Twice-Yearly Lenacapavir or Daily F/TAF for HIV Prevention in Cisgender Women.

BACKGROUND: There are gaps in uptake of, adherence to, and persistence in the use of preexposure prophylaxis for human immunodeficiency virus (HIV) prevention among cisgender women. METHODS: We conducted a phase 3, double-blind, randomized, controlled trial involving adolescent girls and young women in South Africa and Uganda. Participants were assigned in a 2:2:1 ratio to receive subcutaneous lenacapavir every 26 weeks, daily oral emtricitabine-tenofovir alafenamide (F/TAF), or daily oral emtricitabine-tenofovir disoproxil fumarate (F/TDF; active control); all participants also received the alternate subcutaneous or oral placebo. We assessed the efficacy of lenacapavir and F/TAF by comparing the incidence of HIV infection with the estimated background incidence in the screened population and evaluated relative efficacy as compared with F/TDF. RESULTS: Among 5338 participants who were initially HIV-negative, 55 incident HIV infections were observed: 0 infections among 2134 participants in the lenacapavir group (0 per 100 person-years; 95% confidence interval [CI], 0.00 to 0.19), 39 infections among 2136 participants in the F/TAF group (2.02 per 100 person-years; 95% CI, 1.44 to 2.76), and 16 infections among 1068 participants in the F/TDF group (1.69 per 100 person-years; 95% CI, 0.96 to 2.74). Background HIV incidence in the screened population (8094 participants) was 2.41 per 100 person-years (95% CI, 1.82 to 3.19). HIV incidence with lenacapavir was significantly lower than background HIV incidence (incidence rate ratio, 0.00; 95% CI, 0.00 to 0.04; P<0.001) and than HIV incidence with F/TDF (incidence rate ratio, 0.00; 95% CI, 0.00 to 0.10; P<0.001). HIV incidence with F/TAF did not differ significantly from background HIV incidence (incidence rate ratio, 0.84; 95% CI, 0.55 to 1.28; P = 0.21), and no evidence of a meaningful difference in HIV incidence was observed between F/TAF and F/TDF (incidence rate ratio, 1.20; 95% CI, 0.67 to 2.14). Adherence to F/TAF and F/TDF was low. No safety concerns were found. Injection-site reactions were more common in the lenacapavir group (68.8%) than in the placebo injection group (F/TAF and F/TDF combined) (34.9%); 4 participants in the lenacapavir group (0.2%) discontinued the trial regimen owing to injection-site reactions. CONCLUSIONS: No participants receiving twice-yearly lenacapavir acquired HIV infection. HIV incidence with lenacapavir was significantly lower than background HIV incidence and HIV incidence with F/TDF. (Funded by Gilead Sciences; PURPOSE 1 ClinicalTrials.gov number, NCT04994509.).

Sexually transmitted infections in pregnancy and adverse pregnancy outcomes: A retrospective cohort study.

OBJECTIVE: There is a high prevalence and incidence rate of asymptomatic sexually transmitted infections (STIs) during pregnancy in adolescent girls and young women in Africa. The association between STIs and pregnancy outcomes in a hyperepidemic HIV setting has not been well described. METHODS: Pregnant women, HIV-1 negative and <28 weeks' gestation at three primary health clinics in KwaZulu-Natal, South Africa were enrolled from February 2017 to March 2018. Vaginal swabs collected at the first and later antenatal visits were stored and retrospectively tested for HSV-2, Trichomonas vaginalis, Chlamydia trachomatis and Neisseria gonorrhoeae at the end of the study. The association between STIs detected at first and later antenatal visits and pregnancy outcome was assessed using multivariable logistic regression models adjusted for maternal age and treatment received for symptomatic STIs. RESULTS: Testing positive Mycoplasma genitalium at the first antenatal visit was significantly associated with low birth weight (odds ratio [OR] 5.22; 95% confidence interval [CI]: 1.10-15.98). Testing positive for T. vaginalis at the repeat visit was significantly associated with preterm births (OR 2.37; 95% CI: 1.11-5.03), low birth weight (OR 2.56; 1.16-5.63) and a composite adverse pregnancy outcome (OR 2.11; 95% CI: 1.09-4.08). Testing positive for HSV-2 at the repeat visit was also likely associated with experiencing a preterm birth or any adverse pregnancy outcome (OR 3.39; 95% CI: 0.86-13.3) (P = 0.096). CONCLUSIONS: Among predominantly asymptomatic STIs, M. genitalium detected at baseline visit was significantly associated with low birth weight, while T. vaginalis detected at the repeat visit in later pregnancy was significantly associated with preterm birth. Further research is warranted to study the impact of etiological testing of STIs at more than one antenatal visit and empirical treatment on pregnancy outcomes.

Self-care interventions for women's health and well-being.

