier survival analysis of how long the mutations persisted in the sample and what was the probability that they would persist at the end of the study. These data indicate many interesting features of the mutational patterns of HIV-1 in response to ART selective pressure and its subsequent withdrawal that can be applied to research on understanding fitness features of the HIV vis-a-vis genetic composition of the virus.


The diagnostic and therapeutic management of the Coronavirus Disease 2019 (COVID-19) pandemic in the HIV population brought some known criticalities (and opportunities) to the forefront, for both those who are facing their first therapeutic line today, and for those already well viro-suppressed. The clinical, socioeconomic, and psychological impact of the COVID-19 pandemic should not affect the long-term care of people living with HIV, which creates an urgent need to optimize the diagnostic and treatment approach to the first-line or switch regimens. The use of dolutegravir plus a lamivudine two-drug regimen is one of the most promising solutions to ease the management of HIV treatment in this difficult period. In this review, we report the most salient features related to the use of this regimen from real-life cohorts, meta-analyses, randomized clinical trials, and studies presented at international conferences up to March 2021. We focused on the diagnostic and clinical-management implications of its use in real life, and how these comply with the contingent historical situation. The issue of the timing and type of diagnostic procedures and the relevance of classical diagnostic tests (such as genotype for resistance detection) is also discussed. According to the currently available results, dolutegravir plus a lamivudine two-drug regimen represents an outstanding tool, whose expected advantages fulfill the current requirements for optimal daily care of our HIV patients. Over the last year, the HIV treatment scenario has required revision and reassessment, in a way that we could not expect. It was not only the new scientific evidence on the efficacy, safety and manageability of the most recent regimens that required this intervention. The management of the Coronavirus Disease 2019 (COVID-19) pandemic in the HIV population further highlighted some critical issues (and opportunities) that emerged independent of COVID-19 (but were increased by its presence), both for those facing their first therapeutic line today, and for those who are already effectively viro-suppressed. The COVID-19 pandemic created the need for a change in our common recommendations for combination antiretroviral treatment (cART), to fulfill the need to simplify patient visits for diagnosis, medications and tests. This necessitated the use of a simple, well-tolerated regimen with high efficacy, and a high barrier to resistance . T. T15]. TAltogether, these data support the feasibility of such an approach on a large scale, with no need for substantial pre-treatment diagnostic assessments within the limits of the provided indications for use, and a general confidence in its efficacy, regardless of a number of clinical factors.DTG uniformly shows a high genetic barrier to resistance in both 3DR and 2DR, and resistance-associated mutations (RAMs) are rarely observed. In vitro, DTG-resistance was found to develop much more slowly than for raltegravir (RAL), cabotegravir (CAB) and elvitegravir (EVG) , and resAmong drug-naïve patients enrolled in the real-life URBAN study, no virological failures (VF) were observed up to 24 weeks of follow-up . In GEMIHundreds of thousands of patients worldwide have been given first-line treatment with DTG-containing regimens to date, and this represents the sixth case of failure of a first-line DTG-regimen with emerging mutations (the first occurred in a 2DR). Notably, suboptimal DTG levels were recorded in all of them ,22. EmerThe acquisition of a solitary R263K mutation was shown to confer low-level resistance (~2-fold) to both DTG and BIC in vitro, compatible with the high genetic barrier of the two InSTI ,28,29,30Overall, the development of the same M184V+R263K mutational pattern during treatment with two high-barrier, latest-generation InSTI, requires verification of whether the co-existence of these two RAMs could cooperate in conferring significant InSTI-resistance, while preserving viral infectiousness. In the absence of further data, however, this possibility remains a mere hypothesis.as early as the day of HIV diagnosis—or within the following 7 days, was proposed later on .,67.66,67From a diagnostic perspective, it is important to point out that the observed efficacy was consistent within several analyzed subgroups, demonstrating a sustained response regardless of age, sex, race, baseline 3rd agent class, and baseline CD4+ cell count . The conBesides RCTs, real-life studies allowed us to evaluate patient populations that would probably be considered ineligible for a clinical trial, but who represent a significant portion of those treated in our daily practice, as well as those who, probably, would benefit the most from a switch to simpler and better-tolerated therapies.A recent meta-analysis reported random-effects pooled estimates for a viral suppression of 98.6% and 97.5% at 48-weeks and 96-weeks of DTG/3TC, respectively, by combining the results of 11 real-world studies . In factOverall, this evidence in support of the effectiveness of the strategy of simplification to DTG/3TC identifies, and also validates a simple diagnostic procedure to frame the switch in the eligible patients, which is actually limited to a few characteristics . In this regard, it has to be underlined that patients with HBV co-infection should continue taking TAF or TDF (unless contraindicated), as switching to a regimen not including these drugs fails to maintain HBV suppression ,3,4.Virological failure in RCTs is usually defined as one measurement of HIV-1 RNA ≥50 copies/mL, followed by a confirmatory measurement of HIV-1 RNA ≥200 copies/mL. This eventuality leads to the need to perform a genotypic resistance test, by either Sanger or ultra-deep sequencing, to assess the development of resistance mutations.DTG in dual-formulations has a high genetic barrier to resistance development, as evidenced by the extreme rarity of CVW from long-term RCTs , as wellIn the 96 weeks of the TANGO study, no CVWs were observed, not even in patients who contracted COVID-19 . LikewisDespite these results, the GRT at failure remains an indispensable tool of clinical evaluation, in order to better identify the next therapeutic strategy, and, as such, is strongly recommended.An increasing amount of evidence from real-life experiences and clinical trials consistently contribute to the indication of a negligible risk of incomplete control of viral replication, either in terms of the enrichment of HIV-DNA reservoir, residual low-level viremia, or viral rebound/blips, in patients treated with DTG/3TC 2DR vs. standard cART, thus avoiding the need for more stringent monitoring of virological suppression than with 3DR, once the dual regimen is established.In GEMINI-1 and -2 studies, the number of participants reaching TND values at each week visit was similar to DTG+3TC and 3DR, up to the latest follow-up  represents the most widely