The vaginal microbiome plays a complex role in tenofovir's mucosal pharmacology. While the relative contribution of systemic versus local concentrations to PrEP PK/PD is an ongoing debate, the development of effective HIV prevention requires an understanding of events at the site of transmission. Our objective was to further understand the relationship between tenofovir mucosal pharmacology and non-optimal vaginal microbiota using the ex vivo vaginal tissue model. Vaginal and cervical explants were produced from cadaver tissue using a biopsy punch. Explants were incubated with Prevotella bivia (103-105 colony forming units/ml) with and without tenofovir for 24 h. TFVdp and endogenous adenosine triphosphate (dATP) were quantified using liquid chromatography tandem mass spectroscopy. Explants were then challenged with 106 TCID50 the viral concentration where 50% of tissue cultures are infected, of HIVJR-CSF for 3 h. Explants were cultured on gel-foam rafts for 48 h then collected for HIV RNA quantification using RT-qPCR. TFVdp formation in vaginal tissue was approximately 76% lower in anaerobic conditions (p = 0.2) compared to aerobic. While dATP concentrations did not significantly differ between any Prevotella bivia concentrations and the Prevotella-free control, TFVdp and TFVdp:dATP ratio in vaginal tissue decreased as Prevotella bivia concentrations increased, although not statistically. Unexpectedly, tenofovir efficacy increased as Prevotella bivia concentrations increased. The ex vivo tissue model was successful in demonstrating the pharmacology of TFVdp is affected by Prevotella bivia. Viral replication was also affected by Prevotella bivia; therefore, further work is needed to fully understand effects on tenofovir pharmacodynamics.

Background:One month of daily isoniazid and rifapentine (1HP) is an efficacious, well-tolerated, and safe tuberculosis-preventive therapy regimen. Its implementation has been slow due to concern about drug-drug interactions with rifapentine. Methods:This single-arm pharmacokinetic study included 15 adults with advanced human immunodeficiency virus (HIV) disease receiving once-daily 50-mg dolutegravir-based antiretroviral therapy during 1HP therapy, alongside once-daily 800-mg fluconazole for treatment of cryptococcal meningitis. Participants underwent intensive pharmacokinetic sampling on days 1, 5, and 14 of 1HP therapy; sampling was conducted before and 2, 4, 8, and 24 hours after dosing. Dolutegravir, rifapentine, and fluconazole plasma concentrations were measured by means of high-performance liquid chromatography mass spectrometry, and bioequivalence was assessed with standard 90% confidence intervals (CIs). Results:Fifteen participants were enrolled, of whom 9 (60%) were female. The median age (interquartile range) was 37 (27-41) years, and the median CD4 cell count was 11/μL (10-25/μL). Overall, 14 of 15 participants completed sampling on days 5 and 14. The day 14 geometric mean (GM) dolutegravir trough concentration (95% CI) was 0.61 (.32-1.16) mg/L, while the median (interquartile range) was 0.63 (0.34-0.87) mg/L, with 100% of samples above the dolutegravir protein-adjusted 90% inhibitory concentration of 0.064 mg/L, with a GM ratio of 0.76 (90% CI, .4-1.46) compared with day 1. The day 14 maximum plasma concentration and area under the concentration-time curve from 0 to 24 hours for fluconazole were reduced compared with day 1, with GM ratios of 0.73 (90% CI, .57-.94) and 0.77 (.64-.93), respectively. Of 14 participants alive at 12 weeks after 1HP initiation, 11 (78%) had an HIV viral load (VL) <200 copies/mL. No cases of cryptococcal meningitis relapse occurred during the 18-week follow-up. Conclusions:Standard once-daily 50-mg dolutegravir and 800-mg fluconazole dosing may be a safe and effective option for patients receiving 1HP tuberculosis-preventive therapy. These findings support further evaluation in larger studies.

