TBM is the most severe form of tuberculosis. Clinical trial data are required to provide an evidence base for adjunctive dexamethasone in HIV-positive individuals with TBM, and to guide clinical practice. This document details the planned analyses at 12 months post randomisation for the ACT HIV clinical trial (NCT03092817); ‘a randomised double-blind placebo-controlled trial of adjunctive dexamethasone for the treatment of HIV co-infected adults with tuberculous meningitis (TBM)’. The primary endpoint of the ACT HIV trial is death (from any cause) over the first 12 months after randomisation. This statistical analysis plan expands upon and updates the analysis plan outlined in the published study protocol. A further analysis will follow at 24 months post randomisation. This statistical analysis plan expands upon and updates the analysis plan outlined in the published study protocol despite the best available appropriate anti-TB chemotherapy.The ACT HIV clinical trial is a randomised double-blind placebo-controlled trial of adjunctive dexamethasone for the treatment of tuberculous meningitis (TBM) in HIV-positive adults. TBM is the most severe form of tuberculosis, with mortality approaching 50% in people living with HIVMycobacterium tuberculosis, initially acquired via the respiratory route, disseminates via the blood to form a secondary focus in the brain. Rupture of this secondary focus into the subarachnoid space results in TBM, with the consequent host inflammatory response being important for bacterial killing, but also responsible for the pathological complications and often-fatal consequences of the infection.TBM develops when. In 2004, a landmark clinical trial in Vietnam demonstrated that a 6–8 week (duration dependent on Modified Research Council [MRC] TBM severity grade) tapering course of dexamethasone reduced mortality in adults and adolescents with TBM. In this study, only 98/545 (18.0%) individuals were HIV positive, which was too small to determine if dexamethasone benefited this important sub-group of patients. No other trials have been published since 2004 that examine whether corticosteroids improve outcomes from TBM in HIV-positive adults.Adjunctive anti-inflammatory corticosteroid therapy, which may control excessive host inflammation, has long been considered to have a role in TBM treatment. Whether adjunctive dexamethasone is more or less likely to be beneficial in HIV-positive individuals with TBM is not known. There are an estimated 100,000 cases of TBM globally each year, many associated with HIV. Clinical trial data are required to provide an evidence base for adjunctive dexamethasone in HIV-positive individuals with TBM, and to guide clinical practice.How dexamethasone might confer its clinical benefit on those with TBM remains uncertain. A recent study of cerebrospinal fluid (CSF) cytokines in TBM suggested that HIV-positive individuals have higher cytokine concentrations than HIV-negative individualsth May 2017. By April 29th 2021, the predefined sample size of 520 adults with TBM and HIV co-infection had been enrolled from four hospitals in Vietnam and Indonesia: the Hospital for Tropical Diseases (HTD) and Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease (PNT) in Ho Chi Minh City, Vietnam, and Cipto Mangunkusumo Hospital and Persahabatan Hospital in Jakarta, Indonesia. Detailed enrolment criteria for ACT HIV, including consent and ethical approvals, are described in the published trial protocol.The ACT HIV trial commenced recruitment on 25Once enrolled, study participants were randomised to dexamethasone or placebo , with this intervention termed ‘study drug’. Randomisation was stratified by TBM MRC severity score and by hospital. Participants with MRC grade 1 TBM received a 6-week tapering course of study drug, whereas participants with MRC grades 2 or 3 received an 8-week tapering course of study drug. Study drug regimens are shown in. Key follow up outcomes include survival, disability, and severe adverse events; especially neurological events, events requiring corticosteroids, HIV-associated complications , and adverse events considered related to dexamethasone.ACT HIV has numerous sub-studies, as described in the protocolThe primary endpoint of the ACT HIV trial is death (from any cause) over the first 12 months after randomisation.The secondary endpoints of the ACT HIV trial are as follows:1. Neurological disability at 12 months from randomisation2. First new neurological event or death over the first 12 months after randomisation3. Neurological IRIS events over the first 6 months after randomisation4. New AIDS-defining event or death over the first 12 months after randomisation5. HIV-associated malignancy by 12 months from randomisation6. Use of open-label corticosteroid treatment for any reason over the first 12 months after randomisation7. Requirement for shunt surgery by 12 months8. Any serious adverse events reported by 12 months from randomisationThe primary analysis includes all randomised participants. As such, an intention-to-treat (ITT) analysis will be performed for the primary and secondary endpoints. Participants will remain included in this analysis even if no study drug was received after randomisation.. A per protocol analysis will be performed for all primary and secondary endpoints.The per protocol (PP) analysis includes all randomised patients with the exception of those subsequently found to have not met all inclusion criteria, or to have met any exclusion criteria, at the time of enrolment, those with a final diagnosis other than TBM , and those who received less than 7 days of administration of the randomised study drug for reasons other than death, or less than 30 days of anti-tuberculosis drugs for any reason other than death. Trial inclusion and exclusion criteria are listed in the trial protocol, using the most up to date version available at the time of final analysis.Data will be analysed using the program RDefinition: The following baseline characteristics will be summarised by treatment arm for ITT and PP analyses: age, sex, country, site, diagnostic category , history of previous tuberculosis treatment, chest X-ray findings (no TB/miliary TB/pulmonary TB), enrolment MRC TBM grade, enrolment Glasgow coma score (GCS), weight (kg), duration of symptoms, cranial nerve palsy, hemiplegia, para/tetraplegia, urinary retention, history of diabetes, HbA1c, history of intravenous drug use, hepatitis B sAg positivity, hepatitis C Ab positivity, alanine aminotransferase (ALT), bilirubin, full blood count , plasma sodium,leukotriene A4 hydrolase (LTA4H) genotype (CC/CT/TT), routine CSF parameters , duration of anti-tuberculosis chemotherapy (days) before enrolment, enrolment anti-TB regimen, anti-TB drug susceptibility results in culture-confirmed sub-group , HIV infection (new or known diagnosis), anti-retroviral therapy (ART) treatment at enrolment  and time to starting ART (if ART naïve), enrolment CD4 count , HIV viral load. These will be described in a baseline variable table  for continuous data and frequency (percentage) for categorical data. The amount of missing data for each baseline characteristic will also be displayed. We will perform multiple imputation of a variable in case of >5% missing values, and it is assumed that data are missing at random. Otherwise, a complete case analysis will be performed. will be used and subjects will be categorised as ‘definite’, ‘probable’, ‘possible’, or ‘not TBM’. Participants will only be categorised as ‘not TBM’ if they have a confirmed alternative diagnosis  or they recovered without TB drugs.The published TBM diagnostic scoreThe primary endpoint of this trial is death over the first 12 months after randomisation. The primary analysis is a Cox proportional hazards regression model with treatment as the only covariate. Since TBM MRC severity grade at enrolment  is an important risk factor for mortality and was used as stratification variable in the randomisation, we will additionally perform a secondary analysis with MRC grade added as stratum variable. The proportional hazards assumption will be formally tested based on scaled Schoenfeld residuals and visually assessed by a plot of the scaled Schoenfeld residuals versus transformed time. Deaths, by study drug arm, and the results of the analyses will be shown as perSurvivors known to be alive at 12 months will be censored at that time-point and subjects who withdrew or were lost to follow-up before 12 months will be censored at the date they were last known to be alive. Subjects who withdrew or were lost to follow-up before 12 months are estimated to be less than 5% of enrolled participants.LTA4H genotype (CC/CT/TT), drug resistance pattern , ART status at enrolment , and CD4 cell count at enrolment . We will estimate and report the treatment effect by fitting a separate Cox model per subgroup. We will additionally fit a Cox model that includes CD4 count at enrollment as a continuous variable via a restricted cubic spline with four knots and an interaction term with treatment arm.The homogeneity of the treatment effect on overall survival across subgroups will be assessed by formal tests of interaction between treatment and the following pre-defined grouping variables: TBM MRC severity grade at enrolment , diagnostic category , package in R).Kaplan-Meier plots and explicit survival estimates at 3, 6, and 12 months of follow-up will be calculated for the full sample and in the subgroups defined above. We will compare the treatment arms via the absolute risk of death at 12 months  and the restricted mean survival until 12 months  or the competing risks Aalen-Johansen estimates  and report the values of the estimates at 3, 6 and 12 months. We will also fit a Cox proportional hazards model (results are interpreted as relative cause-specific hazards in the presence of competing risks). Analyses will be performed for the full samples (ITT and PP). Subgroup analyses are specified below per secondary endpoint. For the subgroup analyses, we follow the same procedure as for the primary outcome: we fit separate models per subgroup and we fit a model in which we test for interaction by subgroup.Neurological disability will be assessed by the modified Rankin score at months 1, 2, 3, 6, 9, and 12 from randomisation . The maiNeurological disability  at 12 months will be compared between the two arms with a proportional odds logistic regression model with the treatment assignment as the main covariate and adjustment for TBM MRC severity grade. The result will