used indicator of the HIV cellular reservoir size and dynamics . Both MOAlthough still poorly studied, the available data suggest similar dynamics of total HIV-DNA decay in DTG/3TC and in patients treated with cART up to 48 weeks post-switch , and thuOverall, the increasing amount of evidence from real-life experiences and clinical trials consistently contribute to the indication of a negligible risk of incomplete control of viral replication, either in terms of the enrichment of an HIV-DNA reservoir, residual low-level viremia, or viral rebound/blips, in patients who either start on, or switch to, a DTG/3TC 2DR vs. standard cART, therefore supporting the use of diagnostic follow-up protocols with similar timelines to those currently in use.While the data are quite consistent, it should be emphasized that the currently available real-life studies include patients with a long history of virological suppression (years), and, in a relevant percentage, switched while already on other 2DRs. Therefore, further studies are warranted.Some real-life studies have agreed with the RCTs in excluding patients who presented InSTI or NRTI RAMs at BL, or who had previously experienced a VF. A small number of studies, however, used less stringent enrollment criteria, and allowed us to analyze the behavior of DTG/3TC in the presence of RAMs, and/or in patients with an history of failures ,94,95,96Given the rarity of DTG RAMs, the BL resistance  is usually defined by the presence of the NRTI RAMs M184I/V.The MOBIDIP study provided the initial proof-of-concept that a 2DR including 3TC could retain partial efficacy, despite HIV-infected patients harboring a 3TC-resistant virus  . The stuOn the other hand, an initial warning was launched by two real-life studies, which observed an increased risk of VF associated with the presence of M184V ,93. In oAn interesting in vitro study has advanced the hypothesis that the NRTI RAMs K65R, M184I and M184V may have a protective role in the context of DTG-based regimens , antagonizing the development of specific resistance . This phIt is still unknown whether proviral DNA genotyping in virologically suppressed patients could contribute to a prediction of the efficacy of 2DRs, and if it would add significant information to the historical genotype. In the TANGO study, the archived major NRTI RAMs  and INSTI RAMs  were identified in a minority of patients on pro-viral DNA  . Their mIt should be noted that the detection of the M184I in pro-viral DNA is known to be associated with the hypermutation activity of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) ,102. As Overall, with due caution, the data available today seem to exclude the hypothesis that patients with an underlying presence of M184V/I could find themselves in a condition of functional DTG monotherapy . These initial, encouraging data lead us to believe that, on the one hand, the clinical GRT interpretation must be differently calibrated (and reinterpreted) in the context of the switch to highly potent 2DRs. On the other hand, however, there are still many relevant aspects of the mutational virological profile that have not sufficiently declined in terms of their possible impact. Among these, InSTI RAMs could represent a critical element in the future, although this is currently rare. Similarly, the possible correlation between the (higher) number of previous virological failures with a complex resistance pattern, and the viraemic rebound with DTG/3TC is a still largely unexplored aspect, with few data recorded in the literature. These considerations, therefore, do not spare us, in clinical practice, from a carefully evaluation of the clinical history, baseline virological parameters, and historical GRT of virologically suppressed patients in the process of switching to a 2DR.The added value of pro-viral DNA testing in clinical settings is somewhat controversial, and not yet defined. According to international guidelines , drug resistance results on pro-viral DNA must be interpreted with caution . The maiReal-life data consistently report an overall excellent tolerability of DTG/3TC, with an absence/low incidence of toxicity ,106,107,The achievement of a long follow-up in the RCTs important data to be obtained in support of the safety of the DTG plus 3TC regimen, whether it is co-formulated or not. In the 144 weeks of GEMINI-1 and -2 studies, participants in the DTG+3TC group had a significantly lower risk of drug-related AEs (20%) compared with the DTG+TDF/FTC group for the pooled population  . In treaPatients with impaired renal function raise significant issues for cART tolerability, and this type of toxicity is one of the main reasons leading to therapeutic switch, especially from TDF-based regimens. RCTs have demonstrated a good renal safety profile of DTG/3TC in drug-naïve patients, superior to that observed with DTG+TDF/FTC at week-144 , a resulInterestingly, a recent case review reported no virologic failure in 25 patients who switched to DTG/dose-adjusted 3TC once daily, over a median time of 2.5 years of observation . This prThe outbreak of the COVID-19 pandemic has created significant challenges for healthcare systems across the world and has extensively affected the life of all people living with HIV. However, HIV diagnosis, initial treatment and retention-in-care cannot be compromised. This situation calls for the adoption of regimens with: (a) high barrier to resistance (to reduce the frequency of monitoring); (b) favorable safety profile and few major drug–drug interactions ; (c) preferentially in a single tablet without food requirements (to improve adherence and retention in care) [


Health-related quality of life (HRQoL) is considered to be the fourth 90 of UNAIDS 90-90-90 target to monitor the effects of combination antiretroviral therapy (ART). ART has significantly increased the life expectancy of people living with HIV/AIDS (PLWHA). However, the impact of chronic infection on HRQoL remains unclear, while factors influencing the HRQoL may vary from one country to another. The current study aimed to assess HRQoL and its associated factors among PLWHA receiving ART in Pakistan.A cross-sectional descriptive study was conducted among PLWHA attending an ART centre of a tertiary care hospital in Islamabad, Pakistan. HRQoL was assessed using a validated Urdu version of EuroQol 5 dimensions 3 level (EQ-5D-3L) and its Visual Analogue Scale (EQ-VAS).n = 358) reported no impairment in self-care, while 63.1% (n = 380) were extremely anxious/depressed. The overall mean EQ-5D utility score and visual analogue scale (EQ-VAS) score were 0.388 (SD: 0.41) and 66.20 (SD: 17.22), respectively. Multivariate linear regression analysis revealed that the factors significantly associated with HRQoL were: female gender; age  > 50 years; having primary and secondary education;  > 1 year since HIV diagnosis; HIV serostatus AIDS-converted; higher CD 4 T lymphocytes count; detectable viral load; and increased time to ART.Of the 602 patients included in the analyses, 59.5% (The current findings have shown that PLWHA in Pakistan adherent to ART had a good overall HRQoL, though with significantly higher depression. Some of the factors identified are amenable to institution-based interventions while mitigating depression to enhance