BACKGROUND:The female genital tract (FGT) is a unique compartment with physiologically distinct properties complicating the extrapolation of drug efficacy; critical gaps remain in understanding regional variability within the FGT itself. We performed an in-depth investigation across endo- and ectocervical tissues on the utility of the cervical explant model to evaluate pre-exposure prophylaxis (PrEP) efficacy. METHODS:Using normal cervical tissues, we evaluated gene expression of relevant drug metabolizing enzymes and transporters (DMETs) via qRT-PCR and compared ecto- and endocervix. To determine differences in drug phosphorylation and to assess antiretroviral (ARV) efficacy, we incubated explants in tenofovir and emtricitabine then measured intracellular metabolites. Viral infectivity and dose-response with ARVs was measured using viral RNA and p24 following HIV-1JR-CSF challenge. RESULTS:ABCC4 expression was 3-fold lower in ectocervical tissues compared with endocervical, whereas CYP3A5 was 2-fold higher. IL-6 was correlated with ABCB1 (r = 0.52, P = 0.01) and ABCG2 (r = 0.56, P =0.005). Dose-normalized phosphorylation did not differ between endo- and ectocervix (P > 0.5). Infectivity of explants was low (53%) but did not differ by compartment. Intracellular tenofovir diphosphate concentrations were associated with a decrease in ectocervical viral replication (r =0.39, P < 0.05). There was a strong relationship between the proportion of explants infected and emtricitabine dose (P =0.02) but no relationship between intracellular emtricitabine triphosphate and protection. CONCLUSIONS:We identified differences in DMET expression and ARV metabolism between ecto- and endocervical tissues, as well as correlations between DMET and IL-6. Ectocervical explants demonstrated consistent viral infectivity and dose-dependent inhibition. The model is useful in determining tenofovir diphosphate targets.

BACKGROUND:Cryptococcosis is a leading cause of death among people with human immunodeficeincy virus (HIV). Guidelines recommend combination therapy with amphotericin B and flucytosine, but standard flucytosine dosing of 100 mg/kg/day is associated with hematologic toxicity. The FLOOR trial investigated whether lower flucytosine dosing maintained therapeutic efficacy and reduced toxicity. METHODS:This phase 2, single-arm, open-label trial enrolled 48 adults with HIV and confirmed cryptococcal meningitis at 2 hospitals in Uganda. Participants received 10 days of flucytosine at 60 mg/kg/day in 3 divided doses, combined with liposomal amphotericin B (10 mg/kg, single dose) and fluconazole 1200 mg/day for 14 days, followed by standard consolidation therapy. The primary endpoint was the early fungicidal activity (EFA), which measures the rate of Cryptococcus clearance in the cerebrospinal fluid (CSF) in the first 14 days of therapy. Secondary endpoints included CSF sterility at 2 weeks, 18-week survival, and adverse events. RESULTS:The EFA of reduced dose flucytosine was 0.28 log10 colony forming units (CFU)/mL/day (95% CI, .20 to .35), which was lower than historical controls in Uganda receiving flucytosine at 100 mg/kg/day of 0.41 log10 CFU/mL/day (P = .019). At 18 weeks, the estimated survival probability was 77% (95% CI, 66% to 90%). The incidence of rehospitalization (25%) and culture-positive relapse (6.3%) were higher than expected. Grades 3 and 4 hematologic toxicity was similar (10% leukopenia; 21% anemia) to historical incidence. CONCLUSIONS:Reduced flucytosine dose of 60 mg/kg/day for 10 days demonstrated inferior efficacy in CSF fungal clearance. These findings support further investigation of dose optimization strategies. TRIAL REGISTRATION:ClinicalTrials.gov: NCT06414512.

INTRODUCTION:For HIV medications intended for HIV prevention, it is critical to achieve exposures in that will provide reliable protection to the FGT. The female genital tract (FGT) is a complex and heterogenous environment. AREAS COVERED:We reviewed what is known about drug transport and metabolism specific to female genital tissues. We performed a literature search using key words in PubMed and Google Scholar on articles published inclusive of August 2024. We then discuss the impact of sex steroid hormones and vaginal microbiome on the genital tract pharmacology of drugs used for PrEP. EXPERT OPINION:Better characterization of FGT pharmacology can improve PrEP options for women. Better models that can fully capture the complexities of the FGT to evaluate pharmacokinetic-pharmacodynamic relationships in the context of the complex microenvironment of the FGT will need to be developed and validated to move the field forward.

