Accurate routine HIV viral load testing is essential for assessing the efficacy of antiretroviral treatment (ART) regimens and the emergence of drug resistance. While the use of plasma specimens is the standard for viral load testing, its use is restricted by the limited ambient temperature stability of viral load biomarkers in whole blood and plasma during storage and transportation and the limited cold chain available between many health care facilities in resource-limited settings. Alternative specimen types and technologies, such as dried blood spots, may address these issues and increase access to viral load testing; however, their technical performance is unclear. To address this, we conducted a meta-analysis comparing viral load results from paired dried blood spot and plasma specimens analyzed with commonly used viral load testing technologies.Standard databases, conferences, and gray literature were searched in 2013 and 2018. Nearly all studies identified (60) were conducted between 2007 and 2018. Data from 40 of the 60 studies were included in the meta-analysis, which accounted for a total of 10,871 paired dried blood spot:plasma data points. We used random effects models to determine the bias, accuracy, precision, and misclassification for each viral load technology and to account for between-study variation. Dried blood spot specimens produced consistently higher mean viral loads across all technologies when compared to plasma specimens. However, when used to identify treatment failure, each technology compared best to plasma at a threshold of 1,000 copies/ml, the present World Health Organization recommended treatment failure threshold. Some heterogeneity existed between technologies; however, 5 technologies had a sensitivity greater than 95%. Furthermore, 5 technologies had a specificity greater than 85% yet 2 technologies had a specificity less than 60% using a treatment failure threshold of 1,000 copies/ml. The study’s main limitation was the direct applicability of findings as nearly all studies to date used dried blood spot samples prepared in laboratories using precision pipetting that resulted in consistent input volumes.This analysis provides evidence to support the implementation and scale-up of dried blood spot specimens for viral load testing using the same 1,000 copies/ml treatment failure threshold as used with plasma specimens. This may support improved access to viral load testing in resource-limited settings lacking the required infrastructure and cold chain storage for testing with plasma specimens. Lara Vojnov and co-workers report on the use of dried blood spots for HIV viral load testing. Though the preferred method to monitor patients on antiretroviral treatment (ART), there remains limited access to viral load testing, particularly in resource-limited settings suffering from infrastructural challenges.Dried blood spots can be an alternative sample type for viral load testing in resource-limited settings as storage requirements are do not require cold chain or rapid transportation to the laboratory.Several viral load technologies exist with regulatory approvals for use with plasma samples; however, a significant knowledge gap exists in the accuracy of using dried blood spot samples for HIV viral load testing.We conducted a systematic review and meta-analysis and identified 60 studies that fulfilled the inclusion criteria and included data on 6 technologies.Though some heterogeneity was observed in the systematic review, the meta-analysis including 10,871 data points from 40 studies highlighted clear clinical performance metrics for each technology. Four of the 6 technologies had a sensitivity and specificity above 83% using dried blood spot samples for viral load testing with a treatment failure threshold of 1,000 copies/ml.Most studies used dried blood spots prepared in the laboratory with precision pipetting resulting in consistent measurement of blood applied to each spot. Additional studies would be useful to ensure the results observed can be replicated and remain consistent in the field when utilizing other sample preparation techniques.Dried blood spot samples can be used for viral load testing at a treatment failure threshold similar to plasma samples at 1,000 copies/ml.Performance of dried blood spot samples is not perfect; therefore, some misclassification will occur. A small proportion of patients successfully undetectable will be incorrectly considered failing, while a small proportion of patients failing treatment will be considered successfully treated. Furthermore, selection and implementation of technologies for use with dried blood spots may be complicated with heterogenous performance and regulatory approvals.The availability and use of alternative sample types, such as dried blood spots, that require less stringent and robust infrastructure may allow for significant expansion of access to viral load testing in resource-limited settings. Although an estimated nearly 37 million HIV–positive people are currently eligible for antiretroviral treatment (ART) according to the 2016 WHO Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection, only 19.5 million people are on ART worldwide .–69.19–69There was some risk of bias in patient selection, however, low risk of bias with the reference standard and index test . ParticiMean bias was the most commonly reported analytical measurement across all studies included in the systematic review (82%); therefore, forest plots of each study were developed by technology . Half ofme HIV-1 technology [A total of 40 studies provided 45 data sets across the 6 technologies resulting in a total of 10,871 paired dried blood spot and plasma viral load results –66,68,69chnology ,59,63,69chnology ,53,62, 5chnology ,52,64, 1chnology . Approxichnology ,74, of wchnology ,56,59,64chnology ,48,65,69The viral load distribution for the 10,871 plasma specimens tested was relatively equally distributed across all viral load ranges . While ame HIV-1 two-spot, Abbott RealTime HIV-1 one-spot, Biocentric Generic HIV Charge Virale, bioMérieux NucliSENS EasyQ HIV-1, Hologic Aptima, Roche COBAS TaqMan FVE, Roche COBAS TaqMan SPEX, and Siemens VERSANT HIV-1 RNA technologies had a difference between the median dried blood spot and plasma specimen viral loads of 0.09, 0.04, 0.17, −0.30, 0.12, 0.33, 1.99, and −0.13 log copies/ml, respectively. The mean bias for each technology was calculated by pooling all primary data for each technology as though one study (me HIV-1 two-spot (−0.12 log copies/ml), Abbott RealTime HIV-1 one-spot (0.02 log copies/ml), and Roche COBAS TaqMan FVE (0.06 log copies/ml) assay biases were closest to zero, while the bioMerieux NucliSENS EasyQ HIV-1 (−0.41 log copies/ml) and Roche COBAS TaqMan SPEX (1.03 log copies/ml) assay biases were furthest from zero. The Abbott RealTime HIV-1 two-spot, bioMerieux NucliSENS EasyQ HIV-1, and Siemens VERSANT HIV-1 RNA technologies had negative mean biases indicating under-quantification compared to the plasma viral load result, which is expected due to the lower input sample volume. The positive mean biases of Biocentric Generic HIV Charge Virale and Roche COBAS TaqMan SPEX reflect over-quantification compared to the plasma viral load result, likely due to processing and extraction chemistries resulting in amplification of total intracellular and extracellular nucleic acids.The median dried blood spot viral loads were higher than the median plasma viral loads for all but 2 technologies. Overall, the median difference was 1.03 log copies/ml . The Abbne study . The meaWHO and many national clinical guidelines in resource-limited settings recommend using viral load testing as a binary result, above or below a specific threshold to identify treatment failure. We, therefore, compared several treatment failure thresholds for dried blood spot specimens  to the currently suggested 1,000 copies/ml threshold for plasma specimens for correctly classifying patients . Using ame HIV-1 two-spot and Roche COBAS TaqMan FVE protocols had high sensitivities and specificities at all lower thresholds except detectable. The new Abbott RealTime HIV-1 one-spot protocol, however, had high specificities at all lower thresholds, but sensitivity performance was below 85% at the 200 copies/ml and detectable thresholds. The Hologic Aptima had high sensitivities with all thresholds except detectable; however, specificity was lower than 85% at the thresholds of 800 copies/ml and 200 copies/ml. Finally, the bioMérieux NucliSENS EasyQ HIV-1 had sensitivities and specificities greater than 85% at all thresholds.Additionally, to better understand the performance of dried blood spot specimens at lower treatment failure thresholds , we compared the 6 predefined treatment treatment failure thresholds—detectable, 200, 400, 500, 600, and 800 copies/ml—between dried blood spot specimens and plasma for each technology and protocol . The Biome HIV-1 two-spot, Abbott RealTime HIV-1 one-spot, Biocentric Generic HIV Charge Virale, bioMérieux NucliSENS EasyQ HIV-1, Hologic Aptima, Roche COBAS TaqMan FVE, Roche COBAS TaqMan SPEX, and Siemens VERSANT HIV-1 RNA technologies, 59.28%, 68.71%, 38.04%, 52.54%, 50.40%, 62.03%, 33.45%, 47.22% of dried blood spot specimen test results were within the standard deviation of +/−0.3 log copies/ml of the paired plasma test result, respectively , the specificity was reduced compared with venous blood specimens  for dried blood spot specimen viral load testing will result in higher levels of misclassification of treatment failure with most viral load testing technologies.Dried blood spot specimens may increase access to HIV viral load testing for virological monitoring of ART patients in resource-limited settings. This study analyzed the technical performance of laboratory-based viral load technologies for accurate quantification of viral load using dried blood spot specimens compared to plasma specimens. While the performance varied between technologies, the most commonly used viral load technologies, Abbott RealTime HIV-1 one-spot, bioMérieux NucliSENS EasyQ HIV-1, and Hologic Aptima assays using a treatment failure threshold of 1,000 copies/ml. Increasing the treatment failure threshold further reduces the sensitivities of all assays. This performance indicated that most patients with an elevated viral load would be identified by all technologies; however, it is important to note that some patients failing treatment will be missed and only retested a year later when using technologies with lower sensitivity. Additionally, 5 technologies had a specificity above 85% using the treatment failure threshold of 1,000 copies/ml. Two technologies, Biocentric Generic HIV Charge Virale and Roche COBAS TaqMan using the SPEX protocol had low specificities of approximately 55% and 44%, respectively, using a treatment failure threshold of 1,000 copies/ml. This performance would be concerning as significant proportions of patients suppressing their viral load would be incorrectly identified as failing treatment; however, additional viral load measurements are required prior treatment switching and thus this misclassification could be resolved. Though some variability was observed with the Roche COBAS TaqMan using the FVE protocol in the meta-analysis, recent studies have shown consistent performance with sensitivity and specificity greater than 90% [The sensitivity for 5 technologies were above 90%, however, between 80% to 90% for the Abbott RealTithan 90% ,60. FurtFurthermore, though technologies have variable performance when using dried blood spot specimens at lower treatment failure thresholds, several technologies and protocols can be considered if lower treatment failure thresholds are required. While the lower limits of detection of dried blood spot specimens tend to be higher than plasma due to the smaller specimen volume that clearly does not preclude the technologies or protocols from correctly classifying patients as failing or not failing treatment, the most critical clinical measurement used. Therefore, limits of detection should not be the primary metric for consideration when reviewing technology performance or selecting one for routine use. Prior to changing guidelines and putting in the significant work to implement such an algorithm change, it is important to consider a range of factors, including the prevalence and clinical relevance of low-level viremia, especially in relation to implementation and scale-up of optimized antiretroviral drugs such as dolutegravir, access to testing, result utilization, and other scale-up priorities to ensure that patients have access to high-quality testing and necessary follow-up clinical care.The sensitivity analyses performed in this study suggest that low adherence to manufacturer-recommended dried blood spot specimen collection and extraction protocols, study geographies, and technology version used did not contribute significantly to any observed differences in dried blood spot and plasma specimen test results. Though the sensitivity analysis including only data generated