The human right to health is universal and non-exclusionary, supporting health in full, and for all. Despite advances in health systems globally, 3.6 billion people lack access to essential health services. Women and girls are disadvantaged when it comes to benefiting from quality health services, owing to social norms, unequal power in relationships, lack of consideration beyond their reproductive roles and poverty. Self-care interventions, including medicines and diagnostics, which offer an additional option to facility-based care, can improve the autonomy and agency of women in managing their own health. However, tackling challenges such as stigma is essential to avoid scenarios in which self-care interventions provide more choice for those who already benefit from access to quality healthcare, and leave behind those with the greatest need. This Perspective explores the opportunities that self-care interventions offer to advance the health and well-being of women with an approach grounded in human rights, gender equality and equity.

Host genetic variation at a locus near CHD1L impacts HIV sequence diversity in a South African population.

IMPORTANCE: It has been previously shown that genetic variants near CHD1L on chromosome 1 are associated with reduced HIV VL in African populations. However, the impact of these variants on viral diversity and how they restrict viral replication are unknown. We report on a regional association analysis in a South African population and show evidence of selective pressure by variants near CHD1L on HIV RT and gag. Our findings provide further insight into how genetic variability at this locus contributes to host control of HIV in a South African population.

Performance of rapid antigen tests in identifying Omicron BA.4 and BA.5 infections in South Africa.

BACKGROUND: Concerns around accuracy and performance of rapid antigen tests continue to be raised with the emergence of new SARS-CoV-2 variants. OBJECTIVE: To evaluate the performance of two widely used SARS-CoV-2 rapid antigen tests during BA.4/BA.5 SARS-CoV-2 wave in South Africa (May - June 2022). STUDY DESIGN: A prospective field evaluation compared the SARS-CoV-2 Antigen Rapid test from Hangzhou AllTest Biotech (nasal swab) and the Standard Q COVID-19 Rapid Antigen test from SD Biosensor (nasopharyngeal swab) to the Abbott RealTime SARS-CoV-2 assay (nasopharyngeal swab) on samples collected from 540 study participants. RESULTS: Overall 28.52% (154/540) were SARS-CoV-2 RT-PCR positive with median cycle number value of 12.30 (IQR 9.30-19.40). Out of the 99 successfully sequenced SARS-CoV-2 positive samples, 18 were classified as BA.4 and 56 were classified as BA.5. The overall sensitivities of the AllTest SARS-CoV-2 Ag test and Standard Q COVID-19 Ag test were 73.38% (95% CI 65.89-79.73) and 74.03% (95% CI 66.58-80.31) and their specificities were 97.41% (95% CI 95.30-98.59) and 99.22% (95% CI 97.74-99.74) respectively. Sensitivity was >90% when the cycle number value was <20. The sensitivity of both rapid tests was >90% in samples infected with Omicron sub-lineage BA.4 and BA.5. CONCLUSION: Accuracy of tested rapid antigen tests that target the nucleocapsid SARS-CoV-2 protein, were not adversely affected by BA.4 and BA.5 Omicron sub-variants.

High incidence of asymptomatic genital tract infections in pregnancy in adolescent girls and young women: need for repeat aetiological screening.

INTRODUCTION: Sexually transmitted infection (STI) prevalence and incidence estimates for pregnant adolescents are under-reported. We estimated prevalence and incidence of STIs in pregnant adolescents (15-19 years) in comparison with pregnant women 20-24 and >25 years. METHODS: Pregnant women registering at primary care clinics in Umlazi, a periurban subdistrict in KwaZulu-Natal, South Africa, were enrolled in an HIV incidence cohort study during February 2017-March 2018. Women were examined for abnormal vaginal discharge, received empirical treatment, tested for HIV-1 and had vaginal swabs taken at their first and a subsequent visit in the third trimester. Vaginal swabs were stored for STI testing at completion of study and tested for Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium using PCR. RESULTS: A total of 752 HIV-negative pregnant women were enrolled at a median gestational age of 17 weeks: 180 (23.9%), 291 (38.7%) and 281 (37.4%) in the 15-19, 20-24 and >25 years age groups. Pregnant adolescents had an STI prevalence of 26.7% at baseline, not significantly lower than the 20-24 (34.7%, OR 1.4; 95% CI 1.0 to 2.1, p=0.09) and >25 years (33.8%, OR 1.4; 95% CI 0.9 to 2.1, p=0.12) age groups. T. vaginalis (11.1%), C. trachomatis (7.8%) and N. gonorrhoeae (4.4%) were most prevalent in adolescents, a trend similar to the other age groups. Overall, 43.4% were symptomatic and treated at baseline. Overall, 40.7% (118 of 290) of women who tested negative for an STI at baseline tested positive at the repeat visit (incidence 19.5/100 person years). STI incidence in pregnant adolescents was 23.9/100 person years and comparable with older age groups (20.5/100 person years and 16.2/100 person years). At the repeat visit, 19.0% of all women with an STI were symptomatic and treated. Performance of syndromic management was poor at baseline (negative predictive value (NPV) 68.6%, positive predictive value (PPV) 34.0%) and at repeat visit (NPV 58.4%, PPV 34.3%). CONCLUSIONS: Prevalence of asymptomatic curable STIs in pregnant adolescents is high and comparable with women >20 years old. Adolescents remain at substantial risk of asymptomatic incident STIs during pregnancy.