the HRQoL of PLWHA in Pakistan. The HRQoL determined in this study could be useful for future economic evaluation studies for ART and in designing future interventions. Low and middle-income countries (LMIC) like Pakistan experience relatively high rates of new human immunodeficiency virus (HIV) infections , 2. PakiHRQoL is a multidimensional concept  that incHRQoL is an important measure that enables healthcare workers to understand patients' perceived satisfaction and perception of illness . Its assThis was a cross-sectional study using a validated generic HRQoL questionnaire. The study was conducted from July 2019 to September 2019 at the ART Centre in PIMS Hospital, a 947-bed hospital located in Islamabad Capital Territory (ICT), Pakistan. The ART Centre at PIMS is the largest centre of HIV referral cases in Pakistan, serving more than 4200 registered PLWHA who receive free-of-charge HIV treatment. Approximately 20 to 25 patients visit the ART centre daily for their medical needs. The centre was selected because of its geographical location and its capacity in serving a large number of patients from different ethnic backgrounds and various regions of Pakistan.Patients were included in the study if they fulfilled the following criteria: aged 18 years or older, diagnosed with HIV for  ≥ 6 months, have a recent CD-4 T cells count and viral load tests (no more than four weeks at the time of the interview), and can understand and converse in Urdu language . Only PLWHA who were taking ART on a regular basis were included in the study. Adherence to ART was confirmed by checking patients’ hospital pharmacy refill records. Prescription medical claims records can be used to estimate medication adherence based on the refill rate. Patients may routinely be defined as 'adherent patients' if the amount of medication given is at least 80% based on the medication possession ratio (MPR), which is defined as the day-to-day delivery of the medication divided by the number of days the patient should take the medication . Patientn = 599, and d = 5%. The formula provided a required sample size of 628.95 patients (n = 599 + n1 = 29.95). Overall, 630 HIV patients were approached. Out of these, 610 patients voluntarily consented to participate in the study. Six hundred and two patients were included in the final analysis.A simple random sampling technique was used to collect data from PLWHA who fulfilled the eligibility criteria outlined above. Every PLWHA registered at the ART Centre who fulfilled the inclusion criteria had equal chance of being enrolled into the study. The sample size of 599 was calculated using the Raosoft calculator by taking a total of 180,000 HIV patients, 95% confidence interval and 4% margin of error . The samEuroQol quality of life scale EQ-5D-3L and Visual Analogue Scale (VAS) have been widely used for HRQoL assessment . The stuThe data collection was carried out for a period of three months (from July 2019 to September 2019). Each person living with HIV/AIDS attending the ART centre, was approached and invited to participate in the study by the head nurse (M.J.) and HIV physician (N.B.). Those who agreed to participate were informed about the nature and purpose of the study. Each consented patient was assessed against the pre-defined inclusion and exclusion criteria by the principal investigator (A.A.). Data collection was performed at the study setting . The data collection consisted of two parts: the first part covered the socio-demographic and clinical characteristics of the participants, while the second part consisted of the EQ-5D-3L and EQ-VAS. Eligible participants were asked to complete their socio-demographic characteristics  in Part 1 of the data collection instrument, which were confirmed from their medical records. They were also requested to self-assess their HRQoL using EQ-5D-3L and EQ-VAS in part 2 of the data collection instrument. Participants who were unable to understand any part of the questionnaire or were unable to self-administer the survey were assisted by the investigator collecting the data. The participants were instructed to read the brief instruction on the top of the page. Participants’ correct understanding was ensured, and they were encouraged to complete every question in chronological order without skipping any items. The total completion time was approximately 15–25 min. Participants’ clinical parameters, including CD4 lymphocyte count, HIV serostatus, and VL, were retrieved from the patients’ medical records. No monetary benefits or other incentives were given to study participants due to lack of funding for the study.p < 0.05) in HRQoL in the univariate analyses were included in the regression analysis. A multivariate linear regression model was applied to find significant predictors of HRQoL among the PLWHA. Before regression analysis normal distribution of data was assessed using Shapiro–wilk test. Multivariable linear regression models with the same variables of the full and final models were ran only to assess multicollinearity using variance inflation factor diagnostics (VIF). The results are presented as unstandardized beta coefficients accompanied by 95% confidence intervals (CIs). A p-value of  < 0.05 was considered statistically significant.Data were coded in MS Excel 2016 and imported into Statistical Package for Social Sciences (SPSS) version 21  for analysis. Descriptive and inferential statistics were applied as appropriate and depending on the nature of the data. Numerical variables were expressed as means and standard deviations, while categorical variables were presented as frequencies and percentages. HRQoL domains were classified as no problem, some problem and severe problem. The HRQoL utility scores  were calculated using the original 1995 UK population data due to lack of utility score data from the Pakistan population . IndepenThe ethical approval for the study was obtained from the Ethical Review Board (ERB) of ART Centre, Pakistan Institute of Medical Sciences (PIMS) and National AIDS Control Programme (NACP), Pakistan . The objectives and procedures of the study were explained to the patients verbally. Informed consent was obtained from PLWHA who agreed to participate in the study. Moreover, potential participants were informed that participation in the study was voluntary and that they could withdraw at any time. All information collected would be kept confidential and was available to the research team only. All study procedures adopted complied with the principles of the Declaration of Helsinki, Good Clinical Practice and within the applicable laws and regulations of research involving human subjects in Pakistan.n = 393) were male, 41% (n = 247) belonged to the age group 26–50 years, and 37.7% (n = 227) had primary school education. Furthermore, about half  of the participants were symptomatic, 38.9% (n = 234) had CD 4 T cells in the range of 200–500 cells/mm3, more than half  had non-detectable viral load, and 64.6% (n = 389) got infection by injecting drugs  reported no impairment in mobility, 59.5% (n = 358) reported no impairment in self-care, while 53.5% (n = 322) stated no problems in usual activities. Nevertheless, 50.3% (n = 303) of the patients reported moderate pain/discomfort and 63.1% (n = 380) patients were extremely