through compliance with the manufacturers’ recommended protocols did not improve the performance of dried blood spot specimens for viral load testing, manufacturer protocols should be precisely followed for optimal technology performance. Performance variability between test platforms is likely due to different dried blood spot nucleic acid extraction methods and viral load test chemistries employed by each platform. This was observed with the standard Roche COBAS TaqMan SPEX and FVE specimen extraction protocols; the latter protocol was associated with much higher levels of specificity when compared with paired plasma specimen testing. Similarly, the lower specificity of the Biocentric Generic HIV Charge Virale test used in the studies analyzed may reflect a need to optimize specimen extraction for dried blood spot specimens.Viral load testing is known to be inherently variable within limits ,77; howeThis systematic review and meta-analysis had some limitations. Almost all studies in this systematic review and meta-analysis used dried blood spots prepared in laboratories using precision pipetting for consistent measurement of input specimen volumes. Hence, the findings may not reflect performance in the field with less precise specimen collection procedures such as allowing the blood drops to drip from the patient’s finger or heel directly onto filter cards as practiced for HIV early infant diagnosis dried blood spot specimen collection ,54,57,59The sample sizes for each sub-analysis were small; therefore, further studies are warranted to better understand the performance of dried blood spot specimens under these conditions. Abbott has recently changed their dried blood spot protocol to only use 1 dried blood spot and a different dried blood spot extraction buffer. Data on the new protocol are very limited; however, 2 studies reviewed the performance and found sensitivity and specificity of greater than 90% ,59. Whilme HIV-1 one-spot and bioMérieux NucliSENS EasyQ HIV-1 have CE-IVD approval and/or WHO prequalification listing. It is critical, however, for manufacturers to seek regulatory approval to provide better support to countries in need of this additional sample type to further expand access.While this systematic review and meta-analysis informs the use of dried blood spot specimens for viral load testing , it is iThis study raises an important issue of understanding appropriate and acceptable levels of clinical misclassification for current treatment failure thresholds. The acceptable level of misclassification with viral load tests is, however, unclear and further investigation may be warranted to guide technology development and selection.The gold standard biomarker for viral load testing is circulating HIV RNA in plasma ,11,13,80The meta-analysis presented here offers advantages over previous systematic reviews –83, whicme HIV-1 two- and one-spot, bioMérieux NucliSENS EasyQ HIV-1, Roche COBAS TaqMan using the FVE protocol, and Siemens VERSANT HIV-1 RNA technologies can all be considered for use with dried blood spot specimens when prepared using precision pipets or measured micro-capillary EDTA tubes with a treatment failure threshold of 1,000 copies/ml. It is expected that increased interest in and implementation of viral load testing using dried blood spot specimens may encourage manufacturers to acquire regulatory approval for this specimen type for in vitro diagnostic use, while new technologies such as plasma separation cards may further support the testing landscape.In conclusion, though variability was seen between technologies included, several technologies performed best using a treatment failure threshold of 1,000 copies/ml. The Abbott RealTiS1 PRISMA Checklist(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigCircle sizes were relative to the study sample size. The red square was the pooled summary point.(TIF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Isoniazid preventive therapy (IPT) can reduce the risk of tuberculosis (TB) in children living with HIV (CLHIV), but data on the outcomes of the IPT cascade in CLHIV are limited.We evaluated the IPT cascade among CLHIV aged <15 years and newly enrolled in HIV care in eight HIV clinics in western Kenya. Medical record data were abstracted from September 2015 through July 2019. We assessed the proportion of CLHIV completing TB symptom screening, IPT eligibility assessment, IPT initiation and completion. TB incidence rate was calculated stratified by IPT initiation and completion status. Risk factors for IPT non‐initiation and non‐completion were assessed using Poisson regression with generalized linear models.n = 841) underwent screening for TB symptoms and IPT eligibility. Of these, 13  were ineligible due to active TB and 828  were eligible. Five hundred and fifty‐nine  of eligible CLHIV initiated IPT; median time to IPT initiation was 3.6 months (interquartile range [IQR] 0.5–10.2). Overall, 434  IPT initiators completed. Attending high‐volume HIV clinics  was independently associated with IPT non‐initiation. IPT non‐initiation had a trend of being higher among those enrolled in the period 2017–2019 versus 2015–2016  and those who were HIV virally non‐suppressed . Being enrolled in 2017–2019 versus 2015–2016  was independently associated with IPT non‐completion. By 24 months after IPT screening, TB incidence was four‐fold higher among eligible CLHIV who never initiated (8.1 per 1000 person years [PY]) compared to CLHIV who completed IPT , with a similar trend among CLHIV who initiated but did not complete IPT .Overall, 856 CLHIV were newly enrolled in HIV care, of whom 98% ([95% CI 97–99]; Despite high screening for eligibility, timely IPT initiation and completion were suboptimal among eligible CLHIV in this programmatic cohort. Targeted programmatic interventions are needed to address these drop‐offs from the IPT cascade by ensuring timely IPT initiation after ruling out active TB and enhancing completion of the 6‐month course to reduce TB in CLHIV. The impAlthough the Kenya Ministry of Health introduced IPT among CLHIV in 2014, rates of initiation remained low until September 2015 when a massive rollout began . Data arWe conducted this analysis among CLHIV who were newly enrolled in high‐ and moderate‐volume HIV clinics in Kisumu County, western Kenya to (1) determine the proportion of CLHIV completing key steps of the IPT cascade; (2) identify risk factors for IPT non‐initiation and non‐completion; and (3) compare the risk of TB among CLHIV who completed IPT versus those who did not.22.1The annual TB case notification rate in Kisumu County was 209 per 100,000 population in 2019; 52% of the notified cases were co‐infected with HIV . HIV pre2.2This was a retrospective cohort study based on a review of routinely collected data abstracted from the paper‐based registers and electronic medical records of CLHIV aged 12 months to <15 years who were newly enrolled in HIV care at the participating health facilities (including those who were transferred in from other facilities) from September 2015 through July 2019. Data abstraction occurred from November 2019 through November 2020. The data abstraction tool captured the following information: baseline demographic and clinical characteristics . IPT initiation and completion statuses were assessed based on documentation by attending clinicians in the medical records and IPT cards.2.3p‐value of <0.10 on bivariate analysis were included in multivariate analysis. The most recent viral load results before IPT initiation (within 6 months) were included in the analysis. For IPT non‐initiators, viral load results obtained within 6 months of the most recent IPT eligibility evaluation were used. CLHIV with a viral load of less than 1000 copies per millilitre was considered virally suppressed. HIV clinics were categorized into moderate‐volume (<5000 active clients) and high‐volume (≥5000 active clients). Factors with a p‐value less than 0.05 were considered statistically significant in the multivariable analysis.We assessed the cascade estimating proportions completing eligibility screening out of identified CLHIV, IPT initiation out of those who were eligible and IPT completion out of those who initiated IPT with 95% confidence intervals. For this analysis, CLHIV with a negative TB symptom screen, or if symptomatic, had active TB ruled out after further evaluation were considered to be eligible for IPT. Bivariate and multivariable analysis was conducted to determine the risk factors for IPT non‐initiation among eligible children and non‐completion among those who initiated using a modified Poisson regression. We considered both patient‐level and facility‐level risk factors. Factors with a We compared TB incidence rates among three groups of CLHIV: (1) those who were eligible but never initiated IPT (IPT non‐initiators), (2) those who initiated but never completed IPT (IPT non‐completers) and (3) those who completed the 6‐month IPT course (IPT completers) as the reference group. TB incidence rates were calculated as the sum of new cases divided by the person‐years at risk while in care. Person‐years at risk was calculated as the interval from the last date of IPT eligibility screening to the date of TB diagnosis or the date of the last clinic visit or the date they transferred care to another facility, and rate ratios were calculated with 95% confidence intervals.Data were analysed using Stata version 16 .2.4Ethical clearance was obtained from the University of Washington Institutional Review Board, Kenyatta National Hospital/University of Nairobi Ethics Review Committee, and Jaramogi Oginga Odinga Teaching and Referral Hospital Ethics Review Committee. Informed consent was waived since this study only involved abstraction and analysis of data that was collected as part of routine clinical care.33.1n = 465) were female (Table n = 667) of the CLHIV were WHO Stage 1 or 2, 14% (n = 119) were WHO Stage 3 or 4, while 8% (n = 70) were missing information on WHO Stage. Ninety‐nine percent (n = 844) of CLHIV had a date of ART initiation. Of these, 78% (n = 657) were initiated on ART at enrolment. Among those who initiated ART post‐enrolment, the median time to ART initiation was 0.5 months (IQR = 0.2–2.3). Viral load was available for 58% (n = 497) of CLHIV. The median viral load at IPT initiation was 60 copies per millilitre (IQR = 0–1154); 71% (n = 373) were virally suppressed. Among non‐initiators, the median viral load within 6 months of the most recent IPT eligibility evaluation was 229.5 copies per millilitre (IQR = 70–8989.5); 56% (n = 18) were virally suppressed.Overall, 856 CLHIV between the ages of 12 months and 14 years were newly enrolled in HIV care from September 2015 through July 2019. At enrolment, the median age was 5 years (interquartile range [IQR] 1.9–9.2) and 54% (3.2n = 841) of the enrolled CLHIV were screened for IPT eligibility using the ICF tool  of those screened were diagnosed with active TB. Thus, 98%  of the CLHIV who underwent screening were eligible for IPT initiation.Nearly, all  of eligible CLHIV  started within 6 months of enrolment into HIV care, 15%  within 7–12 months and 19%  after 12 months. The date of IPT initiation was unknown for 3% (n = 17) of CLHIV who initiated IPT. Almost all  were on ART at IPT initiation; one participant was not on ART when IPT was initiated, while the ART status at IPT initiation of four participants was unknown.IPT was initiated in 68%  completed the 6‐month course. IPT was discontinued in 2% (n = 10); six had adverse events, while four were diagnosed with active TB. One child died of an unknown cause within 2 months of IPT initiation. IPT outcomes were unknown in 20% (n = 114) of CLHIV who initiated; of these, 86% (n = 98) were lost to follow‐up, while 14% (n = 16) were transferred out to other health facilities. The median number of months on IPT was 1.5 (IQR 1.0–2.8) among those who discontinued, 3.2 (IQR 2.9–6.2) among those who were lost to follow‐up and 2.1 (IQR 1.0–4.9) among those who transferred out.Of 559 CLHIV who initiated IPT, 78% , had a trend to be 90% higher among virally non‐suppressed CLHIV than virally suppressed  and almost thrice as high among CLHIV attending high‐volume HIV clinics than among those attending lower volume clinics .In bivariate analysis, younger age, later year of enrolment, viral load non‐suppression and higher HIV clinic volume were associated with non‐initiation of IPT among eligible CLHIV at a el Table . In multp‐value <0.10 level and thus included in the multivariable model. In multivariable analysis, IPT non‐completion was 40% higher among those enrolled between 2017 and 2019 than those enrolled between 2015 and 2016 .Among CLHIV who initiated IPT, year of enrolment and HIV clinic volume were associated with non‐completion of IPT in bivariate analysis at a 3.6n = 9) occurred among CLHIV who were eligible but never initiated IPT, 24% (n = 4) among those who initiated IPT but did not complete the 6‐month course and 24% (n = 4) occurred among those who completed the 6‐month course. By 24 months after IPT