be summarised as a cumulative odds ratio with corresponding 95% confidence interval and p‐value. Individuals who withdrew or were lost to follow-up before 12 months are excluded.A new neurological event is defined as a fall in GCS by ≥2 points for ≥2 days from the highest previously recorded GCS (including baseline) or the onset of any of the following clinical adverse events: cerebellar symptoms, focal neurological signs, or onset of seizures. A description of all the events will be given, summarising how they met the criteria.Analyses will additionally be performed in the subgroups defined by TBM MRC severity grade, diagnostic category, ART status at enrolment, time from randomisation to start of ART, and baseline CD4 count.. The neurological IRIS rate will be defined as the number of IRIS events divided by the observed person-time of follow-up in each treatment group. A description of all the IRIS events will be given, describing how they met the IRIS diagnostic criteria.The criteria for neurological IRIS diagnosis is defined in the study protocolThe number of IRIS events in each group will be summarised. Death will be interpreted as a competing risk. Analyses will additionally be performed in the subgroups defined by TBM MRC grade, diagnostic category, ART status at enrolment, time from randomisation to start of ART, and baseline CD4 count..Acquired immunodeficiency syndrome (AIDS)-defining illnesses will be defined as per the World Health Organisation (WHO) classification. WHO define AIDS as a clinical diagnosis (presumptive or definitive) of any stage 4 HIV conditionHIV-associated malignancy is defined as new diagnosis of one or more of the three major HIV-associated malignancies; Kaposi sarcoma, high grade B-cell non-Hodgkin lymphoma or invasive cervical cancer.The number of HIV-associated malignancy events in each group will be summarised. Death will be interpreted as a competing risk. Analyses will additionally be performed in the subgroups defined by ART status at enrolment.The number of open-label corticosteroid treatment events in each group will be summarised. The reasons for corticosteroid treatment will be listed. Death will be interpreted as a competing risk.The number of shunts performed in each group will be summarised. Death will be interpreted as a competing risk.. SAE will be sub-grouped into categories. SAE will be grouped and graded as per Common Terminology Criteria for Adverse Events (CTCAE).Serious adverse events (SAE) are defined in the study protocol. Specific adverse events will be summarised, but not formally compared. The total number of serious adverse event episodes per patient will also be summarised and informally compared based on a quasi-Poisson regression model with treatment as the only covariate.The number of patients with any serious adverse event will be summarised and compared between the two treatment arms based on the chi-squared test, or Fisher’s exact test in case the expected count under the null hypothesis in at least one of the cells is smaller than oneThe following subgroups of adverse events will also be separately summarised: clinical grade 3&4 adverse events; serious adverse events possibly, probably, or definitely related to the study drug; adverse events leading to TB treatment or ARV interruptions. Grade 3&4 laboratory abnormalities will be summarised in the same way as clinical adverse events. Adverse events will be shown as perA multivariate model of variables independently associated with death will include: age, enrolment GCS, TBM diagnostic category , study drug allocation (dexamethasone vs. placebo), CD4 count, CSF total leucocytes, CSF total neutrophils, CSF blood:glucose ratio, CSF lactate. Numeric variables will be analysed using restricted cubic splines. We will not include any interactions.No data is associated with this article. Change is just a useful clarification.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Biostatistics and Infectious disease epidemiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Evidence from Clinical trials that have been done this far on disseminated tuberculosis and in Particular TBM have not been conclusive on the role of Corticosteroids in improving outcomes. Corticosteroids have been tried in Tuberculous pericarditis, granulomatous diseases and Leprosy reactions with good outcomes. Their role in bacterial meningitis particularly Waterhouse-Friderichsen Syndrome has been shown to be positive. Given the debate on BCG vaccine and its possible role in preventing Tuberculous meningitis it would be of value if there is a specific way of confirming the number of participants who have had the vaccine. Some of the Secondary end points may be drug related and it is therefore important to know the exact drug regimen used for both TBM and HIV. Otherwise an excellent piece of workIs the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Infectious diseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The issue of potential benefits of corticosteroids in HIV-associated TBM remains unaddressed so far. The authors have planned an almost flawless study to address the issue. The authors are trying the same corticosteroids regimen as had been used in earlier trials which included both HIV-negative and HIV-positive TBM patients. A small paragraph detailing the anti-tuberculosis regimen (drugs and duration) and the ART regimen should be added for better clarity. How will the authors address the issue of "paradoxical reaction"? I find vision loss a major disability among TBM survivors, kindly give due attention to this as well.  Regarding analysis, I feel that the use of median should depend on the distribution instead of assuming an asymmetric distribution. We can, therefore, include 'mean' too and look for its statistical relevance. In my opinion, the corticosteroids regimen that has been planned to use in this study is a bit complicated. With a simpler regimen compliance would be better.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:CNS tuberculosis and other CNS infectionsWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is slick analysis plan by a clearly highly experienced team for a multi-centre randomized clinical trial following highly standardized methodologies. The proposed analyses will lead to a correct, and generally accepted result, as well as guide the future standard of care guidelines for Tuberculous meningitis in HIV+ patients. As the drug on trial is cheap and easy to administer, guidelines will be applicable globally .Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Biostatistics and Infectious disease epidemiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Background: Cryptococcal antigen (CrAg) screening in individuals with advanced HIV reduces cryptococcal meningitis (CM) cases and deaths. The World Health Organization recently recommended increasing screening thresholds from CD4 ≤100 cells/µL to ≤200 cells/µL. CrAg screening at CD4 ≤100 cells/µL is cost-effective; however, the cost-effectiveness of screening patients with CD4 101–200 cells/µL requires evaluation.Methods: Using a decision analytic model with Botswana-specific cost and clinical estimates, we evaluated CrAg screening and treatment among individuals with CD4 counts of 101–200 cells/µL. We estimated the number of CM cases and deaths nationally and treatment costs without screening. For screening we modeled the number of CrAg tests performed, number of CrAg-positive patients identified, proportion started on pre-emptive fluconazole, CM cases and deaths. Screening and treatment costs were estimated and cost per death averted or disability-adjusted life year (DALY) saved compared with no screening.Results: Without screening, we estimated 142 CM cases and 85 deaths annually among individuals with CD4 101–200 cells/µL, with treatment costs of $368,982. With CrAg screening, an estimated 33,036 CrAg tests are performed, and 48 deaths avoided .  While CrAg screening costs an additional $155,601, overall treatment costs fall by $39,600 , yielding a net increase of $116,001. Compared to no screening, high coverage of CrAg screening and pre-emptive treatment for CrAg-positive individuals in this population avoids one death for $2440 and $114 per DALY saved. In sensitivity analyses assuming a higher proportion of antiretroviral therapy (ART)-naïve patients (75% versus 15%), cost per death averted was $1472; $69 per DALY saved.Conclusions: CrAg screening for individuals with CD4 101–200 cells/µL was estimated to have a modest impact, involve additional costs, and be less cost-effective than screening populations with CD4 counts ≤100 cells/µL. Additional CrAg screening costs must be considered against other health system priorities. This includes a sizable population with advanced HIV disease (CD4 ≤200 cells/µL) who are at an increased risk of opportunistic infections such as cryptococcal meningitis (CM). Reflex cryptococcal antigen (CrAg) screening with targeted fluconazole treatment for the prevention of CM was adopted in national HIV guidelines in 2016 at a CD4 count threshold of ≤100 cells/µL. We previously found screening at this threshold to be highly cost-effective  and likely to prevent a significant proportion of CM cases and deaths.Botswana had an estimated adult HIV prevalence of over 20% in 2018, with approximately 350,000 adults living with HIV. Patients with CD4 counts of 101–200 cells/µL are also relatively immunocompromised and at risk for CM, but prevalence of CrAg positivity in this population, estimated at 2.0% : 1.2-2.7%; 21 studies) is substantially lower than prevalence among patients with CD4 ≤100 cells/µL. The impact and cost-effectiveness of increasing the CrAg screening CD4 count threshold have not been systematically evaluated, and a better understanding of the potential impact (in terms of CM cases and deaths avoided), screening program resource needs, and cost effectiveness will inform countries as they consider changes to national screening guidelines. In 2018, the World Health Organization (WHO) conditionally recommended increasing the CD4 count threshold for CrAg screening from ≤100 cells/µL to ≤200 cells/µL for the prevention of CM, we evaluated CrAg screening among patients who are antiretroviral therapy (ART)-naïve (those targeted for pre-emptive treatment in guidelines) as well as ART-experienced patients found to be CrAg-positive through reflex CrAg screening. This ART-experienced population re-engaging in care and treatment now makes up about half of those with incident CM in the region