INTRODUCTION:Dolutegravir is now extensively used in sub-Saharan Africa as a preferred component of antiretroviral therapy (ART). There is a paucity of large studies using routinely collected data from African people living with HIV on dolutegravir-based regimens to inform HIV programmes. We reviewed data in a large programme clinic of people living with HIV on dolutegravir to determine the real-world safety and tolerability of dolutegravir and to describe drivers of treatment discontinuation. METHODS:We carried out a retrospective dynamic cohort analysis of people living with HIV who started on or switched to dolutegravir-based ART at the Infectious Diseases Institute in Kampala, Uganda, between February 2017 and December 2020. Types of adverse events (AEs) were classified according to the Medical Dictionary for Regulatory Activities. Incident rates for AEs and treatment discontinuation were determined using Cox proportional hazard methods. RESULTS:Of 4529 people living with HIV started on or switched to dolutegravir, 2094 (45.9%) were female, and the median age was 49 years (interquartile range [IQR] 41-56). During 8907.93 person-years (PY) of follow-up, 1069 (23.6%; 95% confidence interval [CI] 22.4-24.8) people living with HIV had an AE, at a rate of 10.43 per 1000 PY (95% CI 9.77-11.14). Neuropsychiatric, gastrointestinal, and endocrine AEs were most common. The main AEs driving dolutegravir discontinuation were hyperglycaemia (140/356; 39.3%) and erectile dysfunction (19/356; 5.3%). Only 1.2% (4/356) of the dolutegravir discontinuations were because of neuropsychiatric AEs. Being female (adjusted hazard ratio [aHR] 1.35; 95% CI 1.02-1.78) and previous use of stavudine (aHR 1.46; 95% CI 1.04-2.05) were the main predictors of neuropsychiatric AEs. Risk factors for hyperglycaemia included being overweight (aHR 1.66; 95% CI 1.11-2.47) or obese (aHR 1.84; 95% CI 1.12-3.05), having hypertension (aHR 1.92; 95% CI 1.35-2.73), having diabetes mellitus (aHR 12.6; 95% CI 8.34-19.1), and taking previous ART containing zidovudine (aHR 1.76; 95% CI 1.19-2.59) or stavudine (aHR 1.68; 95% CI 1.15-2.44). These risk factors for hyperglycaemia were also the main drivers of dolutegravir discontinuation. CONCLUSION:AEs were common in this African cohort, and dolutegravir discontinuation was driven by hyperglycaemia and erectile dysfunction. Previous use of older ART with known mitochondrial toxicity was associated with neuropsychiatric AEs and hyperglycaemia. African countries used these drugs for longer periods, and this may contribute to this risk.

Long‐acting injectable (LAI) cabotegravir and rilpivirine for HIV treatment and LAI cabotegravir for pre‐exposure HIV prophylaxis are being rolled out in a multitude of countries worldwide. Due to the prolonged exposure, it can be challenging to undertake ‘traditional’ pharmacokinetic studies and current guidance is derived from their oral equivalents or physiologically based pharmacokinetic studies. This review aims to consider pharmacokinetic characteristics of cabotegravir and rilpivirine and describe anticipated drug–drug interactions (DDIs) with frequent concomitant medications in African settings. Relevant co‐medications were identified from the WHO 2021 List of Essential Medicines. All original human and physiologically based pharmacokinetic studies published in English on PubMed, discussing DDIs with LAI cabotegravir and rilpivirine prior to April 2023, were reviewed. The Liverpool HIV interaction database was also reviewed (https://www.hiv-druginteractions.org/checker). LAI cabotegravir and rilpivirine have half‐lives of 6–12 and 13–28 weeks, respectively. Cabotegravir is primarily metabolized by UDP‐glucuronyltransferase (UGT)‐1A1 and rilpivirine by cytochrome P450 (CYP)‐3A4. LAI cabotegravir and rilpivirine themselves exhibit low risk of perpetrating interactions with co‐medications as they do not induce or inhibit the major drug metabolizing enzymes. However, they are victims of DDIs relating to the induction of their metabolizing enzymes by concomitantly administered medication. Noteworthy contraindicated co‐medications include rifamycins, carbamazepine, phenytoin, flucloxacillin and griseofulvin, which induce CYP3A4 and/or UGT1A1, causing clinically significant reduced concentrations of rilpivirine and/or cabotegravir. In addition to virologic failure, subtherapeutic concentrations resulting from DDIs can lead to emergent drug resistance. Clinicians should be aware of potential DDIs and counsel people receiving LAI cabotegravir/rilpivirine appropriately to minimize risk.