screening, TB incidence was four‐fold higher among eligible CLHIV who never initiated (8.1 per 1000 person‐years [PY]) compared to CLHIV who completed IPT , with a similar trend among CLHIV who initiated but did not complete IPT .Overall, 30 CLHIV were diagnosed with active TB, including 13 with prevalent TB who were diagnosed at screening for IPT eligibility. Seventeen new cases were diagnosed after enrolment with an incidence of 4.9/1000 person‐years  is critical to reducing morbidity and mortality associated with TB in people living with HIV, it remains underutilized, especially in high TB/HIV burden settings, including sub‐Saharan Africa (SSA) . Our resOur findings, based on a large cohort of CLHIV aged 1–14 years from western Kenya , contribute to the limited literature on IPT initiation and completion among CLHIV from high TB/HIV burden countries. The uptake of IPT among CLHIV in Kenya still falls short of the global target of >90%  and coulAdditionally, in this study, a substantial proportion of IPT initiators (40%) were initiated after being on HIV care for over 6 months. Suboptimal or delayed IPT initiation in this study could be due to health system barriers, which have been documented in previous studies, including difficulties in ruling out active TB especially in CLHIV with respiratory symptoms , the feaThere are limited data on outcomes of IPT among CLHIV in other high HIV/TB settings in SSA. In a prospective cohort study that enrolled 66 CLHIV from a paediatric clinic in Tanzania, 74% completed IPT within 10 months . A studyThere are limited data on the effectiveness of IPT on TB incidence in CLHIV. Randomized trials have established the protective effect of IPT for both HIV‐negative children  and adulOur study used routine data collected over several years from eight HIV clinics located within the largest hospitals in the County and was, therefore, suitable for evaluating the outcomes of programmatic IPT in CLHIV in a high burden region in Kenya. Routine data sources are prone to limitations, such as incomplete data, which we minimized by abstracting data from multiple sources, including IPT registers and HIV treatment records. However, we still had several instances of missing data elements on WHO staging (8%), HIV viral load (14%) and IPT initiation dates (3%). Secondly, these routine data sources did not capture some variables that could explain non‐initiation or non‐completion of IPT, such as socio‐economic status, caregiver characteristics , distance from the clinic, INH stockouts and attitudes of providers and caregivers. There are efforts to evaluate these factors in a prospective study. Other than TB status, we did not abstract data on clinical outcomes, such as mortality and loss to follow‐up among IPT non‐initiators, thus these outcomes were not assessed during analysis. TB status was only captured in the medical charts if there was a TB diagnosis but the absence of TB or uncertainty in diagnosis was not outrightly documented in the medical charts.5In this study, we observed that while screening for IPT eligibility was high for CLHIV, there were substantial delays and drop‐offs at initiation and completion of IPT. Targeted programmatic interventions are needed to address these drop‐offs from the IPT cascade by ensuring timely IPT initiation after ruling out active TB and enhancing completion of the 6‐month course, especially in high‐volume hospitals. While shorter course TB prevention regimens are becoming more available, they will likely increase completion but potentially not timely initiation. Virally non‐suppressed CLHIV need additional support to initiate and complete IPT and to achieve viral suppression.DOO, JK, GJ‐S and SML contributed to the conception, design and execution of this study. DOO performed the analyses and drafted the initial manuscript. MABVDS, CMY, EO, JM and DM critically reviewed the paper for analysis and interpretation of the data. All authors revised the manuscript critically and contributed to the final draft and have read and approved the final manuscript.The authors have no competing interests to declare.Research reported in this publication was supported by the Fogarty International Center of the National Institutes of Health , National Institute of Allergy and Infectious Diseases (NIAID) (NIH/NIAID K23AI120793 to SML) and NIH UL1TR000423 for REDCap.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


Objective: The primary endpoint of the study was to determine the proportion of patients with HIV RNA < 50 copies/mL at 48 weeks. Design: Phase IV, multicentric, open-label, single-arm clinical trial of participants recruited in 2018–2019 to evaluate the efficacy and safety of tenofovir alafenamide/emtricitabine/elvitegravir-cobicistat (TAF/FTC/EVG-c) as first-line treatment in HIV-1 infected naïve participants with advanced disease. Methods: Adverse events were graded according to the Division of AIDS scale version 2.0. Quantitative variables were recorded as median and interquartile range, and qualitative variables as absolute number and percentage. T-Student or Wilcoxon tests were used to analyze intragroup differences of the continuous variables. Results: Fifty participants were recruited with a baseline median CD4 lymphocyte count of 116 cells/µL and a viral load of 218,938 copies/mL. The proportion of patients with viral load <50 copies/mL at week 48 was 94% in the per-protocol analysis, with a median time of 1.9 months to achieve it. Three adverse events attributed to the study drug caused trial discontinuation. Conclusions: the use of TAF/FTC/EVG-c in patients with advanced HIV disease in our study demonstrated efficacy comparable to data from pivotal clinical trials with a good safety profile. Advanced HIV disease is defined as the presence of <200 CD4 lymphocytes/µL or an AIDS-defining condition before the start of antiretroviral treatment (ART) . The preThe number of patients with severe immunosuppression included in clinical trials is low, especially in trials of naive patients with the most modern drugs. Traditionally, these patients were preferred candidates to receive protease inhibitors because of their high genetic barrier since treatment was started before receiving the resistance test. However, drugs such as raltegravir, with a lower genetic barrier than protease inhibitors, showed a high degree of virologic efficacy in patients with severe immunosuppression . The comPhase IV, low-intervention, open-label, single-arm clinical trial designed to evaluate the efficacy and safety of TAF/FTC/EVG-c as first-line treatment in severely immunosuppressed HIV-1 infected naïve patients. Recruitment began in April 2018 and ended in May 2019, with the last study visit being recorded in June 2020. The trial was approved by the Ethics Committees of the participating centers and the Spanish Medicines Agency and was carried out in accordance with the Declaration of Helsinki and good clinical practice guidelines. The candidates were informed of the study objectives and procedures, so they signed informed consent before their inclusion. The trial was registered at Clinical Trials.gov (NCT03693508).Patients were recruited from six Spanish centers. Eligible patients needed to be at least 18 years old with positive serology (Western Blot) for HIV-1, naïve to antiretroviral treatment, and <200 CD4 lymphocytes/µL. They had to present serum transaminase levels ≤5 times the upper limit of normality, >1000 neutrophils/µL, >50,000 platelets/µL, >85 g/L of hemoglobin, and serum amylase levels <1.5 times the upper limit of normality. No candidate was excluded because of an active opportunistic infection.After signing the informed consent and extraction of screening samples, the scheduled visits were carried out at baseline and at weeks 4, 8, 12, 24, 36, and 48. At the baseline visit, treatment with TAF (10 mg)/FTC (250 mg)/EVG-c (150–150 mg) in a single daily tablet was initiated. At each visit, clinical data, vital signs, blood count, lipid profile, liver enzymes, kidney function, CD4 lymphocytes, and HIV-1 viral load were collected in the local laboratory.Clinical and laboratory adverse events were evaluated by local investigators and graded according to the AIDS division scale (version 2.0) . The prop < 0.05 (two-tailed).In the intention-to-treat (ITT) analysis, all those patients who received at least one dose of the drug were considered for the analysis, this being the reference population for the safety analysis. In the per-protocol population analysis (PPP), we considered patients who had received at least one dose of the drug without resistance mutations to any of them and did not show major deviations from the protocol, such as treatment dropout, adherence < 80%, or discontinuation of antiretroviral treatment due to prohibited interactions. Quantitative variables are shown as the median and interquartile range (IQR), and qualitative variables as absolute number and percentage. The 95% confidence intervals of the results associated with the primary objective and the main secondary variables are presented. The intragroup differences of the continuous variables were analyzed using the t-Student test or Wilcoxon for paired samples, depending on the normality of the variables. The time to virologic failure or suppression and the cumulative probabilities of these events have been estimated using the Kaplan–Meier method. The statistical package SPSS 26.0  was used for the analysis of the data, and the value of statistical significance was established at P. jiroveci being the most frequent (6 participants). The baseline CD4 lymphocytes were 116/µL (59–157), and the viral load was 218,938 copies/mL .Between April 2018 and June 2019, 50 patients were recruited, 84% males and 90% Caucasians with a median age of 37 years. There was an AIDS event at diagnosis in 12 (24%) participants, The reasons for trial discontinuation are presented in There was a progressive increase in the number of CD4 lymphocytes; thus, the median CD4 at weeks 4 and 48 were, respectively, 201 and 375 cells/µL. The median CD4 lymphocytes increase at week 48 was 220.5/µL (IQR: 143–294). At week 48, 88.1% of the patients had CD4 lymphocytes > 200/µL, and the median time to achieve it was 1.8 months.2 to 25.1 kg/m2 (p < 0.0005 in both cases). There was a slight decrease in eGFR (p < 0.01). At week 48, a statistically significant rise was observed in all lipid fractions, except for triglycerides, but with little change in the total cholesterol/HDL ratio. A total of 260 adverse events were recorded in 46 patients (92.0% of patients with a ratio of 5.7 events per patient). The most frequent was genital herpes in 3.6%. Regarding the relationship with the study medication, five adverse events occurred in five patients. Treatment had to be interrupted due to adverse events in five patients, three of them related to the study drug. No deaths were recorded.The median weight of the patients changed from 68 kg at the baseline visit to 74 kg at week 48, and the body mass index (BMI) changed from 23.8 kg/mOur results show the efficacy of the combination of TAF/FTC/EVG-c similar to that reported in pivotal clinical trials in patients with <200 CD4 lymphocytes/µL . These rDespite the number of reported AIDS events, no deaths occurred in our study. Although the change in BMI in our patients was not very marked, going from normal weight to slightly overweight, it is true that there were two cases of treatment interruption. Weight gain with current treatment regimens is a subject of discussion, especially in severely immunosuppressed patients where “return to health” could play a greater role. Otherwise, the combination of TAF/FTC/EVG-c was well tolerated with few treatment interruptions. There were no renal, hepatic, or bone side effects that led to the discontinuation of treatment. Regarding the lipid profile, although there was an increase in LDL cholesterol and triglycerides, the total cholesterol/HDL ratio decreased slightly, which would argue in favor of the fact that there is no increased cardiovascular risk with the introduction of TAF/FTC/EVG-c.TAF/FTC/EVG-c was a nationally and internationally recommended first-line treatment at the time this study was designed. Despite the difficulty of using an enhancer such as cobicistat and not having a genetic barrier as high as second-generation integrase inhibitors, the results of TAF/FTC/EVG-c in this population were successful, with few viral failures and without resistance mutations developed.There are several limitations to our study. Firstly, it is a single-arm study, so we have to refer to indirect comparisons in terms of efficacy. Secondly, although it provides greater experience in treating this type of patient, the sample size is limited. The difficulty in recruiting and keeping these patients in clinical trials is that some are completed before reaching the projected sample size , and othThe use of TAF/FTC/EVG-c in our study showed comparable results to the data extracted from its pivotal clinical trial in patients with severe immunosuppression with a good safety profile.