and are likely to derive clinical benefit from pre-emptive fluconazole treatment for the prevention of CM.Using data and estimates from Botswana in patients with a CD4 count of 101–200 cells/µL, the objective of this analysis is to expand our CrAg screening models to include those with a CD4 count of 101–200 cells/µL, with an aim of informing policy regarding CrAg screening for patients with higher CD4 counts. As in our previous analysis, but in this analysis focused on those with a CD4 count of 101–200 cells/µL. The model estimates number of CM cases, CM-related deaths and disability-adjusted life years (DALYs) lost, and associated costs of CM management in the absence of screening  develop CM without CrAg screening and (2) with national reflex CrAg screening adoption, as previously describedreening . This isreening ., and local CrAg prevalence and titre data used to predict risk for progression to CM in the CD4 101–200 cells/µL population. Local data were obtained from a 2018–2019 CrAg screening cohort of patients with advanced HIV disease in Gaborone, which included over 900 patients with a CD4 count of 101–200 cells/µL who received reflex CrAg screening and were followed for up to 6 months for incident CM and mortality. In our model, based on local estimates we assume that 650,000 CD4 tests are performed annually for the adult HIV-positive population of 350,000 (around two tests per patient).For these models, CD4 count distribution data were obtained from the Botswana-Harvard HIV Reference Laboratory), adapted from our previous model, estimates the proportion of patients who receive CD4 testing with a CD4 101–200 cells/µL, how many of these patients receive reflex CrAg screening, the proportion who are CrAg-positive, and the proportion previously initiated on ART, i.e. “ART-experienced”  and the target population for pre-emptive fluconazole treatment. Based on data from the prospective 2018–2019 CrAg screening cohort, with a titre >1:160 corresponding with a high risk for incident cryptococcal disease. Approximately 20% of CrAg-positive outpatients with a CD4 101–200 cells/µL had a high CrAg titre, compared to 59% among those with lower CD4 counts of ≤100 cells/µL. For our CD4 101–200 cells/µL models, we assume that patients who screen CrAg-positive and return to clinic are started on pre-emptive fluconazole therapy and none receive a diagnostic lumbar puncture to evaluate for central nervous system infection, given the lower distribution of CrAg titres in the CD4 101–200 cells/µL population compared to ≤100 cells/µL and frequent lumbar puncture refusal in routine-care settings. We used serum CrAg titre data to stratify the risk of CrAg-positive patients progressing to CM Our base model assumes that 5% of patients with CD4 101–200 cells/µL do not receive CrAg screening due to laboratory error or assay stockout and that 10% of patients who screen CrAg-positive do not return to clinic to begin pre-emptive fluconazole, putting them at higher risk for progression to CM. and key parameter assumptions inThebase model treatment module  or low (≤1:160) CrAg titre. For patients who either don’t receive CrAg screening or receive screening but do not subsequently initiate fluconazole therapy, given the comparatively lower CrAg titre distribution in patients with higher CD4 counts of 101-200 cells/µL, we expect a longer delay until progression to CM in the absence of pre-emptive fluconazole compared to CrAg-positive patients with CD4 cell counts ≤100 cells/µL . However, initiation of fluconazole therapy in CrAg-positive patients further reduces the risk of progression to CM in the population with CD4 101-200 cells/µL.Full modeling assumptions are detailed in a Microsoft Excel file accessible online. Patients who are recognized as CrAg-positive and started on pre-emptive fluconazole but subsequent fail therapy and are admitted to the hospital for the management of CM are assumed to have better clinical outcomes  based on limited data from South Africa. Some patients who develop CM and survive hospitalization may develop relapsed CM. Given the small proportion of these patients and small clinical and public health impact, we do not consider them further in our models.Outcomes of patients who develop incident CM are informed by local mortality data from Botswana, with approximately 50% of patients dying within 10 weeks of CM diagnosis under routine care conditions. Very little outcome data exist in this disparate sub-population, which consists of patients: (1) recently started on ART; (2) ART-experienced who defaulted and are now re-establishing care; and (3) ART-experienced but with treatment failure. From local 2018–2019 cohort data in Botswana, approximately 75% of these ART-experienced patients are considered to have recently started on ART , 20% are on ART but with a recent unsuppressed HIV viral load signifying treatment failure, and 5% have a history of recent ART use without a recent HIV viral load signifying likely ART default. For those recently started on ART, we assumed a 33% reduction in risk of CM for those with CD4 101–200 cells/µL compared to our previous estimates for those with CD4 ≤100 cells/µL. In our base model, based on prospective cohort data, those recently started on ART with a suppressed HIV viral load have a low risk of progression to CM without pre-emptive fluconazole therapy (7%), with a greater risk in those with ART treatment failure (60%) and ART defaulters (33%). The combined risk of progression to CM for all ART-experienced patients in the CD4 101–200 cells/µL group is assumed to be 19% without pre-emptive treatment. We estimate an 87.5% reduction in risk of incident CM with pre-emptive fluconazole (factoring in a relatively low baseline CrAg titre distribution in this group). With reflex CrAg screening, patients receive CrAg screening based on CD4 count regardless of prior ART status. However, most (85%) patients with a CD4 count of 101–200 cells/µL are now ART-experienced according to recent cohort data from Botswana 2018–2019). Patients who screen CrAg-positive and receive pre-emptive fluconazole are treated with fluconazole 1200 mg/day for 2 weeks, followed by 800 mg/day for 8 weeks, then 200 mg/day maintenance fluconazole for an average duration of six months pending CD4 count recovery. For patients who progress to CM, hospital bed-day costs, factoring length of hospital admission, were derived using WHO-CHOICE estimates. CM treatment costs are based on two inpatient weeks of amphotericin B deoxycholate with high-dose fluconazole, intravenous fluid and electrolyte supplementation, and laboratory monitoring, followed by consolidation and maintenance fluconazole, as recommended in national treatment guidelines.Costing data for CrAg screening, pre-emptive therapy, and CM treatment costs are derived using local costing data when available  along with associated costs for screening, pre-emptive fluconazole therapy, and CM treatment. We estimate the cost per death averted and cost per DALY saved compared to no screening, assuming an average age of death of 36 years so that alternative sensitivity analyses can be completed by interested readers.Three main sensitivity analyses are reported to account for key areas of parameter uncertainty. The complete Excel-based model is provided as underlying dataSensitivity analysis 1 (SA1): In this analysis, we assume that in some real world settings a lower proportion of CrAg-positive patients are started on pre-emptive fluconazole after laboratory testing (50% versus 90% in the base model) because of programmatic barriers such as inadequate communication of test results to clinics, a lack of fluconazole availability in clinics, lack of provider awareness of treatment guidelines, or for other reasons. This analysis still assumes that 90% of patients attended in outpatient clinics and receiving CD4 testing will stay engaged in health care. Other parameters remain the same as the base model.Sensitivity analysis 2 (SA2): In this model, we assume less benefit of pre-emptive fluconazole in CrAg-positive patients, with a 75% rather than 87.5% reduction in incident CM. This is to account for significant uncertainty in the benefits of pre-emptive fluconazole in this population with a higher CD4 count, and for possible sub-optimal adherence to therapy. Other parameters remain the same as the base model.Sensitivity analysis 3 (SA3): In this model, we test our parameters with a higher proportion of ART-naïve patients receiving CD4 testing and CrAg screening (75% versus 15% in the base model). Other parameters remain the same as the base model. This is to provide estimates applicable to settings with less mature ART programmes where a higher proportion of individuals with CD4 counts of 101–200 cells/µL are likely to be ART-naïve.Sensitivity analysis 4 (SA4): In this model, we expand model 3 which assumes a greater ART-naïve population than observed in Botswana. We also consider lower costs of CM care in other settings. Based on a costing analysis from a cryptococcal meningitis clinical trial that enrolled patients from four countries in sub-Saharan Africa, we use a reduced cost of $2125.00 for two weeks of hospitalization with amphotericin B and fluconazole therapy for incident CM. This model also includes a lower cost of fluconazole therapy used for either CM treatment or targeted preventive treatment for CrAg-positive patients. As in the base model, here we assume a lower cost of care in patients who die during hospitalization (75%). Other costs remain unchanged compared to the base model. . Of patients with incident CM, 60% (85/142) are estimated to die . The total estimated CM treatment costs are $368,982 annually for the health care system.Without CrAg screening , we estiWith implementation of reflex CrAg screening , 33,036 ), with an estimated 56% (60/107) reduction in CM-related deaths at a cost of $1472 per death averted and $69 per DALY saved. For SA4, the number of deaths averted and DALY saved is equivalent to SA3. The lower cost of hospital management of incident CM is offset by the lower cost of fluconazole pre-emptive treatment in this model, with an overall similar cost of $1132 per death averted and $53 per DALY saved.SA1 and SA2 assume a lower proportion of CrAg positive are started on pre-emptive fluconazole and a reduced benefit of pre-emptive fluconazole therapy for CrAg-positive patients with a CD4 101–200 cells/µL, respectively, which may be more realistic under many routine care conditions. Both models will therefore result in a smaller