Background Central nervous system (CNS) compartmentalization provides opportunity for human immunodeficiency virus (HIV) persistence and resistance development. Differences between cerebrospinal fluid (CSF) and cerebral matter regarding HIV persistence are well described. However, CSF is often used as surrogate for CNS drug exposure, and knowledge from solid brain tissue is rare.Methods Dolutegravir, tenofovir, lamivudine, and efavirenz concentrations were measured across 13 CNS regions plus plasma in samples collected during autopsy in 49 Ugandan decedents. Median time from death to autopsy was 8 hours (interquartile range, 5-15 hours). To evaluate postmortem redistribution, a time course study was performed in a mouse model.Results Regions with the highest penetration ratios were choroid plexus/arachnoid (dolutegravir and tenofovir), CSF (lamivudine), and cervical spinal cord/meninges (efavirenz); the lowest were corpus callosum (dolutegravir and tenofovir), frontal lobe (lamivudine), and parietal lobe (efavirenz). On average, brain concentrations were 84%, 87%, and 76% of CSF for dolutegravir, tenofovir, and lamivudine, respectively. Postmortem redistribution was observed in the mouse model, with tenofovir and lamivudine concentration increased by 350% and efavirenz concentration decreased by 24% at 24 hours postmortem.Conclusions Analysis of postmortem tissue provides a unique opportunity to investigate CNS antiretroviral penetration. Regional differences were observed paving the way to identify mechanisms of viral compartmentalization and/or neurotoxicity. Postmortem analysis of CNS tissues reveals heterogenous antiretroviral distribution.

Five long-acting (LA) antiretrovirals (ARVs) are currently available in a limited number of countries worldwide for HIV-1 prevention or treatment-cabotegravir, rilpivirine, lenacapavir, ibalizumab, and dapivirine. Implementing use of LA ARVs into routine clinical practice requires significant changes to the current framework of HIV-1 prevention, treatment, and service provision. Given the novelty, complexity, and interdisciplinary requirements needed to safely and optimally utilize LA ARVs, consensus recommendations on the use of LA ARVs will assist clinicians in optimizing use of these agents. The purpose of these recommendations is to provide guidance for the clinical use of LA ARVs for HIV-1 treatment and prevention. In addition, future areas of research are also identified and discussed.

Background We previously reported the effect of contraception on cervical tenofovir concentrations in Ugandan women with human immunodeficiency virus (HIV). Here we explored the role of cervicovaginal cytokines and drug metabolizing enzymes and transporters (DMETs) to elucidate female genital tract (FGT) drug disposition in a Ugandan cohort.Methods Cervicovaginal fluid and cervical biopsies were collected from Ugandan women with HIV receiving tenofovir/lamivudine-based therapy and intramuscular depot medroxyprogesterone acetate (n = 25), copper intrauterine device (cuIUD; n = 12), or condoms (n = 13) as contraception. Cytokines were measured in cervicovaginal fluid (CVF). Ectocervical tenofovir diphosphate (TFVdp), lamivudine triphosphate (3TCtp), and deoxyadenosine triphosphate (dATP)/deoxycytidine triphosphate (dCTP) concentrations and immune marker/DMET gene expression were measured in cervical biopsies.Results Cervical 3TCtp was not correlated with any CVF cytokines. Cervical TFVdp was correlated with IL-10, IL-7, and IL-17 in CVF. CCR5 mRNA expression in cervical biopsies was higher in cuIUD users versus condom users. Using multivariable linear regression, CVF IL-17, tissue dATP, plasma estradiol, and plasma tenofovir were all significant predictors of cervical TFVdp. Tissue dCTP and plasma lamivudine were significant predictors of cervical 3TCtp.Conclusions TFVdp concentrations in cervix appear to be influenced by local inflammation. In contrast, 3TCtp FGT exposure was not affected by genital inflammation or DMETs. CuIUD users have more immune cells present, which may in turn influence local TFVdp disposition.Main Finding We investigated changes in tenofovir diphosphate and lamivudine triphosphate due to the microbiome and inflammation. While lamivudine triphosphate was not affected by either, tenofovir diphosphate appeared to be affected by local inflammation. Specifically, Th17 cells may influence tenofovir disposition.