Objectives: To determine the real-life effectiveness of 2DR with 25 mg RPV plus 50 mg DTG in a single-tablet regimen (RPV/DTGSTR) and its impact on viral and immune status, lipid profile, and inflammatory markers. Methods: This observational study included 291 treatment-experienced PLHIV, starting 2DR with RPV/DTGSTR between 29 January 2019 and 2 February 2022, who were followed up for at least six months. Participants gave verbal informed consent for the switch in antiretroviral therapy (ART) to RPV/DTGSTR. Results: The mean age of the 291 participants was 51.3 years; 77.7% were male; and 42.9% were in the AIDS stage with a CD4 nadir of 283.5 ± 204.6 cells/uL. The median time since HIV diagnosis was 19.7 years (IQR: 10.6–27). Before 2DR, patients received a median of five ART lines (IQR: 3–7) for 22.2 years (IQR: 14–26), with 34.4% (n = 100) receiving a three-drug regimen (3DR), 31.3% (n = 91) receiving monotherapy, and 34.4% (n = 100) receiving 2DR. The median time on RPV/DTGSTR was 14 months (IQR: 9.5–21); 1.4% were lost to the follow-up. Effectiveness was 96.2% by intention-to-treat (ITT) analysis, 97.5% by modified ITT, and 99.3% by per-protocol analysis. Virological failure was observed in 0.69%, blips in 3.5%, and switch to another ART in 1.4%. The mean lipid profile improved, with reductions in TC/HDLc ratio , LDLc , TG , and CD4/CD8 ratio increase . The cost-effectiveness of 2DR with RPV/DTGSTR was similar to that of DTG/3TC and superior to those of BIC/TAF/FTC and DRV/c/TAF/FTC, with higher virological suppression and lower annual costs. Conclusions: The switch to RPV plus DTG in STR is a cost-effective, long-lasting, and robust strategy for PLHIV, with a very long experience of treatment, which improves the lipid profile without affecting inflammatory markers.Two-drug regimens (2DRs) are emerging in clinical practice guidelines as treatment option for both naive and treatment-experienced people living with HIV (PLHIV).  Since the introduction of high-effectiveness antiretroviral therapy (ART) in the mid-1990s, the life expectancy of people living with HIV (PLHIV) has progressively increased, approaching that of the general population . Two-druSTR) in a cohort of ART-experienced PLHIV. The secondary objectives were to determine the effect of switching on immune and viral status and inflammatory markers and to carry out a pharmacoeconomic study of the change.The main objective of this study was to analyze the effectiveness and safety of changing any ARV regimen  to 2DR with RPV (25 mg/QD) plus DTG (50 mg/QD) in a single-tablet regimen  plus DTG (50 mg/QD) in STR (RPV/DTGSTR). The study was approved by the ethics committee of Virgen de las Nieves University Hospital of Granada, Spain (#1602-N-22), and data were treated in compliance with Spanish legislation on personal data protection.A multicenter, observational, and retrospective study was undertaken in PLHIV treated with RPV/DTGSTR by the attending physician.PLHIV treated with any other ART regimen for at least six months who were switched to 2DR with RPV/DTGSTR , and analytical data for the month before the change to RPV/DTGSTR. Data were also gathered at V0 and at the end of the follow-up on: VL, CD4, and CD8 lymphocyte counts and percentages, CD4/CD8 ratio, total cholesterol, HDL, LDL, triglycerides, GOT, GPT, GGT, bilirubin, alkaline phosphatase, interleukin-6, D-dimer, and glomerular filtration (CKD-EPI). At the baseline visit (V0), data were gathered on age, sex, active co-infections, toxic habits , mode of acquisition of HIV, weight, date of HIV diagnosis, viral load (VL), CD4 and CD8 lymphocyte counts at diagnosis, CD4 nadir, previous ART lines, reasons for change to RPV/DTGTreatment effectiveness was determined by calculating the proportion of participants achieving viral suppression (defined by plasma HIV RNA ≤ 50 cop/mL) at the end of follow-up according to intention-to-treat (ITT) , modifieA blip was defined as VL > 50 copies/mL in one determination and undetectable in the next determination one month later. Virological failure (VF) was defined by VL > 50 HIV-RNA copies/uL at two consecutive determinations in a 6-month period. The interpretation of the genotypic resistance test was performed following the algorithms of Stanford University and the Spanish AIDS Research Network (RIS) .It was estimated that at least 235 treatment-experienced PLHIV were needed to measure the effectiveness of 2DR with 95% confidence interval (CI) and 3% precision, assuming a loss to follow-up of 15%. p < 0.05 was considered significant in all tests. SPSS 20.0 was used for statistical analyses.Statistical analysis: In the descriptive analysis, frequencies with 95% CI were calculated for qualitative variables, means with standard deviation for normally distributed quantitative variables, and medians with IQR for non-normally distributed quantitative variables. The Kolmogorov–Smirnov test was applied to check the normality of variable distributions. The paired samples Wilcoxon test was used to compare quantitative variables. STR by modified ITT analysis . Treatment-cost calculations were based on the laboratory price list for February 2022, without VAT and without considering state discounts. Cost-minimization analysis was performed after one year, to determine the savings generated by switching to RPV/DTG. The cost-effectiveness ratio (CER) was also determined by dividing the cost of each treatment by its effectiveness, and the incremental CER (ICER) of RPV/DTG therapy vs. the others was calculated by dividing the difference in total costs  by the increase in effectiveness.A pharmacoeconomic analysis was performed, comparing therapy with RPV/DTG vs. BIC/TAF/FTC, DRV/c/TAF/FTC, and DTG/3TC. Data obtained from published research in study populations with similar characteristics ,17,18 wen = 100) receiving a three-drug regimen (3DR), 31.3% (n = 91) receiving monotherapy, and 34.4% (n = 100) receiving another 2DR. At baseline (V0), 3.4% (n = 10) had VF and 6.2% (n = 18) had blips. One patient had a chronic VHB infection (HBS antigen positive) and had been under HBV treatment with entecavir for 10 years due to kidney failure after treatment with 600 mg efavirenz, 200 mg emtricitabine, and 245 mg disoproxil tenofovir in STR  were male; and 42.9% had AIDS. The mean CD4 nadir was 283.5 ± 204.6 cells/uL. The median time since HIV diagnosis was 19.7 years (IQR: 10.6–27). Patients had previously received a median of five ART lines (IQR: 3–7) for 15.3 years (IQR: 7.5–2), with 34.4% (r in STR .A genotyping resistance test had been performed in 152 (52.2%) of the participants a median of 13.5 years earlier (IQR: 8.3–17.3); among these, 14 (9.25%) had primary and secondary non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistance mutations conferring either resistance (35.7%) or decreased susceptibility to RPV (42.9%) .n = 203), avoidance of potential long-term toxicity in 26.8% (n = 78), and VF in 3.4% (n = 10) due to poor ART adherence in a multi-tablet regimen (MTR). The results for the remaining study variables are reported in Reasons for the 2DR switch were simplification in 69.8% (STR was 14 months (IQR: 9.5–21); five (1.7%) patients were lost to the follow-up. Effectiveness was recorded in 96.2% (280/291) by ITT, 97.5% (280/287) by modified ITT, and 99.3% (281/283) by PP analyses  had VF, 3.5% (n = 10) had blips, and 1.4% (n = 4) switched from RPV/DTGSTR due to drug interactions (n = 1), adverse events , or by decision of the physician (n = 1)  . One of  (n = 1) . p = 0.0001) and a reduction in the TC/HDLc ratio , LDLc level , and TG level . No statistically significant changes were found in the IL-6, C-reactive protein, fibrinogen, or D-dimer levels. During the follow-up, the following were observed: an increase in the CD4/CD8 ratio .The virological effectiveness ratio was 97.5% for RPV/DTG DTG/3TC b. These STR in ART-experienced PLHIV were aged over 50 years, and around half of them were in the AIDS stage. They had a very long history of ART (median of 22.1 years), with a median of five previous lines. They were switched from different ARV regimens and even included a small percentage of non-suppressed patients. Mutations conferring NNRTI resistance had been detected in 5% over 13 years earlier, and previous exposure to integrase standard transfer inhibitors (INSTIs) was recorded in 30%, but none had resistance mutations to DTG. The majority of participants in SWORD 1 and 2 clinical trials were aged <50 years, while around 11% were in the AIDS stage, and the median time with ART was four years, with a maximum of two previous lines; in addition, they were all suppressed before starting the 2DR, <10% had been exposed to DTG, and none had resistance mutations to NNRTI [Most patients in this real-world study on the use of 2DR with RPV/DTGto NNRTI . In the present study, the reason for switching was simplifying the regimen in more than two-thirds of cases, avoiding long-term toxicity in more than one-quarter, and failing to adequately adhere to their MTR in one-tenth; finally, clinicians even trusted in this regimen in one out of ten patients in failure due to poor adherence to treatments in MTR. Owing to the increased survival and life expectancy of PLHIV achieved by ART, it has been estimated that patients will consume ARVs for a mean of 39.1 years, receiving 57,086 ARVs for 3DR with a booster, 42,815 for 3DR without a booster, and 28,543 for 2DR . The potSTR achieved an effectiveness of 