public health benefit to CrAg screening and a higher incremental cost per death or DALY saved. For SA1, an estimated 25% (21/85) of deaths are averted with treatment of both ART-naïve and ART-experienced with a cost per death averted of $7476 or $349 per DALY saved . For SA2Overall estimated costs, number of CM cases, number of deaths averted, and DALYs saved for the base model and sensitivity analyses are summarized in, the benefit of screening for those with higher CD4 counts, in terms of avoided CM cases and deaths, is less marked. Under base model assumptions compared to no screening for this higher CD4 category of patients, 48 deaths are averted and screening costs of about $156,000 are offset by a $40,000 reduction in treatment costs . The cost per death averted through CrAg screening and pre-emptive fluconazole therapy was estimated at about $2400 ($114 per DALY avoided). If substantially fewer patients who screen CrAg-positive are started on pre-emptive fluconazole therapy , which might better reflect some real-world conditions without focused efforts on providing preemptive treatment, the estimated cost per death averted increases to over $7000 (and $349 per DALY saved).We used local data from Botswana to estimate the cost and impact of laboratory-based CrAg screening for HIV-positive patients with CD4 counts 101–200 cells/µL across a range of assumptions. Compared to screening in patients with very advanced HIV disease (CD4 ≤100)), which reduces the risk of incident CM and failure of pre-emptive fluconazole. In addition, overall CrAg prevalence among the CD4 101–200 cells/µL group is estimated to be less than the CD4 ≤100 cells/µL group. Both of these factors reduce the benefit of screening among patients with higher CD4 counts.  Compared to prior analyses of CrAg screening for patients with CD4 ≤100 cells/µL, fewer CrAg positive patients with CD4 101–200 cells/µL are likely to have high CrAg titres  of patients who received CD4 testing and CrAg screening were ART-naïve, which may inform other health systems with a higher proportion of ART-naïve patients receiving CrAg screening with ART initiation. This sensitivity analysis showed a slightly better impact and cost-effectiveness compared to the base model assuming most patients were ART-experienced although screening was still not cost-neutral or cost-saving. As of 2021, Botswana has an advanced ART program. Whereas CrAg screening guidelines have primarily focused on ART-naïve patients This study is subject to a number of limitations. First, we used local clinical and costing estimates. The relative costs of CrAg screening, pre-emptive fluconazole therapy, and CM treatment between different health systems may impact cost-effectiveness of CrAg screening between settings. We therefore performed sensitivity analysis considering other published estimates of CM management and fluconazole costs in sub-Saharan Africa. Secondly, the base model presents optimistic management assumptions, with about 90% of CrAg-positive patients started on pre-emptive therapy and a nearly 90% reduction in incident CM assuming relatively good adherence. Sensitivity analyses showed that under less ideal assumptions the cost per death averted or DALY saved could increase substantially. Notably, under no model assumptions was CrAg screening in this population estimated to be cost-neutral or cost-saving. Third, there is considerable uncertainty in model estimates, particularly regarding the clinical benefit of pre-emptive therapy in ART-experienced patients. Fourth, we used local CD4 testing practices in Botswana to inform these estimates. Alternative testing practices, such as testing only ART-experienced patients who have treatment failure based on HIV viral load testing or who are newly engaging in care following default, may result in greater cost-effectiveness of reflex CrAg testing.. With current public spending on HIV care of approximately $103 million, added costs from screening under base model assumptions  could contribute marginally to governmental spending in HIV services (~0.1%). The decision of whether or not to adopt CrAg screening in national HIV advanced disease guidelines among patients with higher CD4 counts (101–200 cells/µL) will rely on the availability of these additional resources and competing health system priorities. In summary, nationwide CrAg screening in patients with advanced HIV disease with a CD4 count of 101–200 cells/µL in Botswana is estimated to have a modest impact  for a modest additional cost to the overall HIV/AIDS care and treatment program , with a relatively low cost per DALY saved ($114 base case). With less coverage of pre-emptive treatment for CrAg positive patients, the cost per DALY saved, compared to no screening, is estimated at about $350. Overall, expanding screening to this higher CD4 count population would be estimated to require about 33,000 additional CrAg tests annually, with an estimated cost of about $156,000. These models do not make assumptions about government willingness to pay thresholds. While the Government of Botswana is the ultimate decision maker for the public provision of health care, our results suggest that CrAg screening is cost effective based on a wide range of parameter assumptions, according to published thresholdshttps://doi.org/10.17605/OSF.IO/6QMNH.Open Source Framework: Cryptococcal antigen screening in Botswana, CD4 101–200.This project contains the following underlying data:CD4 100–200 full model data.xlsxCD4 101-200 full model data_27nov21.xlsxhttps://doi.org/10.17605/OSF.IO/6QMNH.Open Source Framework: Cryptococcal antigen screening in Botswana, CD4 101–200.This project contains the following extended data:- Figures S1 (flowchart of screening module) and S2 (flowchart of treatment module)Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the With the modifications I provide my approval for indexing.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This research describes a decision based model to analyse the cost-effectiveness of cryptococcal antigen screening among people living with HIV with an CD4 cell-count between 101-200 cells.  The authors use a previously published adapted model of which all the data is online available. The performed sensitivity analyses for several uncertainties in their data. It is an interesting topic as the WHO changed the CD4 threshold for cryptococcal screening.  It is unclear from the methods how much simulations are run in the model Table 2 says "hotel costs" I think this should be hospitalWhat is the willingness to pay threshold in Botswana. It is hard to make the conclusion whether screening people with a CD4 cellcount between 101-200 is cost-effective or even cost-saving if someone is not familiar with the numbers. Maybe also add the total HIV/AIDS care and treatment program budget just to place the amount in perspectiveLimitation 1 of the study can be addressed by changing this in a sensitivity analysis. That would make the work more useful for other countries too I have some minor comments/considerations:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:medical microbiology, virology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The study title is short and smart and gives a glimpse into what the paper is about. The study is well introduced however I would reorganize the paragraphs to get a better flow, that is: The first sentence of paragraph two can be moved to the beginning of the introduction to draw the picture on how big the HIV problem is in Botswana upfront and then talk about the rationale after. I would merge and reorganize the first two paragraphs. In the methods section, a great deal of work has gone into explaining how the different estimates were arrived at and what type of data was used to arrive at the estimates. This however blurs the description of the actual modeling. It would be beneficial to the reader to get a quick snap short of the final model (could be in a summarized figure placed within the text explaining the methods) This is especially because the figure S1 is very busy and can get confusing, the reader needs to understand what the final model is before trying to understand the smaller details of how the model was arrived at. The screening model clearly talks about the estimates included and how they are arrived at. The viral load(VL) test is used as the proxy for ART experience, it would be good to know if all these VL tests are done after 6 months of ART or at what time point this is done. Pragmatically with all logistical challenges in resource limited settings, a recommendation to do a six month viral load will mean the viral load was done at about  month eight or nine after ART start. Wondering if this choice of defining ART experience could have lumped many ART experience persons as non experienced.  Page 3, the last paragraph talks about the assumptions for those who did not receive CrAG screening, however its not clear where these estimates are derived from. Is this lab data or data from the prospective cohort. Also it seems to belong under the next subheading and yet has been placed under the screening model. The base model, the cost analysis and the sensitivity analysis are presented well and in detail and are supplemented by the tables giving a clear picture of what was being done. The discussion of study findings is comprehensive and puts them in context and the conclusions have been derived systematically from the data presented.