OBJECTIVES/GOALS: An ex-vivo tissue model has been developed to predict target concentrations of tenofovir diphosphate (TFVdp; active metabolite of tenofovir) but has not been utilized to see how vaginal dysbiosis affects TFVdp/dATP exposure in female genital tract (FGT). My central hypothesis is that presence of specific anaerobic bacteria will increase dATP in FGT. METHODS/STUDY POPULATION: De-identified HIV-negative cervical tissues from women undergoing gynecological surgeries will be procured and a punch biopsy will be used to create explants. TFVdp/dATP concentrations were both tested in both aerobic and anaerobic conditions after a 24-hour incubation in tenofovir (TFV) to determine any changes between conditions. TFVdp/dATP in cervical tissue was be measured using LC-MS. Next, media and explants were collected at baseline to characterize donor microbiome for 6 donors. 16S microbiome sequencing was performed on extracted DNA to obtain the relative abundances of each bacteria species present. To test changes in dATP/TFVdp due to the microbiome, explants will be incubated in TFV for 24 hours with Prevotella and Dialister to specifically see how microbiomes dominated by these taxa affect dATP. RESULTS/ANTICIPATED RESULTS: There was no significant difference in TFVdp formation between aerobic and anaerobic conditions after a 24-hour tenofovir incubation (p = 0.2) for 8 donors. dATP was not quantifiable at 24 hours in explants, so explants are being collected before 24hrs during a TFV incubation to determine how quickly dATP depletes after collection. We were able to characterize the donor microbiome in media and tissue at baseline and 24hrs which had inter variability. We did not see any presence of Prevotella or Dialister in any donors. We are working on characterizing bacteria growth over time to see how the donor microbiome would change during a 24 hour experiment in anaerobic conditions. Finally, we anticipate seeing increases in dATP with a Prevotella or Dialister supplemented donor microbiome compared to baseline donor microbiome. DISCUSSION/SIGNIFICANCE: The addition of vaginal dysbiosis to tissue model will increase accuracy of prediction of 100% protective TFVdp concentrations and is likely to provide a translational model that can be used to improve TFV-based PrEP in women and streamline development of future PrEP candidates, bringing more prevention options to women and ending the HIV epidemic.

Background The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B in combination with flucytosine for the treatment of cryptococcal meningitis. When flucytosine (5FC) is used as monotherapy in cryptococcosis, 5FC can induce resistant Cryptococcus mutants. Oral amphotericin B uses a novel drug delivery mechanism, and we assessed whether resistance to 5FC develops during oral LNC-amphotericin B therapy.Methods We enrolled Ugandans with HIV diagnosed with cryptococcal meningitis and who were randomized to receive 5FC and either standard intravenous (IV) amphotericin B or oral LNC-amphotericin B. We used broth microdilution to measure the minimum inhibitory concentration (MIC) of the first and last cryptococcal isolates in each participant. Breakpoints are inferred from 5FC in Candida albicans. We measured cerebral spinal fluid (CSF) 5FC concentrations by liquid chromatography and tandem mass spectrometry.Results Cryptococcus 5FC MIC50 was 4 mu g/mL, and MIC90 was 8 mu g/mL. After 2 weeks of therapy, there was no evidence of 5FC resistance developing, defined as a >4-fold change in susceptibility in any Cryptococcus isolate tested. The median CSF 5FC concentration to MIC ratio (interquartile range) was 3.0 (1.7-5.5) mu g/mL. There was no association between 5FC/MIC ratio and early fungicidal activity of the quantitative rate of CSF yeast clearance (R-2 = 0.004; P = .63).Conclusions There is no evidence of baseline resistance to 5FC or incident resistance during combination therapy with oral or IV amphotericin B in Uganda. Oral amphotericin B can safely be used in combination with 5FC.