95% by ITT analysis and close to 100% by PP analysis in this cohort of ART-experienced PLHIV, and there was no failure in patients with RPV resistance mutations. These effectiveness data are highly similar to those obtained in registered clinical trials [Only four patients were lost to the follow-up of more than 12 months, 2DR with RPV/DTGl trials . Despitel trials , indicatSTR . In addition, the treatment was only suspended in two patients, to avoid drug interactions and for the convenience of the patient as decided by the physician. Finally, VF was observed in two patients, which was attributed to poor treatment adherence, with neither having mutations in the historical genotype, demonstrating the robustness of the treatment and/or that strains with mutations were a very small minority and were completely suppressed after a long time with a previous effective ART. These findings are in agreement with a trial of this 2DR that showed the sustained virological suppression of around 90% after a three-year follow-up, with low VF rates and a well-tolerated safety profile [Only two patients had adverse effects attributable to 2DR with RPV/DTG profile .The treatment improved cholesterol and triglyceride levels and the CT/HDL ratio, as also found in clinical trials  and prevSTR. A recent retrospective, multicenter Spanish study of the switch from 3DR to 2DR with RPV/DTG in virologically suppressed PLHIV also found an increase in CD4 and a decrease in CD8 at 48 weeks [STR [STR [An improvement in immunological recovery, with an increased CD4/CD8 ratio, was recorded at one year of treatment with RPV/DTG48 weeks . Bone meeks [STR . No chanSTR [STR .STR in stable and mostly virologically suppressed ART-experienced PLHIV proved to be a cost-effective alternative to 3DR ART, offering higher effectiveness at a lower cost in comparison to BIC/FTC/TAF and DRVc/FTC/TAF. These results are in line with previous findings of cost saving with DTG/RPV vs. RPV/FTC/TDF, DTG/ABC/3TC, and EVG/c/FTC/TAF and the delivery of comparable efficacy with reduced cumulative drug exposure [The switch to RPV/DTGexposure . FinallyThe main study limitation is its retrospective design. Strengths include the large sample size of this multicenter study. In addition, it is the first to study the switch to this 2DR in PLHIV with long ART experience and no need for treatment suppression for >6 months, including individuals with archived RPV resistance mutations (none failed) and a high rate of exposure to integrase inhibitors. All these characteristics make this a unique study of major interest. In conclusion, 2DR with RPV plus DTG in STR is an effective, safe, long-lasting, robust strategy for patients with prolonged ART experience, improving their lipid profile without affecting inflammatory markers; it is cost-effective compared to the other treatments.


The ARTRM Q174K was associated with increased viral load and decreased CD4+ cell count, while infection with CRF02_AG was associated with a lower likelihood of Q174K. Interestingly, CRF02_AG was positively associated with the ARTRM L10V that, in turn, was observed frequently with darunavir administration. Infection with CRF02_AG was positively associated with the ARTRM S162A that, in turn, was frequently observed with the administration of nevirapine, also associated with lower CD4 counts. Zidovudine or Nevirapine receipt was associated with the development of the ARTRM E138A, that, in turn, was associated with lower CD4 counts. Determination of a patient’s HIV variant can help guide ART choice in Kazakhstan. For example, PLHIV infected with CRF02_AG will benefit less from darunavir and nevirapine, and emtricitabine should replace zidovudine.In Kazakhstan, the number of people living with HIV (PLHIV) has increased steadily by 39% since 2010. Development of antiretroviral therapy (ART) resistance mutations (ARTRM) is a major hurdle in achieving effective treatment and prevention against HIV. Using HIV  However, Eastern Europe and Central Asia are exceptions and between 2010 and 2020, an estimated 43% increase in new HIV infections was reported2. In Kazakhstan, the number of PLHIV was 22,000 in 2016, increasing to an estimated 24,000 in 2017, 26,000 in 2018, 33,000 in 2019, and 35,000 in 2020. Kazakhstan’s newly reported HIV cases grew 73% between 2010 and 20203.The estimated number of newly diagnosed persons living with HIV (PLHIV) declined from 2.8 million in 2000 to 1.5 million in 20204. In accordance with WHO recommendations, the Ministry of Health in Kazakhstan has authorized a clinical protocol for HIV treatment to begin universal ART administered no later than 14 days from the date of diagnosis, regardless of the clinical stage of the disease or CD4+  cell count5. The first line regimen in Kazakhstan offers both a preferred ART combination or an alternative6. If one of the components of the preferable treatment is not suitable for PLHIV for any variety of reasons, then it can be changed to another—the alternative treatment. For instance, patients with exceptional circumstances, such as pregnant women, patients with low (≤ 50 cells/μL) CD4+ cells counts, PLHIV with co-morbid conditions like neurocognitive disorders, chronic kidney disease, cardiovascular diseases, or chronic hepatitis, may be prescribed tailored alternative ART regimens. Since 2017, the list of ARV drugs in Kazakhstan has been expanded: tenofovir (TDF), atazanavir (ATV) with ritonavir or cobicistat, bictegravir (BIC), elvitegravir, as well as these combination drugs: TDF/lamivudine (3TC)/ dolutegravir (DTG), TDF alafenamide/emtricitabine (FTC)/bictegravir, TDF/emtricitabine/elvitegravir/cobicistat, DTG/rilpivirine, and DTG/3TC. Preferred ART regimens for adults were changed from TDF/FTC/EFV to TDF/FTC + DTG or TDF/FTC/BIC, and for pregnant women was changed from TDF + 3TC (or FTC) + EFV to TDF / FTC + DTG. Similarly, the post-exposure prophylaxis regimen (TDF + 3TC (or FTC) + LPV/r was replaced by TDF (or TAF) + 3TC (or FTC) + DTG). Additionally, the treatment effectiveness indicators have been revised recently; in adults (instead of VL < 1000) and in children (instead of VL < 500) a new treatment efficacy indicator VL < 50 has been introduced that will allow timely testing of resistance and, based on that, a change in treatment regimen. Changes in the perinatal diagnostic algorithm now require the first biomaterial sampling to be done within 48 h after birth to accelerate diagnosis and initiation of early ART in the newborn7.The antiretroviral (ARV) treatment is recommended by the U.S. Department of Health and Human Services (DHHS) and the World Health Organization (WHO) for treatment of all PLHIV8. To achieve the 2020 goal, 90% of all people living with HIV (PLHIV) would need to know their HIV status, 90% of all people with diagnosed HIV will have received sustained ART, and 90% of all people receiving ART will have been virally suppressed, i.e., viral load < 1000 copies/mL9. According to UNAIDS reports, as of 2020, in Kazakhstan, the efforts towards the 90–90–90 target have slowly increased to an estimated 78–73–8410.The Joint United Nations Program on HIV/AIDS (UNAIDS) has established a 90–90–90 strategy that sought to reduce the number of new HIV infections to 500,000 per year by 2020, with even more ambitious 95–95–95 goals for 203014. Factors that give rise to ART resistance mutations (ARTRM) include, sub-optimal treatment adherence, duration of viral replication maintained during incompletely suppressive therapy, effect of ARTRM on drug susceptibility and virus replication, and the ease of acquisition of a particular ARTRM8. ART resistance can be caused by the presence of acquired drug resistant (ADR) mutations in treatment-experienced PLHIV and/or transmitted drug resistance (TDR) mutations in treatment-naïve PLHIV16. Among populations receiving non-nucleoside reverse transcriptase inhibitors (NNRTI)-based ART, the reported levels of drug resistance can vary widely, NNRTIs from 50 to 97% and nucleoside reverse transcriptase inhibitors (NRTIs) from 21 to 91% in one review17.To achieve UNAIDS 95–95–95 targets by 2030, it is imperative to control the development of ART resistance by maintaining adherence to treatment among PLHIV receiving ART. During replication, HIV produces a high rate of mutational variants that pose challenges to achieving effective ART therapy and viral suppressionIn this study, we present the analysis of HIV drug resistance mutations among 602 PLHIV in Kazakhstan. We analyze associations between ARTRMs and contributing factors, such as CD4 count, viral load, ART, and infecting HIV subtype.HIV pol sequences from 602 PLHIV receiving ART were analyzed. All the PLHIV were registered with Kazakh Scientific Center of Dermatology and Infectious Diseases, Almaty, Kazakhstan. All patient information, including demographic data, was collected at the time of registration. Blood samples from the participants were collected between 2017 and 2019. Analyses for CD4+ cell counts were performed using BD FACS Count Reagent Kit  and for viral load using AmpliSens HIV-Monitor-FRT kit . The AmpliSens HIV Resist-Seq kit  was used for preparation of samples for sequencing. The kit includes reagents for RNA extraction from blood samples, RT-PCR, and specifically amplifies the Pro (protease) and RT (reverse transcriptase) genes using random primers provided with the kit. Following amplification, the samples were sequenced on Applied Biosystems 3100 Genetic