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Cryptococcal meningitisI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Since the development of antiretroviral therapy (ART) with TDF plus 3TC plus EFV, this specific regimen has not been studied enough with long-term lipid and uric acid monitoring.A prospective follow-up cohort study was performed. Sixty-one treatment-naive male patients with human immunodeficiency virus (HIV) were divided into three groups based on their baseline CD4+ cell count . The lipid and purine metabolism parameters of the patients over 144 weeks were analyzed.P = 0.001, 0.000, respectively). Moreover, the percentages of hyper-cholesterolemia, hyper LDL cholesterolemia, hyper-triglyceridemia and low HDL cholesterolemia also gradually increased, especially low HDL cholesterolemia significantly increased (P = 0.0007). The lower the baseline CD4+ cell counts were, the higher the TG levels and the lower the TC, LDL-c and HDL-c levels were. But there was significant difference of only baseline LDL-c levels between the three groups (P = 0.0457). No significant difference of the UA level and the percentages of hyperuricemia was found between the different follow-up time point groups or between the three CD4+ cell counts groups . The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load.Within 144 weeks, TG, LDL-c, TC and HDL-c gradually increased, especially TC and HDL-c (Gradual increasing dyslipidemia was found in male patients with human immunodeficiency virus primarily treated with tenofovir plus lamivudine plus efavirenz for 3 years. There-fore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen. In recent years, the numbers of patients with human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) have sharply increased. By the end of 2019, approximately 38 million people worldwide lived with HIV, and 33 million people died of HIV-related diseases [3 and the viral load reaches undetectable levels within the first year of starting treatment, AIDS patients are predicted to have a normal life expectancy [The most effective treatment for AIDS is antiretroviral therapy, which can prolong life expectancy and improve quality of life , 5. Whenpectancy , 5. The pectancy –8. As ofpectancy –12.HIV infection itself and antiretroviral therapy (ART) treatment drugs can cause dyslipidemia. As a first-line ART program launched since the National Twelfth Five-Year Plan in China, the tenofovir (TDF) plus lamivudine (3TC) plus efavirenz (EFV) regimen has a weaker effect on lipid metabolism. Our previous study showed that newly diagnosed male AIDS patients had decreased total cholesterol (TC) levels, uric acid (UA) levels and high-density lipoprotein cholesterol (HDL-c) levels as well as increased triglyceride (TG) levels, especially patients with CD4+ counts < 200/µL. The dyslipidemia and decreased UA levels gradually returned to normal at 4 weeks after initial ART with the TDF plus 3TC plus EFV regimen. Due to the scarcity of female HIV-infected patients who come to the infection clinic of our hospital and have not received antiviral treatment, our previous study lacked the data on the dynamic changes of lipid metabolism and uric acid metabolism in newly diagnosed female AIDS patients after the application of TDF+3TC+EFV. Therefore, this study also did not include female HIV-infected patients. This specific regimen has not been studied enough using long-term lipid and uric monitoring, which is the focus of the current study.A prospective cohort study was conducted on sixty-one male patients with HIV who were treatment-naive with the TDF plus 3TC plus EFV regimen at the Public and Health Clinic Centre of Chengdu from October 1, 2012, to December 31, 2017.The inclusion criteria were as follows: age from 18 to 65 years old; gender is not limited; HIV-1 antibody positive according to enzyme-linked immune-sorbent assay and confirmed by Western blotting; CD4+ T cell count 500 µL within 30 days before enrollment; voluntarily signed informed consent and agreed to undergo follow-up analyses; no plan to move away from current address during the trial; no history of antiretroviral therapy.The following exclusion criteria were used in this study: patients with acute infections; patients with opportunistic infections or AIDS-related malignant tumors at the time of enrollment; patients with opportunistic infection occurring within 3 months before enrollment and still in unstable condition within 2 weeks before enrollment; patients with hemoglobin <9 g/dL, white blood cell count < 2000/µL, neutrophil count < 1000/µL, platelet count < 75,000/µL, serum creatinine > 1.5-fold upper limit of the normal value (ULN), aspartate aminotransferase/alanine aminotransferase/alkaline phosphatase > threefold ULN, total bilirubin > twofold ULN, serum creatine phosphokinase > twofold ULN, or creatinine clearance rate < 60 mL/min; women who were pregnant or lactating; current drug users; patients with severe mental or neurological diseases; patients with a history of alcoholism; and patients with severe digestive tract ulcers.AIDS, dyslipidemia and hyperacidemia diagnostic criteria were applied according to the corresponding guidelines –15. AccoThe participants were divided into three groups according to their baseline CD4 T cell counts: there were 26, 12, and 23 patients in the < 200, from 200 to 350 and > 350 cells/µL groups , respectThe subjects fasted overnight for at least 12 h. At 8:00 a.m. the next day after emptying stool and urine, anthropometric parameters, including height, body weight (BW), body fat weight, lean body mass weight, body mass index (BMI) and body fat percentage, were measured by specially trained researchers using a body fat measuring instrument.The subjects fasted overnight for at least 12 h. At 8:00 a.m. the next day, the venous blood of those patients was drawn to measure TC, LDL-c, HDL-c, TG, UA, HIV viral nucleic acid (HIVRNA), and T lymphocyte subsets.TC, TG, HDL-c, LDL-c and UA levels were measured by the enzymatic method of an automatic biochemical analyzer purchased from Zhejiang Eastern European Biological Products Company. HIVRNA was detected by fluorescent quantitative PCR; T lymphocyte subsets  were measured by flow cytometry using a Beckman flow cytometer.The follow-up time points were 0, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 weeks after patients underwent ART with the TDF plus 3TC plus EFV regimen [Databases were established according to the needs of the research by two researchers simultaneously collecting and entering data. All of the data were checked by the researchers to assess data integrity, authenticity, and accuracy.Patients and the public were involved in the development of the research question or in the design of the study. Patients received oral and written information about this study; however, they were not involved in the recruitment and implementation of the study. In addition, the burden of the intervention was assessed by the investigators. After signing an informed consent form by the participants, they were assessed for eligibility prior to data collection.The Statistical Package for the Social Sciences software version 17.0  and GraphPad Prism 8 (GraphPad) software were used for statistical analysis. TC, LDL-c, HDL-c, TG and UA levels normally distributed, and statistical analysis was conducted directly. Nonnormally distributed HIVRNA levels were subjected to natural logarithmic transformation before statistical analysis. Quantitative data were expressed as χ ± SD, and categorical data were expressed as rates or percentages. One-way ANOVA was used to compare metabolism parameters from baseline to 144 weeks, and a paired t-test was used to compare metabolism parameters between baseline and some follow-up time points. The Kruskal-Wallis H (K) test for K independent samples was used to compare the percentage of dyslipidemia and hyperuricemia from baseline to 144 weeks. The Mann-Whitney test for two independent samples was used to compare the percentage of dyslipidemia and hyperuricemia between baseline and some follow-up time points. One-way ANOVA was used to compare metabolism parameters between the three different CD4+ T cell count groups at the same time point. Two-way ANOVA was used to compare metabolism parameters between the three different CD4+ T cell count groups from baseline to 144 weeks. A p value < 0.05 was considered statistically significant.The study was approved by the hospital ethics committee of the Public and Health Clinic Centre of Chengdu (PJ-K2012-012-01). All patients gave written informed consent.Sixty-one treatment-naive male patients with HIV in the Public and Health Clinic Centre of Chengdu from October 1, 2012, to December 31, 2017, were divided into three groups according to their baseline CD4+ T cell count: there were 26, 12, and 23 patients in the < 200, 200 to 350, and > 350 cell/µL groups, respectively. There were 42 cases of infection through homosexual contact, 13 cases of infection through heterosexual contact and 5 cases involving both types of sexual contact. The general information, baseline immunity and virological indicators, and lipid metabolism parameters of 61 patients are shown in Table In 61 patients, the average CD4+ T cell count . Compared with baseline, there were significant differences in TC levels at 96 and 144 weeks . There were no significant differences in LDL-c .TC, LDL-c, HDL-c, TG and UA levels Figs. A all graeks Fig. A and in eks Fig. C (P = 0.P > 0.05). In contrast, the percentage of hypo-HDL cholesterolemia  and at 24, 48, 96, 144 weeks compared with baseline . The percentage of hyperuricemia .The percentages of hypercholesterolemia Fig. A, hyper mia Fig. C gradualmia Fig. C (P = 0.P > 0.05). The difference of the LDL-c levels . TC levels  , Tanzani3% , TanzIn this prospective 3-year follow-up cohort study was the first to report the use of long-term lipid and uric monitoring to assess the efficacy of the TDF+3TC+EFV regimen. The results showed that in patients treated with the TDF+3TC+EFV regimen for 3 years, TC, LDL-c and HDL-c levels gradually increased, especially TC and HDL-c levels. TG levels first gradually decreased and then gradually increased. Moreover, the percentages of hypercholesterolemia, hyper-LDL cholesterolemia and hypertriglyceridemia all gradually increased, while the percentage of hypo-HDL cholesterolemia gradually decreased. This shows that in the early stage of ART treatment, disordered lipid metabolism was improved, especially hypo-HDL cholesterolemia. However, along with prolonged ART, the proportion of hyperlipidemia and hypertriglyceridemia gradually increased, especially hyper-LDL cholesterolemia and hypertriglyceridemia after 48 weeks and hypercholesterolemia after 120 weeks, which ranged from 9.84%, 8.2%, and 24.59% at baseline to 21.43%, 21.43%, and 50.00% at 144 weeks, respectively.This alternate cause of early lipid metabolism may be partially due to the changes in not weight gain but appetite improvement among patients who contracted AIDS after ART treatment. Because within 48 weeks after ART, all anthropometric parameters, including body weight, body fat weight, lean body mass weight, body fat percentage and BMI, did not increase. While the long-term characteristics of lipid and purine metabolism may be partially due to the changes in weight gain among patients who contracted AIDS after ART treatment. Because among those patients body weight increased by 2.81 kg, and body fat weight increased by 2.74 kg from 48 to 144 weeks, but lean body mass weight did not increase over the 144 weeks.This finding was consistent with