anxious/depressed. The overall mean EQ-5D 3L visual analogue scale (EQ-VAS) score was 66.20 (SD: 17.22) . Of the 602 participants, more than half 56.0%,  of the respondents reported a complete healthy state  with no impairment in any domain, 86 (14.3%) indicated a moderate problem in the first four domains and severe impairment in the fifth domain , while 23 (3.8%) reported worst health state  with extreme impairment in all the five domains of EQ-5D-3L (Table % of the P < 0.001). One-way ANOVA test revealed that EQ-5D scores varied significantly by gender (P < 0.001), age (P < 0.001), marital status (P < 0.001), and level of education (P < 0.001). The findings indicated that asymptomatic HIV patients had significantly higher mean EQ-5D score (0.58  ±  0.37) than symptomatic HIV patients (0.26  ±  0.34) and AIDS-converted patients (−0.38  ±  0.22). In addition, HRQoL score significantly differed according to the participants’ HIV serostatus (P < 0.001), CD4 count (P < 0.001), viral load (P < 0.001), time to ART (P < 0.001), and the route of HIV infection (P < 0.001) , gender (P < 0.001), age (P < 0.001), marital status (P < 0.001), and level of education (P < 0.001). Furthermore, asymptomatic HIV patients had the highest mean VAS score (74.75  ±  15.27) compared to symptomatic HIV patients (59.15  ±  15.02) and AIDS-converted patients (45.10  ±  5.12). Additionally, patients with CD4 count > 500 cells/mm3 had significantly higher VAS score (86.46  ±  8.25) than patients with CD4 count 200–500 cells/mm3 (62.13  ±  12.06) and CD4 count < 200 cells/mm3 (52.21  ±  9.83). VAS scores also significantly differed by HIV serostatus (P < 0.001), viral load (P < 0.001), time to ART (P < 0.001), and route of HIV infection (P < 0.001) . The regression model results  revealed that factors significantly associated with HRQoL were: female vs. male ; age > 50 years vs. 18–25 years ; primary education vs. illiterate , secondary education vs. illiterate ; non employed vs. employed ; years with HIV 1–5 years vs. < 1 year , 6–10 years vs. < 1 year , > 10 years vs. < 1 year ; HIV serostatus symptomatic vs. asymptomatic , AIDS-converted vs. asymptomatic ; CD4 count 200–500 cells/mm3 vs. < 200 cells/mm3 , > 500 cells/mm3 vs. < 200 cells/mm3 ; viral load detectable > 20 copies/mL vs. non-detectable ; time on ART 12–48 months vs. < 12 months , > 48 months vs. < 12 months ; no idea vs. sex with a man , blood products vs. sex with a man .Standard multiple regression analysis was used to assess the ability of socio-demographic and clinical characteristics to predict HRQoL in PLWHA in Pakistan as indicated by EQ-5D index , low education, living with a partner/spouse, unemployed, being HIV symptomatic, AIDS-converted, lower CD 4 lymphocyte count, and detectable viral load were the factors that could predict poor HRQoL in our population and need special attention to improve HRQoL. Adherence to ART significantly improved HRQoL, except for psychological component, suggesting a need for training of healthcare professional caring for PLWHA on the screening and management of anxiety and depression among HIV patients. Psychoeducation should be conducted during initial evaluation to reduce negative beliefs regarding ART. The findings of the present study provide support for future work to design effective and holistic interventions that are culturally sensitive for Pakistan. Further studies on the determinants of health outcomes in this vulnerable population are also warranted.


Among pregnant women, the most frequent RAMs were K103N , M184V , Y181C/V/I , P225H , and K219N/E/Q/R . Protease and integrase inhibitor-RAMs were relatively few or absent. Based on the genotype susceptibility score distributions, 73%, 88%, and 14% of children, adolescents, and pregnant women, respectively, were not susceptible to all three drug components of the WHO preferred first-line regimens per 2018 guidelines. These findings suggest that routine HIVDR surveillance and access to better ART choices may improve treatment outcomes in Sierra Leone.Human immunodeficiency virus (HIV) drug resistance (HIVDR) is widespread in sub-Saharan Africa. Children and pregnant women are particularly vulnerable, and laboratory testing capacity remains limited. We, therefore, used a cross-sectional design and convenience sampling to characterize HIV subtypes and resistance-associated mutations (RAMs) in these groups in Sierra Leone. In total, 96 children , 47 adolescents , and 54 pregnant women  were enrolled. Median treatment durations were 36, 84, and 3 months, respectively, while the sequencing success rates were 45%, 70%, and 59%, respectively, among children, adolescents, and pregnant women. Overall, the predominant HIV-1 subtype was CRF02_AG , with minority variants constituting 12%. Among children and adolescents, the most common RAMs were M184V (76.6%,  According to the World Health Organization (WHO) 2019 report on HIV drug resistance (HIVDR), the emergence and spread of HIVDR in low- and middle-income countries (LMICs) was a major factor limiting the success of antiretroviral treatment (ART) programs in achieving the 90–90–90 global targets for 2020 [Sierra Leone is a low-income West African country of 7.4 million people. The HIV epidemic in the country is described as generalized, with an estimated national HIV prevalence of 1.5% in 2018, i.e., 70,000 people living with HIV, of whom 6600 were children aged 0–14 years . The NatDespite the welcome improvements in the areas of HIV care outlined above, barriers persist in other areas of the national AIDS response in Sierra Leone. For example, the UNAIDS 2019 report cited earlier observed that there was uneven progress across the 90-90-90 targets. In 2018, fewer than half (49%) of PLWH in Sierra Leone knew their HIV status, and 41% of HIV-diagnosed patients were receiving ART, while only 26% of those on treatment were virally suppressed, the latter being likely due to a combination of poor treatment adherence and high prevalence of HIVDR in this setting . In addin = 64) and an acquired HIVDR (ADR) prevalence >95% (n = 151) among adult HIV-infected individuals aged >18 years who were receiving routine HIV care at Connaught Hospital, the country’s main referral health facility in Freetown [In the first study characterizing HIVDR in Sierra Leone, we previously observed a pre-treatment HIVDR (PDR) prevalence of 37%  children aged 2–9 years inclusive, (2) adolescents aged 10–18 years inclusive, and (3) pregnant women aged >18 years. The children and adolescents were enrolled at the HIV Clinic at Ola during Children’s Hospital, while the pregnant women were recruited at the HIV and Antenatal Clinic at the Princess Christian Maternity Hospital located in Freetown, Sierra Leone. Both hospitals serve as the national referral health centers for obstetric and pediatric cases and are major teaching affiliates of the College of Medicine and Allied Health Sciences of the University of Sierra Leone. The children and pregnant women were not biologically related.Due to lack of HIVDR testing capability in Sierra Leone, none of the study participants had ever had baseline HIVDR testing at ART initiation; study participants were, therefore, selected consecutively using convenience sampling. At the time of study enrollment, the Sierra Leone National HIV Treatment Guidelines from 2017  recommended a dual nucleos(t)ide reverse transcriptase inhibitor (NRTI) backbone, i.e., tenofovir/lamivudine (TDF/3TC), abacavir/lamivudine (ABC/3TC), or zidovudine/lamivudine (AZT/3TC) for plus the non-nucleotide reverse transcriptase inhibitors (NNRTIs) efavirenz (EFV) or nevirapine (NVP) as the preferred first-line regimen for all age groups. The ritonavir-boosted protease inhibitors (PIs) lopinavir (LPV/r) or atazanavir (ATV/r) were recommended as preferred second-line treatment for adults, or as alternative first-line for children <3 years who were exposed to nevirapine for prevention of MTCT.The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Case Western Reserve University/University Hospitals Cleveland Medical Center (approved 16 July 2019) and the Sierra Leone Ethics Scientific and Research Committee (approved 28 February 2019). Written informed consent was obtained from participants aged ≥15 years, whereas parental or guardian consent was obtained for participants <15 years.