Analyzer  with a usage of BigDye Terminator v1.1 Cycle Sequencing Kit .Ethical approval for this study was obtained from Institutional Research Ethics Committee, Nazarbayev University, Kazakhstan (# 141/06052019). All experiments were performed in accordance with relevant guidelines and regulations. All participants gave informed consent to participate in the parent study from which 18. Recombinant forms were further analyzed using the RIP tool19. The FASTA format file with sequences was uploaded to the HIVdb Program provided by the Stanford HIV Database21. Level of resistance was assigned to the ARTRM as follows: "Susceptible" implied no evidence of decreased ARV susceptibility. "Potential low-level resistance" implied presence of mutations that suggested previous ARV exposure or of mutations that were associated with drug resistance only when they occurred in the presence of other mutations. "Low-level resistance" was used to describe mutations that confer lower ARV susceptibility in vitro, or mutations that led to poor virological response to ARV treatment in a patient. "Intermediate resistance" signified high probability of a drug's activity being decreased, whereas "high-level resistance" meant the expected level of resistance was close to that seen in viruses with the highest levels of in vitro drug resistance, or that clinical evidence existed showing that PLHIV infected with these viruses had little to no virological response to ARV treatment21.All sequences were subtyped using REGA-HIV-1 automated subtyping toolSTATA version 15.0 statistical software22. Continuous variables were summarized in means and standard deviations (SD) when normal distributions were noted or medians and interquartile ranges when distributions were skewed. Wilcoxon rank-sum test was used to evaluate the associations of these continuous variables: CD4 count, viral load, and the presence of ARTRMs. We used simple logistic regression to assess the relationship between the administration of a specific ARV drug or drug combination or a specific HIV subtype with the presence of ARTRMs. The association between the administration of specific ART component and CD4 count or viral load was analyzed using the Wilcoxon rank-sum test. Simple and multiple logistic regression analysis was performed to determine the direction and strength of association between independent variables and the occurrence of virologic failure. Crude and adjusted odds ratios (OR) with 95% confidence interval (CI) were calculated from univariate and multivariable analysis, respectively. A p-value < 0.05 was considered statistically significant. Among all ARTRMs, those drug resistance mutations possessing a significant association with continuous or categorical variables were analyzed.Statistical analyses were performed using Study participants comprised almost equally of men and women, coming from all regions of Kazakhstan: including Akmola, Aktobe, Atyrau, East Kazakhstan, Karaganda, Kostanay, Kyzylorda, Mangystau, North Kazakhstan, Pavlodar, Turkistan, West Kazakhstan, Zhambyl regions; and 3 cities with State status, i.e., Almaty, Shymkent, and Nur-Sultan. Samples, collected in 2017 (38%), 2018 (43%) and 2019 (19%), were all from ARV-experienced PLWH  while the most common alternative is two NRTIs plus an NNRTI. Among our 602 study participants, only 37.5% were taking a Ministry of Health ART regimen  of study participants at the time of enrolment. Medium CD4 counts of 200–500 cells/mm3 were seen in 316 (52.5%), and > 500 cells/mm3 in 111 (18%) participants  sequences showed the presence of drug resistance mutations. The distribution of ART resistance was: 11 (2%) PI, 21 (3%) NRTI, 108 (18%) NNRTI, 1 (0.2%) PI and NRTI, 3 (0.4%) PI and NNRTI, 47 (8%) NRTI and NNRTI.Drug resistance mutations were divergent in two viral subtypes: A6 and CRF02_AG  in A6 subtype patients. Mutation K103N was found to less often (15%) among the A6 subtype compared to CRF02_AG (40%)  of M184V mutation and similar distributions of Q174K: 90% occurrence in A6, and 83% in CRF02_AG. As seen in an earlier study in KazakhstanAmong our study group, fewer PLHIV were found resistant to PI than those to NRTI or NNRTI Fig. . There wWe found 59 10%) PLHIV with high level resistance to FTC or 3TC. Other resistance was far less common: 15 (3%) to DDI, 13 (2.2%) to ABC, 6 (1%) to D4T, 4 (0.7%) to ZDV, and 4 (0.7%) to TDF , EFV (20%), RPV (4%), DOR (1.7%), ETR (1%)  and Nevirapine (NVP), respectively . On the other hand, we found an interaction between the simultaneous administration of ZDV with NVP and the presence of E138A mutation, which would decrease the odds of possessing this ARTRM . Moreover, the prescription of Darunavir (DRV), a protease inhibitor, was associated with a 4.9-fold increase in the odds of acquiring L10V mutation . The occurrence of RT ARTRM, S162A (other), was 1.46 times higher among PLHIV receiving NVP . Comparing various subtypes of the HIV-1 virus with the presence of ARTRMs, infection with CRF02_AG was found to increase the likelihood of L10V, G16E  and S162A ARTRMs , while reducing the rate of Q174K mutation . There was a negative association with the administration of NVP and CD4 count, while the prescription with Lopinavir/ritonavir (LPV/r) had a positive association with CD4 count . In contrast, the presence of I13V, N37D, Q174K mutations increased, while I93L and V77I mutations reduced the odds of having virologic failure among the PLHIV  may have associations with specific ARV therapy response28. No such reports are available about the prevalence of ARTRM, or their association with the HIV variants prevalent, in Kazakhstan. A 2007 publication reported that 59% of HIV subtype A sequences derived from Russia and Kazakhstan PLHIV carried a secondary mutation, V77I, in protease23. Consistent with this earlier observation, 93 (15%) of our study participants carried the V77I mutation in the protease gene, with 89 (96%) of them infected with subtype A6 subtype.Among our study participants 56% were infected with HIV subtype A6, 24% with CRF02_AG, 1.5% with subtype G, and 0.7% with other variants. Subtype A6 evolved as an indigenous HIV variant in former Soviet Union countries and is still predominantly prevalent in this region, including Kazakhstan6. Among out study participants, 32% were receiving the alternative combination, TDF FTC EFV regimen, while 20%, 15%, and 1.5% received other regimens, respectively, ZDV 3TC NVP, ZDV 3TC EFV, and TDF FTC NVP, which were neither preferable, nor alternative , was found to be decreased by an increase in the duration of ART, higher CD4 count, and the presence of a V77I mutation , PI + NNRTI 0.4%), PI (2%), NRTI (3%), NRTI + NNRTI (8%), and NNRTI (18%). Recent studies report that rates of ARV resistance rise with time on ARV%, PI + N48. The A62V mutation is known to frequently co-occur with K65R and Q151M, giving rise to NRTI multi-resistance50. A62V was previously reported to be prevalent in HIV subtype A patients from Russia51; we observed a high occurrence of the same mutation in our study participants infected with subtype A6. Overall, there were 133 PLHIV carrying A62V mutation, 126 (95%) of whom were infected with subtype A6.We found that in our study cohort, some mutations occurred especially often: NRTI accessory A62V (22%), NNRTI major K103N (17%), and NRTI major M184V (8%). M184V is an NRTI mutation known to occur frequently among ARV experienced individuals. It induces high level resistance to 3TC. K103N is another persistent NNRTI mutation that gives rise to high-level resistance to NVP and variable resistance to EFV52.We found levels of resistance to NRTI and/or NNRTI to be higher than resistance to PI Fig. , likely 20. Surprisingly, the simultaneous administration of these drugs was negatively associated with the presence of E138A  validity of our results based on potential sources of bias: selection, measurements, confounding, and analysis. Since ART access is relatively centralized and the Kazakh Scientific Center of Dermatology and Infectious Diseases in Almaty provided samples from patients throughout the nation, we believe that geographic generalizability is substantial. However, the preponderance of our participants were persons who inject drugs or persons with likely heterosexually acquired HIV infection. Underrepresentation of men who have sex with men should be noted, and we cannot discount misclassification among men who may deny same sex contact when they might, in fact, have occurred. Our data from 2017 to 2019 may not reflect current trends. Fortunately, measurements were state-of-the-art and subject to best laboratory practices. Use of multivariable models in our statistical analysis enabled examination of confounding factors and interactions. However, we acknowledge that 602 subjects are not enough to completely disentangle the relationships of sociodemographic and behavioral factors and ARV drug combinations with the many ARV mutations studied. Still our study contributes substantially to the Central Asia data in this sphere.In conclusion, to achieve effective treatment and prevent further transmission of HIV in Kazakhstan, it is imperative that current treatment protocols are optimized to eliminate components of ARV combination that may promote ARTRM. For efficient ART optimization, complete patient profiling, including the existence of ARTRM and the infecting HIV subtype or variant, needs to be taken into account.Supplementary Tables.