the literature report that among patients living with HIV for a mean duration of 17.4 years, 35.6% had ASCVD, and of those without ASCVD, 53–86% had intermediate or moderate-to-high 10-year ASCVD risk scores, cardiovascular risk factors including HIV, 31.9% had low high-density lipoprotein cholesterol levels, and 79.3% needed to receive statin therapy .3 increased the risk of hypercholesterolemia [In this prospective 3-year follow-up cohort study we assessed baseline CD4+ cell count, age, anthropometric parameters, and immunological and virological indicators impacting on lipid and purine metabolic parameters in male patients with HIV undergoing primary treatment, and found that the lower the baseline CD4+ cell count was, the higher the TG levels were and the lower the TC, LDL-c and HDL-c levels were. The risk factors for lipid and purine metabolic parameters included age, anthropometric parameters, and immunological and virological indicators. After comparisons between different time points over the 144 weeks and comparisons with baseline levels, no significant difference in UA levels was found. The lower the baseline CD4+ cell count was, the higher the UA levels were, and these changes were maintained throughout the follow-up period after ART treatment; however, there was no statistical significance in the change from baseline to 144 weeks between the three different CD4+ T cell count groups. That is, the TDF+3TC+EFV regimen and CD4+ T cell count at baseline had no long-term dynamic effects on purine metabolism. This finding was inconsistent with the literature report that a high CD4+ cell count was a risk factor for hypertriglyceridemia, while a CD4+ cell count less than 200 copies/mmerolemia . Regardlerolemia .To our knowledge, this prospective follow-up cohort study is the first to investigate this specific regimen with TDF plus 3TC plus EFV using with long-term lipid and uric acid monitoring and the risk factors for lipid and uric acid. The results showed that gradual increasing dyslipidemia was found in male patients with human immunodeficiency virus primarily treated with tenofovir plus lamivudine plus efavirenz for 3 years. The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. There-fore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen.In this cohort study the proportion of patients with abnormal lipid metabolism during the whole study period was less than that reported in the literature. The reasons may be related to the younger age of patients in this cohort, the duration of follow-up was not too long, and the TDF+3TC+EFV regimen had little effect on lipid metabolism.The present study had some limitations. The sample size was small, and this was a single-center cohort study that only involved the TDF+3TC+EFV regimen and male patients, and the duration of follow-up was not too long. Further multicenter studies, more ART regimens, increase the number of female patients and large-sample randomized controlled trials and longer the duration of follow-up are necessary.Gradual increasing dyslipidemia was found in male patients with human immunodeficiency virus primarily treated with tenofovir plus lamivudine plus efavirenz for 3 years. The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. Therefore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen.


Dolutegravir has replaced efavirenz in most low‐ and middle‐income countries, due to better tolerability and formidable resistance profile, but dolutegravir side effects suggest alternatives are needed. We evaluated doravirine resistance in South Africa as a first step to assess whether doravirine may replace dolutegravir.Pol sequences were generated using validated population‐based in‐house genotyping and submitted to Stanford HIVdb v8.9.A retrospective dataset was analysed for predicted doravirine susceptibility, including sequences obtained from three patient groups. First, data from 277 patients initiating antiretroviral treatment (ART) were collected between February 2013 and October 2014 as part of a national survey. Second, data from 788 patients experiencing NNRTI‐based ART failure were obtained between February 2013 and October 2014 as part of a national survey. Third, data derived from 584 patients who had genotypic drug resistance testing requested after NNRT‐based failure as part of individual patient management between January 2016 and December 2019. Less than 5% of patients initiating ART presented with genotypic doravirine resistance, whereas most patients experiencing NNRTI‐based ART failure presented with predicted intermediate (41.0%) or high‐level resistance (43.8%) to doravirine. High‐level resistance to doravirine was commonly predicted by the presence of at least three DRMs (79.7%). The predicted resistance profile to doravirine in ART‐naïve patients is promising, but less so in those experiencing failure to first‐generation NNRTIs. Accumulation of NNRTI DRMs seems to be an important factor in the poor resistance prediction for doravirine.Although doravirine is approved as initial therapy in patients who are ART‐naïve, it is currently recommended to obtain a genotype prior to the initiation of ART. Clinical studies are needed to ascertain whether predicted resistance profiles in ART naïve and NNRTI‐treated patients translate into poor clinical outcomes, especially in settings where genotypic resistance testing is not available. The preHowever, with wider use of dolutegravir, concerns about significant weight gain among African women treated with integrase inhibitors (InSTIs) have been raised , 7. It iIn vitro experiments indicate a different resistance pathway, and possible clinical efficacy to isolates with several NNRTI mutations [Doravirine, a third‐generation NNRTI, may be an alternative to dolutegravir, with comparable virological suppression for doravirine compared to ritonavir‐boosted darunavir , 10 and utations , 14, 15.We assessed the level of predicted resistance to doravirine in ART‐naïve and patients experiencing NNRTI‐based ART failure in South Africa.22.1Genotypic sequence data from two sources were included for this analysis. First, a well‐characterized cohort of patients initiating ART (n = 277)  and pati2.2Pol sequences were generated using validated population‐based in‐house genotyping methods, depending on the time of sampling [sampling , 20.https://hivdb.stanford.edu/).The 2019 IAS‐USA drug resistance mutation tables were used to identify HIVDR mutations , and thepol nucleotide sequences were submitted to GenBank using Bankit (https://www.ncbi.nlm.nih.gov/WebSub/); accession numbers: KU127587‐KU128374, KT892975‐KT893251 and MW125724 ‐ MW126307.The 2.3http://www.graphpad.com/quickcalcs). Two‐sided Fisher’s exact tests were used to compare mutation prevalence between groups, considering a p < 0.05 as statistically significant.Statistical analyses were performed using GraphPad QuickCalcs  Committee at the University of the Witwatersrand (Clearance Number M181158 and M190593).3pol subtype was HIV‐1 subtype C (98.6%). Overall, 47 out of 277 surveyed patients presented with at least one NNRTI drug resistance mutation  had a median age of 34 years and most were women (58.8%). The most common p = 0.0451). Only four patients (1.4%) with doravirine resistance harboured high‐level resistance, compared to 8.3% for nevirapine and 7.6% for efavirenz. Among patients with doravirine resistance, three patients presented with a single NNRTI mutation, whereas in 10 patients, two to four NNRTI mutations were detected  presented with genotypic doravirine resistance compared to 9.4% (n = 26) with efavirenz/nevirapine resistance (3 presentpol subtype was HIV‐1 subtype C (98.5%). Patients were predominantly treated with efavirenz‐based regimens ; duration of treatment was not available. At least one NNRTI DRM was detected in 95.1% of patients (n = 1305). The most common NNRTI DRMs were K103N/S , V106A/I/M , G190A/S/E  and Y181C/I/V . Exposure to efavirenz or nevirapine did not affect the prevalence of most DRMs, except for L100I, K103N/S and P225H, which were more frequently detected in efavirenz‐exposed patients ; whereas Y181C/I/V were more commonly detected in patients experiencing nevirapine‐based regimen failure (p = 0.0009). Figure Patients experiencing NNRTI‐based ART failure n = 1372) were mostly female (63.6%), had a median age of 37 years, and a median HIV viral load of 4.8 log copies/mL (IQR: 4.3 – 5.3 log copies/mL). The most common 372 were p < 0.0001). However, 41.0% and 43.8% of patients were predicted to harbour intermediate and high‐level resistance to doravirine respectively.Only a minority of patients retained full susceptibility to efavirenz and nevirapine  compared to 15.2% (n = 209) of patients who remained fully susceptible to doravirine (Predicted high‐level resistance to doravirine (n = 601) was commonly caused by at least three DRMs , whereas intermediate resistance (n = 562) was most often caused by the presence of one or two DRMs . The DRM patterns among patients with at least three DRMs were extremely varied, and no link between the treatment group or DRM pattern was found.To our knowledge, this is the first study looking at predicted doravirine resistance profiles in our setting. Doravirine follows a distinct resistance pathway compared to commonly used NNRTIs such as efavirenz and nevirapine .In our ART‐naïve cohort, 8.3% of patients presented with at least one NNRTI DRM, but predicted resistance to doravirine (4.7%) remained significantly lower compared to efavirenz/ nevirapine resistance (9.4%). Studies in Europe showed lower doravirine resistance in ART‐naïve patients, with only 1.4% in France, Italy and Greece  and 1.8%Although the predicted doravirine resistance profiles may look favourable in the ART‐naïve cohort, there are no clinical outcome data from settings without baseline genotyping. Real‐life data would be required to confirm these findings, as baseline genotyping is generally not available in LMICs. Only 15.2% of patients experiencing NNRTI‐based ART failure retained full predicted susceptibility to doravirine in our cohort. DRIVE‐BEYOND attempted to recruit patients with transmitted NNRTI mutations (K103N and G190A) and assess the efficacy of doravirine, but only 10 patients were recruited, and eight obtained virological suppression by week 48; one patient was lost to follow‐up at week 16 and one patient presented with virological failure at week 24 due to non‐adherence . A smallet al. found subtype C accounted for the highest prevalence of doravirine resistance [in vitro