® K2 EDTA, Waltham, MA, USA) and centrifuged at 2000 g for 10 min. Plasma was collected into cryogenic vials  and stored at −20 °C in Sierra Leone before transporting on dry ice (−80 °C) to the Galicia Sur Health Research Institute in Vigo, Spain, for molecular analysis. HIV reverse transcriptase (RT), protease (PR), and integrase (IN) were amplified from virus-derived plasma using an in-house Sanger sequencing protocol as previously described [http://hivdb.stanford.edu, accessed on 21 June 2020). For each patient, 3 to 5 mL of venous blood were collected into tubes (BD Vacutainerescribed . FASTA sThe genotypic susceptibility score (GSS), which estimates the number of HIV active drugs in a given combination ART regimen, was calculated as follows: (1) susceptible and potential low-level resistance were scored as 1, (2) low-level and intermediate resistance were pooled as intermediate and scored as 0.5, and high-level resistance was scored as 0. The GSS was assigned by summing the individual resistance scores for each first-line drug in the combination ART regimen as we previously described . p < 0.05 was considered statistically significant.Statistical analysis was performed using the Statistical Package for the Social Sciences software . Categorical variables were presented as frequency (percentages) and were compared using Pearson’s chi-squared test. Continuous variables were expressed as median  and compared using the non-parametric Mann–Whitney U-test or Kruskal–Wallis test, where appropriate. 3 and 544 (IQR 260–849) cells/mm3, respectively, with 100% of children and 93.6% of adolescents having acquired HIV through MTCT. All children and adolescents (100%) were ART-experienced, with a median treatment duration of 36 (IQR 12–60) months and 84 (IQR 60–108) months, respectively. The majority of children  and adolescents  were on first-line ART.A total of 197 PLWH were enrolled in the study, consisting of 96 children, 47 adolescents, and 54 pregnant women. For the pregnant women, the median age were 26 years (IQR 23–31) and the median CD4 count was 526 cells/mm3 (IQR 459–698). The majority  were ART-experienced with median ART duration of 3 months (range 1–96) and were receiving tenofovir/lamivudine/efavirenz, TDF/3TC/EFV . Over a quarter of the pregnant women  were new HIV diagnosis, with the majority of them  diagnosed during the second or third trimester of pregnancy. Plasma samples were analyzed from all 197 patients, and the HIV pol region was successfully sequenced in 108 patients, distributed as follows: 43 children, 33 adolescents, and 32 pregnant women. This yielded an overall sequencing success rate of 54.8% (108/197). The sequencing success rates by demographic groups were as follows: children, 44.8% (43/96); adolescents, 70.2% (33/47); and pregnant women, 59.3% (32/54).The majority of patients were infected with the HIV-1 subtype CRF02_AG , followed by subtypes G , A , and C .Genotypic resistance testing was performed on all 96 children (100%) and 47 adolescents (100%), which yielded 64 RT, 57 PR, and 67 IN sequences.Nearly 80% (51/64) of RT sequences from children and adolescents harbored RAMs to NRTIs and NNRTIs. n = 21/64), or in combinations of two , three , or five RAMs . On the other hand, 133 NNRTI RAMs were identified, either as a single RAM , or in combinations of two , or three  RAMs.A total of 117 NRTI RAMs were identified from these two groups and were distributed as follows: as single RAMs , K103N , Y181C/V/I , T215F/Y , V108I , A98G , G190A , K101E/H , M41L , L74I/V , and H221Y   a,b. n = 2), M46I/M (n = 1), and I84I/V (n = 1). The combination mutation I54V+V82A confers intermediate resistance to all the currently approved PIs with the exception of darunavir, which retains full activity. Interestingly, there was no documented record of prior exposure to PI-based ART in these 6 patients.We observed 4 PI-associated RAMs in 6 children/adolescents, as follows: the combination mutation I54V + V82A (n = 2), E157Q (n = 5), and S230SR (n = 1).Polymorphic accessory INSTI-selected mutations, which have minimal effect on INSTI susceptibility were observed in 8 children/adolescents, as follows: T97A ; and (2) 7 RT, 6 PR, and 9 IN from the newly diagnosed ART-naïve patients (n = 15). Genotypic resistance testing was performed on all 54 pregnant women (100%). A total of 34 RT, 27 PR, and 33 IN sequences were obtained, which were distributed as follows based on treatment status: (1) 27 RT, 21 PR, and 24 IN from ART-experienced patients (n = 26/34) of RT sequences harbored RAMs to NRTIs or NNRIs in pregnant women, which were distributed as follows: (1) ART-experienced , and (2) newly diagnosed ART-naïve . Overall, 76.5% (n = 1/39) or as a combination of two mutations ; and (2) 2 RAMs in the newly diagnosed ART-naïve, which occurred as a combination of two mutations . On the other hand, 18 NNRTI RAMs were identified, as follows: (1) 5 RAMs in the newly diagnosed ART-naïve patients, which occurred as a combination of two  or three mutations ; and (2) 13 RAMs in the ART-experienced patients, which occurred as a single mutation  or in combinations of two , three  or four mutations . A total of 9 NRTI RAMs were identified, as follows: (1) 7 RAMs in the ART-experienced group, which occurred as a single mutation , M184V , Y181C/V/I , P225H , K219N/E/Q/R , and K238T   a,b. n = 1), E157Q (n = 2), and G163R (n = 1), which have minimal effect on INSTI susceptibility, were observed in four pregnant women.No PIs RAMs were observed, whereas three accessory INSTI-selected mutations T97A  and NNRTIs (nearly 50%) in the three demographic groups, whereas the prevalence of RAMs to PIs and INSTIs was relatively low or absent. Our findings confirm the situation previously described in the WHO 2019 HIVDR report ,16,17,18Current international ART treatment guidelines recommend a dual NRTI-backbone in combination with an INSTI or a boosted-PI with a high genetic barrier to developing resistance such as darunavir/ritonavir (DRV/r) in the treatment of HIV-infected individuals ,19,20,21Despite its high potency, good tolerability, and high genetic barrier to developing drug resistance, safety concerns persist around the peri-conception use of DTG in women of child-bearing age and during the