HIV-1 drug resistance remains a global challenge, yet access to testing is limited, particularly in resource-limited settings. We examined feasibility and limitations of genotyping using dried filter analytes in treatment-experienced Kenyan youth with HIV. Youth infected with HIV perinatally were enrolled in 2016–2018 at the Academic Model Providing Access to Healthcare in Eldoret, western Kenya. Samples were shipped in real-time at ambient temperature to the US, and those with viral load (VL)>1,000 copies/mL were tested based on convenience. Dried blood spots genotyping was attempted when unsuccessful from Hemaspots. Multiple logistic regression was used to examine predictors of genotyping success. Samples from 49 participants  were shipped after median 7 days from collection, arrived in 20 shipments after median 5 days, and extracted after median 2 days . Overall, 29/49 (59%) samples with VL > 1,000 copies/mL and 25/32 (78%) with VL > 5,000 copies/mL were genotyped by either Hemaspots or DBS. Successful genotyping was associated with higher Hemaspot volume and higher VL. Real-life HIV-1 drug resistance testing from dried filter analytes is feasible, even in settings with constrained resources. Findings, particularly relevant where resistance testing is limited for clinical care, raise awareness to implementation practicability of this guidelines-recommended test in care of more individuals and populations. Further optimization of filter analytes is needed to overcome related challenges.IMPORTANCE In this manuscript we use dried filter analytes shipped from Kenya to the US in real time, to demonstrate the real-life feasibility of conducting HIV drug resistance testing in a vulnerable population of young children and adolescents with HIV in a resource limited setting. Such testing, which is recommended in resource-rich settings, is unavailable in most resource limited settings for individual clinical care. We show that real-life HIV drug resistance testing from dried filter analytes is feasible, even in settings with constrained resources. These findings raise awareness to the importance of HIV drug resistance for individual care, even in such settings, and emphasize the implementation practicability of this guidelines-recommended test. HIV drug resistance (DR) challenges sustainable antiretroviral therapy (ART) , 2. TreaDried filter analytes (DFAs) like dried blood spots (DBS) can be stored and shipped at ambient temperatures, are considered plasma-equivalent, and are used for DR surveillance in resource limited settings (RLS) –11. Limi–To address limited DR testing access in RLS, our objective was to establish the proof-of-concept feasibility for the primary outcome of successfully obtaining a sequence from any DFA in treatment-experienced Kenyan CAWH. Accomplishing this process in settings with limited infrastructure could support using this sequence for DR interpretation and improving the care of this and other vulnerable populations.Of 65 participants with VL > 1,000cmL who had Hemaspot and/or DBS prepared, 49 had both analytes, and were shipped. In those 49, median age was 15 years, 43% were female, with median CD4 496 cells/μL (18%), and median 8 years on ART. The median VL was 11,827cmL and 32/49 (65%) had VL > 5,000cmL. P-value = 0.422). Similarly, among 27 samples with VL > 5,000cmL, 5/7 (71%) frozen and 9/20 (45%) processed immediately were successfully genotyped .Overall, 29/49 (59%) samples with VL > 1,000cmL and 25/32 (78%) with VL > 5,000cmL were successfully genotyped from any DFA . Genotypes were obtained from 14/45 (31%) Hemaspots, all 14 with VL > 5,000cmL . The 12 100 μL Hemaspots had median VL 2,432cmL, including only five with VL > 5,000cmL. The 37 200 μL Hemaspots had median VL 16,844cmL, including 20/24 handled immediately with VL > 5,000cmL, and 7/9 frozen with VL >5,000cmL (Table S2). A larger (nonsignificant) percentage of frozen Hemaspots were successfully genotyped versus those processed immediately , and 11/18 (61%) with VL > 5,000cmL were successfully genotyped from DBS.P = 0.009), as well as from those prepared with 200 μL Hemaspots that were frozen . In multiple logistic regression analysis, obtaining a genotype from either DFA was associated with higher VL. Hemaspot genotyping success was associated with higher VL and shorter time in transit. DBS genotyping success was also associated with higher VL  was less successful than from 200 μL Hemaspots  or 78% , though low, are within the range of reports using DBS in not fully controlled settings , 15–17. 15–Third, increased genotyping success was associated with higher VL, a known concept for DFAs , 13, 18.Fourth, longer transit time was associated with less successful Hemaspot genotyping, but not DBS. This observation, extending reports in more controlled settings , 19, is Limitations of this study, which mandate interpretation caution, include small sample size, heterogenous parameters that could influence genotyping, no use of skin pricks, and lack of DFA validation. However, this study was not designed as validation, but rather proof-of-concept to demonstrate feasibility of real-life transcontinent DFA DR testing.In summary, despite complexity and divergence in analytes (Hemaspots/DBS), storage temperature (frozen/not), Hemaspot volume (100/200 μL) and VLs, we demonstrate real-life feasibility of HIV DR testing from DFAs in RLS, which could be used to inform clinical care. Findings are particularly important for vulnerable populations like ART-experienced CAWH, as they mature into adulthood with accumulated DR . This fePerinatally infected CAWH at the Academic Model Providing Access to Healthcare (AMPATH) in Eldoret, western Kenya were enrolled in 2016–2018 , 23. BloUpon arrival, if feasible considering time constraints, Hemaspots with VL > 1,000cmL were left at ambient temperature and attempted for genotyping within 1 day. If not feasible, samples were frozen at −80°C and attempted for genotyping when convenient. DBS were stored at −80°C and attempted if Hemaspot genotyping was unsuccessful. The last four samples with VL < 5,000cmL were genotyped from DBS only, since Hemaspot success for such samples was low.Proportions and exact 95% confidence intervals (CIs) were calculated for three outcomes: Hemaspot genotype success, DBS genotype success among Hemaspot genotype failures, and genotype success by either DFA, defined as overall genotyping success.P-value < 0.05 considered significant)  were used to examine relationships between continuous predictors and log odds of genotype success. Analyses were performed in R version 4.0.2 (ificant) . See Sup


Results: In this review, 57 patients were identified, with a female  and Black African  preponderance. Females were more likely to present with headache (p = 0.006), and headache was more likely to be present in those who died (p = 0.02). There was no statistically significant association between baseline CD4 count (p = 0.079) and viral load (p = 0.72) with disease outcome. Overall, 77% (41/53) of patients had classical imaging findings, including bilateral gadolinium-enhancing punctate and perivascular white matter lesions. However, many patients (23/57) required a brain biopsy as part of their diagnostic workup. Corticosteroid treatment was commonly prescribed in patients  and had a mortality benefit, with an overall survival in this group of 71% (p = 0.0008). In those who died, median survival was 5.5 months. In rare instances, recurrence of the disease was noted, which responded poorly to treatment. Discussion: CD8E represents a new and complex condition with few risk factors identified for its occurrence. The presenting symptoms are broad, but headache appears to be more common in females and more significantly associated with death. Though rare, CD8E is likely under-diagnosed, possibly due to overlapping features with other illnesses and lack of physician experience in its recognition and management. Corticosteroids demonstrate a clear mortality benefit, but more studies are required to determine their optimal dosing and duration, as well as the use of steroid-sparing agents. Further reviews should help to better determine the risk factors for the condition, as well as non-invasive biomarkers, to aid in diagnosis and help to predict poor prognosis and disease recurrence.Introduction: Encephalitis is a life-threatening neurological condition with multiple causes in the setting of Human Immunodeficiency Virus (HIV). CD8 Encephalitis (CD8E) is a newly recognised condition which can present in an acute manner, with pertinent features including classical radiological findings with an intense brain parenchymal infiltration of CD8+ T cells. This review attempted to clarify the symptomatology, distribution and determinants of this condition, as well as to examine its vast unknowns. Methods: A literature review was undertaken in July 2022, utilising the PubMed and Google Scholar databases. Papers published between 2006–2022 were reviewed. Eighteen papers, totalling 57 patients, were found and analysed. Statistical analysis was undertaken using Chi-squared and Wilcoxon rank-sum tests as appropriate, with  Human Immunodeficiency Virus (HIV) is the virus responsible for Acquired Immunodeficiency Syndrome (AIDS), and despite concerted efforts, it remains a global health issue, with ~37 million people infected worldwide. Despite major advances in the diagnosis and management of HIV, it remains a leader in morbidity and mortality, particularly in Sub-Saharan Africa, where the majority of infections reside . During Disorders of the CNS associated with HIV represent a complex facet of HIV disease management, with the aetiology depending on the degree of immunosuppression, stage of disease and treatment history . They maEncephalitis is defined as the inflammation of the brain parenchyma, leading to neurological dysfunction that manifests as changes in cognition and consciousness. Though viral causes predominate, bacterial, autoimmune and fungal aetiologies can cause encephalitis in both immunocompetent and immunosuppressed patients . CD8+ T A literature review was conducted for this narrative review in July 2022. Papers were identified by utilising the PubMed database and attempted to examine CD8E concerning its aetiology, presentation and treatment in HIV-positive populations. Papers were selected if they were written in English, published between 2006–2022 and had undergone peer review.Search terms included ‘CD8+ T cells’, ‘HIV’ and ‘Encephalitis’. These terms were used in combination to identify search results. All authors independently participated in the evaluation of identified abstracts to ensure they fulfilled the study aims. All paper types, including reviews and case series/reports, were included in this analysis. Papers were excluded from analysis if information was inadequate, duplicated or incomplete, or if there were concerns of conflicts of interest or selection bias. From this initial search, 14 papers were found that met our inclusion criteria. A second literature search was undertaken in September 2022 including a screening of references, identifying an additional 5 papers. All selected papers were fully extracted and analysed. Data regarding patient demographics, symptomology, radiological and histological characteristics (where brain biopsy had been performed), cerebrospinal fluid (CSF) findings, treatment strategies, long-term outcome and preceding HIV control  were obtained where available and collated for analysis. In cases in which data were incomplete or not available, they were excluded from a given variable analysis. In cases of recurrence, data pertaining to the first incidence of CD8E were collected. A patient death was attributed to CD8E if either explicitly concluded so by the author or if clearly occurring during their presentation with CD8E.p-values < 0.05 were considered significant. For comparison of group differences, the Chi-squared test and Wilcoxon rank-sum test were used where appropriate.For independent comparisons, We identified 19 papers in this review, which consisted of 57 patients . Of idenThough only a minority of patients (3/57) presented on admission with CD8E as a presenting illness of undiagnosed HIV, 79% (45/57) of cases were on an established ART regime (range 0–17 years). Of the remaining cases , five had recently stopped therapy due to poor compliance and patient choice up to 5 months prior to their presentation.p = 0.006). Cognitive decline was the most commonly reported symptom in male patients, ranging from confusion to coma, being reported in 41% (9/22) of cases versus 12% (4/34) of females (p = 0.99). Overall features of vomiting and focal neurology were rarely reported. Importantly, constitutional symptoms were not described in any of the identified cases. Of those patients who died, headache was the most predictive, present in 64% (16/25) of patients (p = 0.02).The presenting features of patients were variable, with