experiments with doravirine in subtype A and C [in vitro data suggest that the accumulation of NNRTI mutations increases doravirine resistance, despite the absence of doravirine‐specific mutations [The proportion of patients experiencing NNRTI‐based treatment failure and presenting with resistance to doravirine in our cohort (84.8%) was significantly higher compared to the proportion observed in two European studies (42.0% and 18.8%) , 28. Alt A and C . Specifi A and C . Anotherutations .The findings from our NNRTI‐failure cohort suggest that routinely switching patients with existing NNRTI resistance to doravirine may not be advisable until we can confirm that virological suppression is greater in a clinical context than suggested from the resistance data.et al. [These are observational data, and prone to a wide variety of potential biases, including referral bias. However, the demographic data reflect the overall South African ART cohort, which is predominantly female, and in an approximate age range. The ART‐naïve dataset used in this study is somewhat outdated. Moyo et al.  recentlyet al. . To date4Doravirine seems a suitable antiretroviral for evaluation in first‐line patients in our region who cannot tolerate or do not want to take dolutegravir‐based regimens, as both an initiation or switch option in virologically suppressed patients, with a predicted >90% efficacy. More real‐world data and/or clinical trial data are required to ascertain the clinical outcomes of doravirine in patients with pre‐existing NNRTI DRMs, especially from LMICs with circulating non‐B subtypes, before doravirine can safely be used to switch patients experiencing NNRTI‐based regimen failure.Although doravirine is approved as initial therapy in patients who are ART‐naïve, it is currently recommended to obtain a genotype prior to the initiation of ART, which might not be feasible in many LMICs with large ART programmes. However, with the increased rollout of genotyping in some LMICs, baseline genotyping for a target population might be achievable. Alternatively, regular surveillance of pre‐treatment resistance, including doravirine, should be performed to inform the feasibility of using doravirine in first‐line treatment as has been done for first‐generation NNRTIs. On the other hand, the predicted resistance profiles in patients experiencing NNRTI‐based ART failure do not by itself substantiate the use of doravirine, until we have compelling data documenting viral suppression in the presence of these mutations.W.D.F.V is doing registration and investigator‐led studies that utilize doravirine, as well as several other antiretrovirals. M.M. started a full‐time role at ViiV Healthcare after the conceptualization and analysis of this study, but prior to publication. All others have no conflicts of interest to declare.K.S. performed the research, analysed the data and wrote the paper. M.M. and W.D.F.V. conceptualized the study, interpreted the data and critically reviewed the paper. A.M.J.W and L.H. interpreted the data and critically reviewed the paper.None declared.No specific funding was received to perform this research.


Cox proportional model and propensity score matching were used to identify variables that affect the possibility of achieving IR. The tropism characteristics of virus were compared using the coreceptor binding model. In addition to baseline CD4+ counts and age implications, CRF01_AE cluster 1 was associated with a poorer probability of achieving IR than infection with cluster 2  and other subtypes . The mean time from cART initiation to achieve IR was much longer in patients infected by CRF01_AE cluster 1 than other subtypes/sub-clusters (P < 0.001). In-depth analysis indicated that a higher proportion of CXCR4 viruses were found in CRF01_AE clusters 1 and 2 (P < 0.05), and showed tendency to favour CXCR4 binding to V3 signatures. This study indicated the immune restoration impairment found in patients were associated with HIV-1 CRF01_AE cluster 1, which was attributed to the high proportion of CXCR4-tropic viruses. To improve the effectiveness of cART, more efforts should be made in the early identification of HIV-1 subtype/sub-cluster and monitoring of virus phenotypes.There are great disparities of the results in immune reconstruction (IR) of the HIV-1 infected patients during combined antiretroviral therapy (cART), due to both host polymorphisms and viral genetic subtypes. Identifying these factors and elucidating their impact on the IR could help to improve the efficacy. To study the factors influencing the IR, we conducted a 15-year retrospective cohort study of HIV-1 infected individuals under cART. The trend of CD4 Human immunodeficiency virus type 1 (HIV-1) is characterized by extensive genetic diversity and evolved into various clades and circulating recombinant forms (CRFs) ,3. Phylo+ cell, coreceptor usage, and viral genotype, but most previous studies have focused on subtype B    than CRF01_AE cluster 2 (223 weeks) and non-CRF01_AE subtypes (140 weeks) respectively (< 0.001) A. The an+ cell count was a major variable affecting immune reconstitution, we assessed the effect of the HIV-1 genotype on immune recovery in three subgroups of CD4+ cell count . As shown in + cell count in the CRF01_AE cluster 1 was significantly lower than those in CRF01_AE cluster 2 and non-CRF01_AE group (P < 0.05). Furthermore, Cox regression analysis was used to determine the factors that affect the completion of immune reconstruction. After adjusting the baseline characteristic, the multivariate analysis demonstrated that the completion probability of CRF01_AE cluster 1 in the subtype group was significantly lower than others (P < 0.05) (Table S3).As the baseline CD4P < 0.05) (Tables S1 and S4). Moreover, the recent infection subgroup analysis of patients revealed the negative effects of X4 tropism on immune reconstitution probability. Therefore, we assessed the distribution of tropism in different subtypes and clusters. As shown in P < 0.001). Meanwhile, a higher proportion of X4 tropism was found in CRF01_AE cluster 1 (48.0%) compared with other subtypes. As X4 tropism was reported to be associated with poor CD4+ cell recovery . In. Inenv-VIn addition, residue I8 in the V3 loop was surrounded by hydrophilic amino acids, indicating that a hydrophilic environment favours residue I8 more. In contrast, residue T13 in the X4 V3 loop surrounded hydrophobic amino acids D. Theref+ T-cell regeneration during long-term cART. According to the molecular epidemiological study, the CRF01_AE cluster 1 and 2 are dominant clusters circulating among the heterosexual population in Southeast Asia and Southern China [+ cell count. The reason may be that the ratio of X4 tropism in CRF01_AE cluster 1 was significantly higher [There are increasing evidences indicating that certain HIV clades or clusters have a faster disease progression. It is understandable considering HIV-1 is faster evolving virus jumped from animal to human about 100 years ago. The previous studies have demonstrated that CRF01_AE infected individuals were associated with fast HIV progression and advanced immunodeficiency ,37. Howern China . The prey higher ,18,39. T+ cells and eventually complete the individual's immune reconstruction within a certain period [+ cell count and infection period, respectively. Meanwhile, we also carried out propensity score matching on the baseline variables. Ultimately, similar results to the overall cohort were obtained in all subgroups. The effectiveness of antiretroviral therapy will not only be affected by the HIV-1 subtypes, but also by its sub-cluster. Although baseline CD4+ cell count and age factors may also play a role [+ cell count recovery of CRF01_AE and cluster 1 individual should be monitored more closely because these patients may develop AIDS more quickly than patients who are not infected with this genotype.Previous studies have shown that most patients who can respond to viral suppression through antiviral therapy will continuously increase the number of peripheral CD4n period ,40,41. Ty a role ,42,43, c+ cell count decline and HIV-1 progression, leading to less effective cART [In terms of the distribution of viral tropism, in our cohort, we observed that the proportion of X4 tropism in CRF01_AE was much higher than that in other subtypes, which is an agreement with previous studies conducted in China that a high proportion of X4 tropism was reported in CRF01_AE subtype and demonstrated that X4 tropism is associated with lower CD4 counts and increased progression to immunosuppression ,44. Inteive cART ,35,36. Cive cART ,45.+ cell count increase among patients. Still we cannot fundamentally elucidate its biological mechanism. It is necessary to further study the relationship between IR and other gene-encoded viral proteins. Second, although we have made great efforts to collect recent infection patients and potential factors, the group’s size and analysis model adjustment factors in our study were still limited. We believe that the sample size of infected participants should be increased, and more available clinical follow up data should be accessible.Some research limitations should be noted. First, we only observed that CRF01_AE and CRF01_AE cluster 1 was related to poor IR and the viral tropism is an influential factor for low CD4+ cell count gains ability and longer time required to complete the immune reconstruction. The underlying reason may be attributed to the significantly high proportion of X4-tropic virus in CRF01_AE and cluster 1. It also explained why the HIV epidemic in Guangxi was under greater pressure and patients were more difficult to treat. Nowadays, there are at least two changes have facilitated our capacity to do genotype and phenotype surveillance: the advancement of “next-generation” sequencing technology and the abundance of sequencing data generated by routine drug resistance monitoring. These progresses have improved the efficiency for such surveillance activities. Consequently, this study strongly emphasized the significance of timely genotyping and phenotyping surveillance to reduce the possibility of immune reconstruction failure and ensure patient’s quality life.In summary, our study had provided an up-to-date evaluation of the relationship between the subtypes/sub-clusters. and immune reconstruction progression after cART among diagnosed HIV patients. We determined that CRF01_AE and its cluster 1 were the critical risk factors associated with the lower CD4Click here for additional data file.