first trimester of pregnancy, due to the reported higher incidence of neural tube defects ,26,27. AWe found that M184V was the most frequent NRTI RAM, occurring with a prevalence of nearly 77% among children/adolescents and 11% of pregnant women. This mutation is selected by 3TC and emtricitabine (FTC), which significantly reduces their activity  while conferring low-level resistance to ABC ,37,38. IThe marked disparity in frequency of M184V-selected viruses among children and adolescents (77%) and pregnant women (11%) was noteworthy. Studies have shown that M184V is associated with reduced replicative activity in the absence of drug pressure, compared with the wild-type (WT) virus ,43,44. TThe prevalence of the NNRTI mutation K103N, which is selected by EFV and NVP was high in all three study groups. This finding is in line with reports from the SSA, where the frequency of K103 has exceeded 10% in most LMICs reporting HIVDR surveillance data to the WHO . There aOur study had several limitations. Firstly, our study enrolled a small sample size from a single clinical site, which limits the generalizability of study findings. Secondly, although the issues of pretreatment (PDR) and/or transmitted drug HIV drug resistance (TDR) remain important barriers to reducing vertical HIV transmission, we were unable to assess this fully due to the relatively small number of ART-naïve pregnant study population. Thirdly, due to insufficient plasma samples, VL measurement could not be performed for all patients. Finally, because the majority of the patients were on ART for a long period of time , we suspect that a substantial proportion of these patients were either virologically suppressed or had low viremia, which may have contributed to the low rate of sequencing success, especially among children. Nonetheless, our findings contribute to our current understanding of circulating HIV strains and HIVDR among three vulnerable populations that are disproportionately affected by the growing HIV epidemic and may have some policy implications for PMTCT program activities. Larger studies are needed to assess the countrywide HIVDR prevalence rates and associated factors to help inform treatment strategies and public health approaches in the national AIDS response in Sierra Leone.We observed a high prevalence of circulating RAMs to NRTIs  and NNRTIs (EFV and NVP) among HIV-infected children, adolescents, and pregnant women in Freetown, Sierra Leone. In contrast, the prevalence of RAMs to PIs and INSTI was low or absent. Although the three populations were not biologically related, our findings may have implications for the success of PMTCT efforts in the country. Furthermore, our findings suggest the need for a national HIVDR surveillance program to help guide ART choice and improve treatment outcomes for patients.


People living with human immunodeficiency virus (HIV) with immuno-virological discordant responses are at an increased risk to develop acquired immunodeficiency syndrome (AIDS) and severe non AIDS events which are risk factors for death. This study was aimed to assess prevalence of immuno- virological discordant responses and associated risk factors among highly active antiretroviral therapy (HAART) users in Tigray, Northern Ethiopia.+ count and viral load measurement were collected during data collection period. Fisher’s exact test, bivariate and multivariate logistic regressions were used for data analysis. P-value < 0.05 with 95% CI was considered as statistically significant.A cross sectional study was conducted from September to December 30, 2016 on 260 people living with HIV who started first line HAART from January 2008 to March 2016 at Mekelle hospital and Ayder comprehensive specialized hospital. Baseline and follow-up clinical data and CD4+ result were collected from patient charts. Besides, socio-demographic data and blood samples for CD4 p = 0.007)), male gender ((Adjusted Odds Ratio (AOR) (95% CI) =1.71(1.13–1.10), p = 0.029)), type of regimen given ((AOR(95% CI) = 0.30 (0.10–0.88), p = 0.028)) and good treatment adherence ((AOR (95% CI) = 0.12 (0.030–0.0.48), p = 0.003)) were associated risk factors for virological discordant response. Likewise, immunological discordant response was associated with tuberculosis co-infections (p = 0.016), hepatitis B virus co-infections (p = 0.05) and low CD4+ count (≤100 cells/μl) at baseline (p = 0.026).Among the 260 study participants, 8.80%  =8.77–8.84%) and 2.70% (95% CI = 2.68–2.72%) had virological and immunological discordant responses, respectively with an overall immuno-virological discordance response of 11.50% (95% CI = 11.46–11.54%). The median age of the study participants at HAART initiation was 35 (IQR: 28–44 years). More than half (58.1%) of the study participants were females. Age at or below 35 years old at HAART initiation (AOR ((95% CI) = 4.25(1.48–12.23), Over all, immuno-virological discordance response was 11.5% in the study area. Males, low baseline CD4+ count, poor/fair treatment adherence, and TB and HBV co-infections were significantly associated with higher immuno-virological discordance. We recommend that decision of patient treatment outcome, regimen change and patient management response should be done using trends of both viral load and CD4+ count concurrently.The online version contains supplementary material available at 10.1186/s12879-021-06206-4. The use of highly active antiretroviral therapy (HAART) has substantially improved the survival of people living with human immunodeficiency virus (HIV) by suppressing the viral load to undetectable levels and providing a consistent increase in the number of T lymphocytes both of which lead to slow progression of the infection towards AIDS –3.Although, the goal of HAART is to suppress viral load to undetectable level and restore CD4+ count , 6.Studies on immuno-virological discordant responses among people living with HIV have reported from 29 to 33% , 7. ImmuIt is reported that people living with HIV with discordant responses were found to be at an increased risk to develop AIDS events such as opportunistic infections and severe non AIDS events such as stroke, liver and kidney failure, meningitis and endocarditis; risk factors for death , 16, 17.n = 86) and did not address factors associated with immunological and virological discordant responses. Moreover, this study cannot represent the national level prevalence of immunological and virological discordant. The limitations of the previous study calls for more data to be generated from each region with representative sample to forward reasonable findings and recommendations to help policy makers and implementers in order to plan and design proper intervention strategies to control HIV. The aim of this study was therefore, to fill the existing knowledge gap on the prevalence of immunological and virological discordant responses and associated predictors among people living with HIV on first line HAART in Tigray regional state, Northern Ethiopia.While immuno-virological response plays a key role to address treatment outcome, regimen change and management for people living with HIV using HAART, there is scant data in Ethiopia. To the best of our knowledge, there is a single study in Southern region of Ethiopia . HoweverThe study was conducted