onsets over days to months. Common symptoms at presentation included headache, seizure and confusion; however, these occurred at different rates between males and females. In females, the most common symptom was headache, occurring in 62% (21/34) of females and only 27% (6/22) of males, respectively  (p = 0.0Of those with baseline serum CD4 performed at presentation (50/57), the median CD4 count was 315 cells/µL (range: 64–876 cells/µL), with a median serum HIV viral load (49/57) of 600copies/mL .Males (20/56) had higher baseline CD4 counts (median: 315 cells/µL) than females (median: 238 cells/µL). Baseline viral load was on average lower, but with a greater range in females  compared to males .p = 0.72). Patients that died demonstrated higher CD4 counts (median: 320 cells/µL) than those who survived (median: 227 cells/µL) (p = 0.079) . When considering serum investigations where available by outcome (47/57), those who died (20/47) had higher peripheral viral loads (median: 1676 copies/mL) versus those who survived (median: 660 copies/mL) (= 0.079) .CD4+ nadir was only reported in 20 cases, with a median value of 23 cells/µL (range: 5–516 cells/µL). Only three cases reported a CD4 nadir >200 cells/µL.p = 0.947). However, in comparison to those who died, survivors demonstrated lower median protein levels (0.93 g/L versus 1.12 g/L) and CSF viral load (1100 copies/mL versus 4968 copies/mL) . The median CSF white cell count was 29 cells/µL (range: 1–220 cells/µL) and was typically lymphocyte predominant. In cases in which the CSF lymphocyte count was subtyped, 90% demonstrated predominantly CD8+ cells with inverted CD4:CD8 ratio (18/20). The median CSF protein was moderately elevated at 0.92 g/L (range: 0.4–2.19 g/L). CSF HIV viral load was seldom performed (22/57), with a median value of 1200 copies/mL , and it was often in excess of the paired peripheral HIV viral load. Those who survived had higher markers of neuroinflammation versus those who died, as indicated by a higher median CSF cell count  .In the 18 patients in whom a simultaneous CSF viral load and serum viral load assessment was made, there was a trend towards higher CSF viral loads in peripherally suppressed patients .Of those with available imaging, 77% patients (41/53) had typical findings on MRI imaging, including bilateral, confluent and symmetrical high-signal-intensity lesions that were gadolinium contrast enhancing throughout the white matter and sometimes the grey matter, with preferentially perivascular distribution. Cases reporting only bilateral white lateral lesions without more specific features were labelled as semi-typical in nature and represented less than a quarter of cases (12/53). Overall, 40% (23/57) of patients received a brain biopsy as part of their diagnostic workup.p = 0.0008) with treatment length varying considerably between 1–30 months. In those who did not receive steroids, death occurred in 60% cases. The overall case fatality rate was 45% (25/55). Relapse rate was low (n = 4), but occurred multiple times in one instance. Time to death was rarely explicitly reported (10/25), but varied from 2 days to 5 years post diagnosis, with a median survival time of 5.5 months. Overall follow-up time (21/35) ranged from between 3 months–8 years, with a median of 10 months’ follow up. Summary characteristics are described in In 64% of patients (35/55), corticosteroids were used as part of a treatment regime. In those who received corticosteroids, the survival rate was 71% (25/35)  . Two casp = 0.006), whereas cognitive decline was more common in males. The symptomatology of CD8E, however, is markedly differentiated from HAND, in which a spectrum of cognitive impairment, including a dementia-like process with marked changes in executive function and impulse control, is seen. Typically, this occurs in elderly patients in a chronic manner, even amongst those with good viral control [As shown in our review, CD8E often presents in an acute/subacute manner with headache, confusion and seizures, and rarely with ataxia and drowsiness. Emergent findings in the demographics of our cases are the wide range of ages amongst cases (19–69 years) and clear female and Black African predominance, which has been commented upon previously . Though  control ,38.As our data show, CD8E can occur within both ART-naïve and well-controlled HIV patients, with no clear preponderance to treatment status or degree of immunosuppression (CD4 count range: 64–876 c/µL). Though the majority of our cohort was well established on ART at the time of presentation, three patients presented as a new diagnosis of HIV with CD8E. This would suggest that CD8E should be considered an indicator condition for HIV testing, as it has not been described in any other immunocompromised population group to date. Moreover, this makes the risk of an iatrogenic pathophysiology underlying CD8E somewhat less likely.In the 35% (20/57) of our cohort with available CD4+ nadir measurements, the average value was 96 cells/µL. A lower CD4+ nadir has been observed to be a predictor of neurocognitive impairment and HAND, and it has been linked to the establishment of a viral reservoir in the CNS and subsequent CSF escape ,40. Accop = 0.02), which, if linked with increased ICP, would explain the paucity of CSF investigations undertaken in our cohort. Electroencephalogram (EEG) studies were rarely reported, but often showed diffuse and generalised slowing, with no signs of epileptiform activity. Though these are non-specific findings, it is important to note that the value of EEGs is in diagnosing conditions mimicking encephalitis such as non-convulsive status epilepticus. They can also provide aetiological clues, such as in HSV encephalitis, and so should be encouraged [Lumbar puncture (LP) is an essential investigation in the diagnosis and management of encephalitis in HIV and non-HIV patients. Rather strikingly, CSF analysis data were not available in a quarter of cases (15/57). The most common reason for not performing an LP was the concern or presence of symptoms of raised intracranial pressure (ICP). This is a commonly cited reason for not performing an LP, given the risks of cerebral herniation. However, LP should be performed in all patients upon ruling out raised ICP on neuroimaging, as it is critical in establishing aetiology by means of serology, culture and molecular methods . In factcouraged .n = 21), our data show that CSF viral load can be highly raised (median 1200 copies/mL), even in the setting of a suppressed plasma viral load. The significance of CSF viral load is important, given its correlation with neurological dysfunction, although the significance of low-level CSF viraemia is uncertain in patients on existing ART [Though rarely performed in conjunction with peripheral viral load  found at the time of symptoms within the CNS and not in the periphery. In conjunction with the raised CSF viral load, this is highly suggestive of viral escape, which may be a trigger for CD8E development ,19. Indep = 0.947). Where CSF lymphocyte subsets were tested, 90% demonstrated predominantly CD8+ lymphocytes, often with a significant CSF CD8+/CD4+ ratio up to 5:1 in nature [CSF analysis in CD8E clearly demonstrates a marked lymphocytic pleocytosis (29 cells/mL) alongside a significantly raised protein (0.92 g/L) in the setting of normal CSF: plasma glucose ratio ,18,19,20n nature ,23,30. Tn nature  and myasn nature , the caun nature . This suOf interest, in one case by Kerr et al. , low-levn = 340), 57% of confirmed encephalitis cases had no cause identified [A notable observation is the high rate of brain biopsy as part of the diagnosis and management of patients with CD8E. In our case series, 23 (40%) cases had biopsy undertaken as part of their initial management. Though this may seem appreciably high, it is important to consider that in retrospective cohort studies  encephalitis . TherefoPeriventricular and deep white matter hyperintensities are also seen in HAND, which may be enhanced upon contrast imaging . HoweverOther common diagnoses to consider in the setting of predominant white matter lesions of the CNS in the setting of HIV include PCNSL, Progressive Multifocal Leukoencephalopathy (PML) and Acute Disseminated Encephalomyelitis (ADEM). PCNSL is a non-Hodgkin lymphoma that is intricately related to EBV infection, an important consideration in severely immunosuppressed patients, and it occurs in 2–6% of HIV patients with advanced disease (CD4 < 100 cells/uL). However, it classically presents with rapidly acute symptoms of personality change and aggression, which are commonly described, but no seizure or signs of increased ICP, such as in CD8E, with imaging often showing single/multiple focal lesions with central necrosis and haemorrhage .PML, caused by reactivated JC virus, is linked to secondary immunosuppression from medications , but is most commonly described in the setting of HIV infection, with an incidence of 0.7 per person-year of follow up . ClassicADEM, like PML, is typified by demyelination throughout the CNS, but it ordinarily has a trigger, which may include preceding viral infection or vaccination receipt, but it can be secondary to HIV infection. Unlike in CD8E, typical features include predominant motor dysfunction and ataxia with concurrent fever and seizures. Like CD8E, symptoms can be subacute, although hyperacute forms complicated by haemorrhage are recognised ,56. MRI As such, amalgamating radiological, clinical and biochemical correlates can help the attending physician in the recognition of CD8E.p = 0.0008), making this a useful intervention in suspected cases. Commonly employed regimens have involved intravenous methylprednisolone pulsed over 3–5 days, followed by an oral steroid taper over durations ranging from 1 month to 30 months, most commonly with prednisolone. Importantly, there was variability in the times until treatment initiation, which was up to 33 days in one case [Across reported cases in the literature, it appears that CD8E is a steroid-responsive disease, with marked improvements in neurological deficits and radiological findings seen amongst recipients ,18. In oPublished cases show varying recovery timeframes, which range from a few days to several months, with as of yet no discriminating factors for responders versus non-responders. Although follow up was performed in most survivors, it was done so for a relatively short period of time (median of 10 months). As such, we presently cannot be sure of any long-term consequences of CD8E in the follow up of survivors ,19.Given the plethora of side effects from chronic steroid use, the use of a steroid-sparing regime is an important consideration, as is the general use of immunosuppression in already immunosuppressed individuals. Mycophenolate Mofetil (MMF) has been employed for this purpose and seems to be a good choice given its relatively good CSF penetration and tolerability. In three reviewed case reports of its use, it allowed for sustained improvements in neurology and steroid dose weaning, but it did not necessarily alter the outcome, with two of these cases dying ,22,30.Of note, MMF has been used in two cases of relapsing CD8+ with contrasting outcomes. Salam et al. reported success in employing MMF as a means of offsetting steroid dependency in a case of multiple recurrences of symptomology . HoweverCD8E is very rarely observed to follow a polyphasic course following treatment, though Salam et al. reported multiple relapses over a 3-year period, directly relating to steroid weaning. Interestingly, in two papers reviewed, CSF viral load was seen to rise between recurrences despite prior treatment and alongside stable peripheral viral loads, raising the question as to whether CSF viral escape may be the driving pathophysiology behind relapsing disease and could be a marker to predict recurrence ,22.CD8E represents a new and poorly understood condition which has the potential to affect HIV patients independent of viral control. Though typically associated with abrupt symptoms, it remains clear that it can lead to protracted disease with a high case fatality ratio and an overreliance on brain biopsy to diagnose. It is apparent from our review that more robust information is needed to understand the natural history of this condition. Certainly, wider use of CSF analysis, including CSF viral load and CD8+ T lymphocyte count, as well as a better understanding of patient characteristics will be needed to allow for advances to be made. Importantly, though few cases exist in the literature, it is likely that many of the undifferentiated encephalitis diagnoses found in clinical practice in HIV patients could be ascribed to this. Given the high fatality rate, CD8E should be considered in all HIV patients presenting with encephalitic disease, regardless of premorbid status, with the timely initiation of immunosuppressive therapy alongside a review of ART and genotypic resistance testing.