The HIV epidemic continues to pose a significant burden on the healthcare system. Although the incidence of annual new infections is decreasing, health disparities persist and most new infections remain concentrated into different racial, ethnic, and minority groups. Pre-exposure prophylaxis (PrEP), which involves those at high risk of acquiring HIV to take chronic medications to prevent acquisition of the virus, is key to preventing new HIV infections. The purpose of this article is to review medication therapies for PrEP and examine their role in personalizing PrEP in different patient populations. Additionally, new medications currently under development for PrEP are reviewed, as well as treatment as prevention (TasP) and post-exposure prophylaxis (PEP). There are currently four medications available for PrEP: the oral options of co-formulated emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) or emtricitabine/tenofovir alafenamide (FTC/TAF); injectable long-acting cabotegravir (CAB-LA); and the vaginal ring dapivirine (DPV-VR). FTC/TAF is not currently indicated for persons at risk for HIV through vaginal sex due to lack of studies, but trials are currently ongoing. DPV-VR is available in Zimbabwe and South Africa and has been endorsed by the World Health Organization but is not currently available in the United States. Several agents are also in development for use in PrEP: the novel long-acting injectable lenacapavir, a first-in-class capsid inhibitor, which has no cross-resistance to any existing HIV drug class; the subdermal implant islatravir, a first-in-class translocation inhibitor; and VRC01, a broadly neutralizing antibody (bnAb) which has been evaluated in proof-of-concept studies that may lead to the development of more potent bnAbs. Overall, PrEP is highly effective at preventing HIV infection in high-risk populations. Identifying optimal PrEP regimens in different patient populations is complex and must consider patient-specific factors and medication cost and access considerations. Lastly, providers should consider individual patient preferences with regard to prevention to improve access, retention in care, and adherence.

OBJECTIVE:The aim of this study was to understand how vaginal microbiota composition affects antiretroviral concentrations in the setting of hormonal contraception initiation. METHODS:Cervicovaginal fluid (CVF) concentrations of tenofovir, lamivudine, and efavirenz from 73 Malawian women with HIV were compared before and after initiation of depot-medroxyprogesterone acetate (DMPA) or levonorgestrel implant. We evaluated antiretroviral concentrations and vaginal microbiota composition/structure in the context of contraception initiation and predicted genital shedding using multivariable repeated measurements models fit by generalized estimating equations. RESULTS:Mean lamivudine CVF concentrations decreased 37% 1 month after contraception initiation. Subgroup analyses revealed a 41% decrease in women 1 month after initiating levonorgestrel implant, but no significant difference was observed in DMPA group alone. Tenofovir, lamivudine, and efavirenz CVF concentrations were positively correlated with anaerobic bacteria associated with nonoptimal vaginal microbiota. Risk of genital HIV shedding was not significantly associated with tenofovir or lamivudine CVF concentrations [tenofovir relative risk (RR): 0.098, P = 0.75; lamivudine RR: 0.142, P = 0.54]. Lack of association between genital HIV shedding and efavirenz CVF concentrations did not change when adjusting for vaginal microbiota composition and lamivudine/tenofovir CVF concentrations (RR: 1.33, P = 0.531). CONCLUSION:No effect of hormone initiation on genital shedding provides confidence that women with HIV on either DMPA or levonorgestrel implant contraception will not have compromised ART efficacy. The unexpected positive correlation between antiretroviral CVF concentrations and certain bacterial taxa relative abundance requires further work to understand the mechanism and clinical relevance.