from September 1 to December 30, 2016 in Mekelle Hospital (MH) and Ayder comprehensive specialized hospital (ACSH), both located in Mekelle city. Mekelle, the capital city of Tigray Regional Sate is located 783 Km north of Addis Ababa, the capital city of Ethiopia. Of the five governmental hospitals found in Mekelle city, two hospitals namely MH and ACSH were selected purposely owing to their high patient flow. In 2016, around 8727 people were living with HIV (PLHIV) ever enrolled in MH, of these, 6789 were ever started HAART, and 4189 were actively on HAART. Similarly, in ACSH there were 1564 PLHIV ever enrolled of these, 1387 patients ever started with 1222 were actively on HAART.A cross sectional study was conducted to determine the immunological and virological discordant responses among people living with HIV who were taking first line HAART. The study participants were selected consecutively using convenient sampling technique.The study participants were patients 10 to 64 years of age who were on first line HAART for 7 months to 9 years. Patients who were less than 6 months on HAART follow up, who did not have current CD4+ count, who had been on second line regimen, lost to follow up, febrile, and transfer in individuals were excluded from the study. Of the total 441 people living with HIV who visited the hospitals during the study period, 154 were excluded as they did not fulfil the inclusion criteria, and 287 were included in the study  and laboratory data .All people living with HIV who was on first line HAART for at least 6 months and visiting these health facilities during data collection period were included consecutively. All participants were requested for their willingness and included after written informed consent, and for minors consent from parents/guardians and assent from children. Baseline and follow-up clinical related data such as WHO clinical stage, type of regimen given, presence or absence of opportunistic infections, presence of chronic non-communicable diseases, nutritional status, HBV and HCV co-infections, any prophylaxis given, time interval from HIV diagnosis to HAART initiation), age at HAART initiation, and baseline and current (i.e. performed 3–4 months before data collection period) CD4+ count were collected from charts. In addition to recording socio demographic characteristics , blood samples for CD4+ count and viral load measurements were collected during the data collection time.Seven milliliter of blood was collected from each participant for CD4 + count and viral load test using vacutainer and poured in to two EDTA test tubes during data collection period. A test tube containing 3 mL of blood stored at room temperature was sent to ACSH ART laboratory for CD4+ count. The second test tube containing 4 mL of blood was centrifuged at 1600 rpm for 20 min using Sigma centrifuge  in ACSH and the separated plasma was transported to Tigray Health Research Institute (THRI) using dry ice box and stored at − 80 °C until analyzed for viral load measurement.m2000sp automated sample preparation system  in THRI according to manufacturer’s instructions during data collection period (at 36 months median time of HAART follow -up). Extracted RNAs were measured using Abbott m2000rt quantitative Real Time HIV-1 assay  with HIV-1 RNA detection level of 150 to 10 million copies/mL based on the manufacturer’s procedures.HIV-1 RNA was extracted from 0.2 mL of plasma using Abbott Baseline and current CD4+ count was collected from medical records. In addition, we collected blood samples from each participant and did last CD4+ count (to have persistent CD4+ count) and done at ACSH ART laboratory using the BD FACS Count™  following the manufacturer’s protocol during data collection period (at 36 months median time of HAART follow up). Then, immunological failure was determined using the current and last (latest) CD4+ count.> 1000 copies/ml after at least 6 months of HAART initiation [Virological failure: is defined as plasma viral load itiation .Viral suppression: Viral load level below < 150 copies/ml after at least 6 months of HAART initiation .Immunological failure: defined as CD4+ count falls at or below250 cells/μl following clinical failure or persistent levels below100 cells/μl after 6 months of HAART initiation .Clinical Failure: New or recurrent clinical event indicating severe immunodeficiency  after 6 months of HAART initiation .Immunological discordant response: defined as plasma viral load level below 1000 copies/mL with immunological failure.>1000copies/mL).Virological discordant responses: defined as good immunological response with virological failure , “fair adherence” , and “poor adherence”  based on self-report and/or pill count .p-value < 0.05 with 95% confidence interval.To maintain quality, sample collection, handling, transportation, and laboratory work was done using the standard operational procedures. Moreover, the expiry date of reagents, completeness, and consistency of filled questionnaires was checked. We used Epidata version 3.1 to enter data and SPSS version 21 for statistical analyses. Median and interquartile ranges (IQR) were used to analyze continuous variables and counts with percentages for categorical variables. Fischer’s exact test was used to see statistical association of the variables with immunological discordant responses, whereas, binary and multiple logistic regressions were used to determine the associations of independent variables with virological discordant responses. Statistically significant was set at a n = 9), invalid viral load result (n = 15) and insufficient samples (n = 4). Hence we only included the data of 260 participants in the data analysis. From these, more than half (58.1%) of them were females. The median age at HAART initiation and at 36 months median time of HAART follow up was 35 (Interquartile range (IQR): 28–44) and 39 (IQR: 32–48), years, respectively. Similarly, median CD4+ count at 36 months median time of HAART follow-up was 384 (IQR: 239–572) cells/mm3. About 63% of the total participants had history of opportunistic infections at least once at baseline/follow up and 51% of them took isoniazid (INH) prophylaxis. Majority (74%) of them started HAART at 11 mean time interval (standard deviation (SD): + 19) of HIV diagnosis. Of the total 239 patients, who took Cotrimoxazole prophylaxis, 48% of them used for a median time of 30 months (IQR): 18–48)  (data not shown). Study participants with no formal education were 4.47 times more likely to experience immuno-virological discordant responses ((AOR (95% CI) = 4.47) compared to these who attended secondary school and above. In addition, immuno-virological discordant responses were 80% less likely to occur in study participants who had viral load less than1000 copies/mL in a 36 median time of HAART initiation (AOR(95% CI) = 0.20 (0.05–0.86), p < 0.001). Immuno-virological discordant response were also statistically associated with HBV (p = 0.037) and HCV (p = 0.027) co-infections ) patients had immuno-virological discordant responses. Among the discordant responders, 23/30 (76.7%) of them who had virological failure were immunologically recovered (p > 0.05), and all were on TDF based regimen. More than half (57.1%) with immunologically discordant responses had history