Data on the burden of dyslipidaemia among people with HIV undergoing antiretroviral therapy (ART) in sub-Saharan Africa are limited and little is known about the factors contributing for poor lipid profiles. The aim of this study was to determine the prevalence of dyslipidaemia and factors associated with lipid levels among HIV-infected patients receiving first-line combination ART in North Shewa, Ethiopia.A cross-sectional study was conducted between April and December 2018 among 392 HIV-infected adults receiving first-line ART for at least six months at the ART clinic of Mehal Meda Hospital in North Shewa, Ethiopia. Blood samples were collected for determination of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and CD4 counts. Logistic regression analysis was used to determine factors associated with lipid abnormalities.The prevalence of dyslipidaemia was 59.9% (95% CI 55.0–64.7%). High TC, high TG, low HDL-c, and high LDL-c were obtained in 47.3%, 30.9%, 19.4% and 29.6%, respectively. Fifty-four participants (13.8%) had high ratio of TC/HDL-c (TC/HDL-c ratio ≥ 5). Older age was independently associated with high TC , high TG , low HDL–c , and high LDL–c . Living in an urban area  and smoking  were associated with low HDL–c. Participants with longer duration of ART use were more likely to have high TG , low HDL–c , and high LDL–c . High BMI was independently associated with higher TC , high TG  and high LDL–c .We found a high prevalence of dyslipidaemia among HIV-infected patients receiving first-line ART in North Shewa, Ethiopia. There is a need for monitoring of blood lipid levels in patients with HIV on long term first-line ART with a special attention to be focused on older age, urban residents, longer duration of ART use, high BMI and smokers. The introduction of combined active antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection has dramatically reduced morbidity and mortality from AIDS-related causes and increased the life expectancy of HIV-infected people . With imDyslipidaemia associated with combination ART use is characterized by increased levels of serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and triglycerides (TG) and a decreased high density lipoprotein-cholesterol (HDL-c) level , 5, 6. TThere is evidence demonstrating an increased risk and prevalence of dyslipidaemia among HIV infected persons in Africa . FurtherThis was a cross-sectional study conducted between April and December 2018 at the ART clinic of Mehal Meda Hospital (MMH). MMH; located in Mehal Meda town of Menz Gera Midir in North Shewa, Eastern Amhara state, Ethiopia, provides HIV/AIDS interventions including free diagnosis, treatment and monitoring. HIV-infected patients attend the ART clinic once in a month for clinical evaluation and prescription refill for combination antiretroviral (ARV) regimens. All HIV-infected patients aged 18 years or older who had been receiving first-line combination ARV regimens for a minimum of six months were consecutively enrolled. Critically ill patients, patients already on anti-dyslipidemic drugs, pregnant women, known diabetes mellitus and renal failure patients were excluded from the study. A total of 422 eligible patients visiting the ART clinic were recruited for this study. This was based on sample size calculation using 80% power, 5% level of significance, 50% prevalence and non-response rate of 10%. A total of 30 patients were excluded from this study (due to faulty laboratory results and not being fasting at the time of appointment), leading to a total of 392 patients.2). Blood sample was collected from each participant in the mornings after an overnight fasting and centrifuged at 3000 cycles/ minute, and then serum was obtained for lipid profiles. Serum total cholesterol, triglycerides and HDL-c concentrations were assessed by specific colorimetric assays, using an automated analyzer system . LDL-c concentration was determined using the Friedewald et al. formula ) age of patients was 41.2 ± 14.4 years (ranging from 18–80 years). The mean BMI of patients was 20.4 ± 3.1 kg/m= 0.001) .P > 0.05).The mean lipid levels of HIV infected adults on ART are summarized in Dyslipidaemia prevalence was 59.9% (95% CI 55.0–64.7%), and the main type of lipid abnormality was high TC (TC ≥ 200 mg/dl); which was found in 185 (47.3%) participants . High TGP values ≤ 0.25 were included in multivariable logistic regression analysis   .P = 0.001), high BMI  and lower CD4 cell count  were significantly associated with higher TC. Older age , longer duration of ART use  and high BMI  were associated with a high risk of raised TG. A lower HDL–c was independently associated with older age , living in an urban area , longer duration of ART use  and smoking . In addition, participants with older age , who had longer duration of ART , and high BMI  were more likely to have high LDL–c.The multivariable logistic regression analysis revealed that older age  in HIV-infected patients receiving ART at a hospital in Northeast Ethiopia. The most frequent form of dyslipidaemia was high TC (47.3%), followed by high TG (30.9%) and high LDL-c (29.6%). The overall prevalence of dyslipidaemia (59.9%) in this study was relatively lower than that reported in HIV-infected patients receiving first-line HAART in Southern Ethiopia (82.3%) . It was Nearly 14% of our participants on ART had a high TC/HDL-C ratio. Previous studies have reported prevalence of high TC/HDL-C ratio ranging from 16.7% to 45.1% in patients on standard combination first line ART , 16. ThiWe found that older age was significantly associated with poor lipid profiles , and this was consistent with findings of previous studies , 22, 23.3). A study from a South African population on ART also reported unfavorable lipid profile changes, with lower CD4 count being risk factor for elevated cholesterol [In the current study, being on ART for five or more years was associated with higher levels of TG and LDL-c but with lower levels of HDL-c. This is probably related to a combination of the effects of an aging HIV-infected population coupled with improved health due to suppression of viral load and the effect of ARV drugs on lipid metabolism. This was comparable to previous findings, which showed that long term exposure to ART was associated with poor lipid profiles , 18, 19.lesterol . This islesterol , 16, 18.2) were significantly associated with poor lipid profiles . This is in line with the findings of the study in Southern Ethiopia [2 with raised levels of TC and LDL-c. Changes in body fat distribution expected to occur with continued exposure to ART could offer a potential explanation for these findings. This study also found an association between smoking and decreased levels of HDL-c. Though the proportion of smokers in our sample population was very small and made up exclusively of men, the association is in agreement with previous reports, which indicated that ART-exposed smokers had poorer lipid profiles compared with non-smokers [The present study revealed that patients with high BMI Click here for additional data file.