Abstract Background The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B for treatment of cryptococcal meningitis. Oral LNC amphotericin B applies nanotechnology, which hopes to improve intracellular drug delivery to affected tissues while reducing extracellular concentrations and thereby adverse events. When flucytosine (5-FC) is used as monotherapy, it can induce stable, highly resistant mutants of Cryptococcus. Since LNC amphotericin is a novel drug delivery mechanism, we assessed whether resistance to 5-FC develops in this context. Results from the ACTA and AMBITION Clinical Trials 10-week mortality by antifungal regimen for cryptococcal meningitis from the clinical trial by Molloy et al. (ACTA) in top section and Jarvis et al. (AMBITION) in bottom section. Mean survival for each group is represented by diamonds and 95% confidence interval represented by error bars. Encapsulation of Amphotericin B in a Lipid nanocrystal Particle Lipid nanocrystal particle encapsulates amphotericin B molecule and delivers the drug to antigen presenting cells. Methods The trial enrolled subjects with HIV who were diagnosed with cryptococcal meningitis and randomized to receive 5-FC and either standard IV amphotericin or LNC amphotericin. Subjects had a lumbar puncture (LP) performed on screening and days 3, 7, and 14. We used broth microdilution methods, standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to assess the MIC of isolates in each arm of the trial. We tested cryptococcal isolates from the cerebrospinal fluid (CSF) on the day of screening and then on the last positive CSF to contain cryptococcal growth. Statistical analysis included chi-square to test for a difference in MIC between the control and LNC amphotericin groups; and linear regression to compare MIC with early fungicidal activity (EFA), which is a surrogate endpoint. Intervention and Control Gropus for EnACT Phase 2 The EnACT trial phase 2 was completed in 4 cohorts to determine the ideal combination of IV and LNC amphotericin. The MIC analysis was completed in the 4th cohort. In the 4th cohort, participants randomized to the intervention group started LNC amphotericin B from the day of enrollment. Broth Microdilution for 5-Flucytosine Susceptibility Testing A 96-well plate was prepared with serial dilutions of 5-FC ranging from 256 µg/mL to 0.5 µg/mL in columns 1-10. Columns 11 and 12 were drug-free controls. The first and last CSF isolate from each unique participant was inoculated into rows B-D and E-G, respectively. The first row is a negative control and the last row is a positive control. Results The MIC50 was 4 µg/mL and MIC90 was 8 µg/mL for both the control and LNC amphotericin B groups. There was no evidence of 5-FC resistance in any Cryptococcus isolate tested after 2 weeks of therapy. 73% (n=8) of participants in the control group and 73% (n=19) in the LNC amphotericin group had no change or a decrease in MIC from the first to last Cryptococcus isolate. 27% (n=3) in the control group and 19% (n=5) in the LNC amphotericin group had a 2-fold increase in MIC. 9% (n=2) in the LNC amphotericin group had a 4-fold increase in MIC. There was no association with MIC and EFA. 5-FC CSF levels were above the MIC50. Distribution of MIC for flucytosine The MIC for each CSF isolate in the control and oral amphotericin B arms of the trial with the percents of each MIC level in each trial arm. Chi-squared statistic testing for a difference in MIC between each trial arm. Early Fungicidal Activity Compared to MIC for Each Trial Arm The EFA is compared to MIC in the control and oral amphotericin groups. Linear regression comparing EFA and control group shows that there is no correlation between MIC and EFA. Change in MIC During Treatment with Flucytosine and Amphotericin B A) Percent of participants in each trial group who had an increase in MIC over the treatment course. B) Individual participants are represented by a line with the MIC represented by a dot on the end of each line. Conclusion There is no evidence of baseline resistance to 5-FC or incident resistance during therapy in individuals presenting with cryptococcal meningitis in Uganda. LNC amphotericin B can safely be used in combination with 5-FC. Disclosures All Authors: No reported disclosures