of TB co- infections (p = 0.016). At baseline, all of the patients were at WHO clinical stage III/IV and 71.4% of them had CD4+ count less than 100 cells/μl (p = 0.026) (data not shown).Immunological discordance was found in 2.70% (95% CI = 2.68–2.72%) of the study participants. Though not statistical significant, about 57.1 and 71.4% of the immunologically discordant study participants were females and greater than 35 years age at HAART initiation, respectively (p = 0.05). In addition, 11.1% of the study participants with poor/fair treatment adherence experienced immunological discordant responses compare to their counter parts (p = 0.08)  (data not shown).Virological discordant responses were seen in 8.80% (95% CI = 8.77–8.84%) of the study participants. Among these virologically discordant responders, 69.6% (16/23) and 56.5% (13/23) of them were males and aged less than or equal to 35 years old at HAART initiation, respectively. Furthermore, 69.6% (vs. 48.1%) of them had CD4+ count less than 384 cell/μl at 36 median time of HAART initiation. The majority (86.9%) with good virological responses were with good treatment adherence. However, 38.9% (vs 6.6%) of these with poor/ fair treatment adherence had virological discordant responses (p = 0.029) compared to females. Patients whose, HAART initiating at less than or equal to 35 years of age) were 4.25 times more likely to experience virological discordance ((AOR (95% CI) = 4.25(1.48–12.23), p = 0.007). Furthermore, the odds of virological discordant response was 70% less likely to occur among individuals who took TDF based regimen ((AOR (95% CI) =0.30 (0.10–0.88), p = 0.028) compared with these on AZT based. Poor/fair treatment adherence was also predictor for virological discordant responses ((AOR (95% CI) =0.12 (0.03–0.48), p = 0.003)  =1.711.13–1.10), –1.10, p > 1000 copies/mL) with immunological success [This study described immunological and virological discordant responses and associated risk factors among persons on HAART at 36 months median time of HAART follow up. Generally, 11.5% of the study participants experienced immuno-virological discordant responses. Our finding were comparable with a study conducted in Brazil, 9% . However success . However success .3 at 12 months compared to baseline in spite of full virological suppression (VL < 40 copies/mL) [In median time of 36 month of HAART follow up, immunological discordant response was found 2.70%. Our result was much lower than the studies conducted in Southern Ethiopia, 15.1–20.9% , Oman, 2pies/mL)  and CD4+pies/mL) , 12.p = 0.016). This finding is also in line with a study conducted in South Korea [M. tuberculosis induced apoptosis of CD4+ cells which subsequently lead to depletion of CD4+ cells and results in poor immunological recovery despite viral suppression [TB co-infection, HBV co-infection and CD4+ count below 100 cells/μl at baseline were factors associated with immunological discordant responses. Multicenter cohort study conducted in Germany indicated that individuals who developed TB had slow immune recovery despite viral suppression as compared with these mono-infected individuals . Our stuth Korea . This mipression .p = 0.05) compared to non HBV co-infected individuals, 2.8%. This finding in lines with a study conducted in Switzerland [It was statistically significant that 18.2%of HBV co-infected individuals had immunological discordant response (tzerland  and Chintzerland . These sp = 0.026). Association of baseline CD4+ count with immunological discordance was also reported by studies conducted in Nigeria [Immunological discordant responses was also associated with low CD4+ count (< 100 cells/μl) at baseline . In other hand, impact of types of regimen on immunological discordance was also reported by a study conducted in Italy [Furthermore, the current study indicated that all those immunological discordant responses were on TDF based regimen. Though not statistically significant, other study from Oman  also indin Italy .Virological discordant responses in this study were found 8.8% at 36 months median time of HAART follow up. Our finding was comparable to other studies conducted in Southern Ethiopia, 9.3–13.9% , Rwanda,p = 0.029) compared to females. This was in line with the study conducted in Rwanda which reported lower odds of virological discordant responses among females as compared with males [In this study, virological discordance was 1.71 times more likely to occur in males  compared to initiating above 35 years old. This current finding was also supported by the study conducted in Nigeria, where the odds of virological discordant responses was 1.58 times more likely to occur (p = 0.08) in these who initiated HAART at younger age (< 35 years old) [Another variable which was found as an independent predicator of virological discordance was age at initiation of HAART. Accordingly, in a multiple logistic regressions, initiation of HAART at age of less than or equal to 35 years old was 4.25 times more to experience virological discordance p = 0.00 comparedp = 0.028) compared with study participants on AZT based. This finding was also consistent with the study conducted in Nigeria where 89% were less likely to have virological discordant even though significant difference not observed [Our study also indicated that study participants on TDF based regimens were 70% less likely to experience virological discordant responses (p = 0.003) compared with these with poor/fair treatment adherence. This was also supported from the study conducted in Nigeria (p = 0.001) [Furthermore, it was statistically significant that odds of virological discordant responses was 88% less likely to occur among study participants with good treatment adherence (= 0.001)  which in> 1000 copies/mL with immunological recovery. Second, we couldn’t do prospective cohort study to see the cause and effect of the associated factors with immuno-virological discordance response.The strength of this study was that this is the first study in the region which can give information on immuno-virological discordance response for the regional and national government and other stakeholders. However, our study had limitation. First due to budget constraints we did not perform HIV drug resistance test for these who had viral load level Over all, immuno-virological discordance response was found in 11.5% in the study population at 36 months median time of HAART follow up. TB co-infection, HBV co-infection, low CD4+ count at baseline and WHO clinical stages III/IV were associated factors for immunological discordant responses. Whereas, virological discordant response was associated with age at HAART initiation, type of regimen given, male gender and poor/fair treatment adherence. Therefore, due attention should be given to the identified factors. Moreover, given evaluation of immuno-virological response is important to address patient treatment outcome, regimen change and patient management, we recommend that decision of patient treatment response should be done using trends of both viral load and CD4+ count concurrently.Additional file 1: Supplement Table 1. Immuno-virological discordance responses & associated risk factors among HAART users in Mekelle Hospital & Ayder comprehensive specialized hospital (n=260).