Aberrant phosphorylation and subsequent aggregation of the trans-activation response (TAR) element DNA binding protein 43 (TDP-43) is a common feature of multiple neurodegenerative disorders and contributes to disease severity. Here, we investigated whether pathologic phosphorylation of TDP-43 (pTDP-43) is a hallmark of human immunodeficiency virus (HIV)- infected brains. We evaluated pTDP-43 immunoreactivity and TDP-43 kinases in HIV-infected (HIV + ) and seronegative post-mortem brain samples. We then used an inducible transgenic mouse model of the HIV-1 protein Tat and primary neuronal cultures, to decipher the underlying mechanism of the proteinopathy. Since opioid use disorder (OUD) can exaggerate HIV neuropathology, we explored interactions between HIV-1 Tat and morphine, a prototypical opioid, for all outcome measures. Cytoplasmic pTDP-43 and TDP-43 immunoreactivities were increased in neurons of the basal ganglia of post-mortem, HIV+ human tissues   compared to seronegative controls. An evaluation of TDP-43 kinases revealed an increase in the levels of cytoplasmic casein kinase 2 (CK2) in HIV-positive   human tissues but not CK1δ. There was a significant positive correlation between pTDP-43 and CK2 levels. Eight weeks of Tat induction and 2-week subcutaneous morphine exposure (10–40 mg/kg, increasing by 10 mg/kg/b.i.d.) independently produced similar outcomes for cytoplasmic pTDP-43 and CK2 levels in the mouse striatum. In primary, mouse striatal neuronal cultures, co-exposure to Tat and morphine for 24 h increased pTDP-43 levels and CK2 activity. Co-treatment with the CK2 antagonist CX-4945 prevented the Tat- and morphine-induced increases in pTDP-43 levels. Our results demonstrate that CK2 may be a viable therapeutic target for treating pTDP-43 proteinopathy in neuroHIV and OUD. Summary Statement HIV/HIV-1 Tat and morphine independently increase pathologic phosphorylation of TAR DNA binding protein 43 in the striatum. HIV- and opioid-induced pathologic phosphorylation of TAR DNA binding protein 43 may involve enhanced CK2 activity and protein levels.

The HIV epidemic remains a significant public health burden. Women represent half of the global HIV epidemic, yet there is an urgent need for a variety of prevention options to meet the needs of more women. Pre-exposure prophylaxis (PrEP) is a valuable prevention tool that uses antiretrovirals before a potential HIV exposure to prevent virus transmission. Development of effective preventive drug regimens for women is dependent on convenient dosing schedules and routes of administration, and on identifying defined target concentrations in mucosal tissues that provide complete protection against HIV transmission. There is a critical need for a translational model that can accurately predict in vivo target concentrations that are completely protective against HIV infection. There is no gold-standard preclinical model to predict PrEP efficacy. In this study, we review the strengths and limitations of three different preclinical models and their utility in predicting target concentrations in the female genital tract: humanized mice, non-human primates, and the ex vivo tissue model.

BACKGROUND:Optimal penetration of anti-infectives in the female genital tract (FGT) is paramount in the treatment and prevention of infectious diseases. While exposure of anti-infectives in lower FGT tissues (e.g. cervix, vagina) has been described, little data exist on upper genital tissues (e.g. ovary, uterus). METHODS:Autopsies were performed and post-mortem tissues were collected within 24 h of death for female participants with advanced HIV in Uganda (n = 27). Tenofovir, lamivudine, efavirenz and fluconazole concentrations were measured using LC-MS/MS in plasma, ovarian, uterine, cervical and vaginal tissues. Tissue penetration was calculated as tissue-to-plasma concentration ratios (TPRs). RESULTS:TPRs of tenofovir, lamivudine and fluconazole were highest in vaginal tissue (medians 1.86, 1.83 and 0.94, respectively), while the TPR of efavirenz was highest in ovarian tissue (median 0.65). With cervix as a reference compartment, vaginal TPRs were significantly higher than cervical for all four drugs; TPRs of efavirenz in uterine and ovarian compartments were also significantly higher than cervical. Most of the post-mortem FGT samples had a TPR of greater than 1 for tenofovir and lamivudine, while less than 50% had a TPR of greater than 1 for both efavirenz and fluconazole. CONCLUSIONS:Penetration of anti-infectives was not homogeneous among the FGT compartments. Approximately 70% of FGT tissues had a TPR of greater than 1 for tenofovir and lamivudine, favouring the prevention of local HIV replication and transmission in the FGT.