Safety and pharmacokinetics of escalating doses of neutralising monoclonal antibody CAP256V2LS administered with and without VRC07-523LS in HIV-negative women in South Africa (CAPRISA 012B): a phase 1, dose-escalation, randomised controlled trial.

BACKGROUND: Young women in sub-Saharan Africa continue to bear a high burden of HIV infection. Combination anti-HIV monoclonal antibodies are a potential HIV prevention technology that could overcome adherence challenges of daily oral pre-exposure prophylaxis. In this phase 1 clinical trial we aimed to determine the safety and pharmacokinetic profile of the broadly neutralising monoclonal antibody CAP256V2LS. METHODS: CAPRISA 012B, a first-in-human dose-escalation phase 1 trial evaluated the safety, pharmacokinetics, and neutralisation activity of CAP256V2LS alone and in combination with VRC07-523LS in young HIV-negative women in Durban, South Africa. Groups 1 and 2 were open label with CAP256V2LS administered at 5 mg/kg and 10 mg/kg intravenously and 5 mg/kg, 10 mg/kg, and 20 mg/kg subcutaneously. In group 3, participants were randomly allocated to receive a combination of CAP256V2LS and VRC07-523LS at 10 mg/kg and 20 mg/kg subcutaneously comixed with ENHANZE, a recombinant human hyaluronidase. Once safety was established in the first three participants, dose escalation took place sequentially following review of safety data. Primary endpoints were the proportion of participants with mild, moderate, and severe reactogenicity or adverse events, graded as per the Division of AIDS toxicity grading. The trial is registered on the Pan African Clinical Trial Registry, PACTR202003767867253, and is recruiting. FINDINGS: From July 13, 2020, to Jan 13, 2021, 42 HIV-negative women, aged 18-45 years, were enrolled. All 42 participants, eight with intravenous and 34 with subcutaneous administration, completed the trial. There were no serious adverse events or dose-limiting toxicities. Most commonly reported symptoms following intravenous administration were headaches in seven (88%) and nausea in four (50%) participants. Commonly reported symptoms following subcutaneous administration were headache in 31 (91%), chills in 25 (74%), and malaise or fatigue in 19 (56%) participants. Adverse events included transient lymphocytopenia in eight (19%), proteinuria in nine (21%), elevated aspartate aminotransferase in ten (24%), and alanine aminotransferase in five (12%) participants. INTERPRETATION: CAP256V2LS administered alone and in combination with VRC07-523LS was safe with favourable pharmacokinetics and neutralisation activity, supporting further assessment in larger clinical studies. FUNDING: European and Developing Countries Clinical Trials Partnership, South African Medical Research Council, and South African Department of Science and Innovation.

HIV prevention for the next decade: Appropriate, person-centred, prioritised, effective, combination prevention.

UNAIDS and a broad range of partners have collaborated to establish a new set of HIV prevention targets to be achieved by 2025 as an intermediate step towards the sustainable development target for 2030.The number of new HIV infections in the world continues to decline, in part due to the extraordinary expansion of effective HIV treatment. However, the decline is geographically heterogeneous, with some regions reporting a rise in incidence. The incidence target that was agreed for 2020 has been missed.A range of exciting new HIV prevention technologies have become available or are in the pipeline but will only have an impact if they are accessible and affordable and delivered within systems that take full account of the social and political context in which most infections occur. Most new infections occur in populations that are marginalised or discriminated against due to structural, legal, and cultural barriers.The new targets imply a new approach to HIV prevention that emphasises appropriate, person-centred, prioritised, effective, combination HIV prevention within a framework that reduces existing barriers to services and acknowledges heterogeneity, autonomy, and choice.These targets have consequences for people working in HIV programmes both for delivery and for monitoring and evaluation, for health planners setting local and national priorities, and for funders both domestic and global. Most importantly, they have consequences for people who are at risk of HIV exposure and infection.Achieving these targets will have a huge impact on the future of the HIV epidemic and put us back on track towards ending AIDS as a public health threat by 2030.

Vaginal microbial shifts are unaffected by oral pre-exposure prophylaxis in South African women.

Vaginal microbiota have been shown to be a modifier of protection offered by topical tenofovir in preventing HIV infection in women, an effect not observed with oral tenofovir-based pre-exposure prophylaxis (PrEP). It remains unclear whether PrEP can influence the vaginal microbiota composition. This study investigated the impact of daily oral tenofovir disoproxil fumarate in combination with emtricitabine for PrEP on the vaginal microbiota in South African women. At baseline, Lactobacillus iners or Gardnerella vaginalis dominant vaginal communities were observed in the majority of participants. In cross sectional analysis, vaginal microbiota were not affected by the initiation and use of PrEP. Longitudinal analysis revealed that Lactobacillus crispatus-dominant "cervicotypes 1 (CT1)" communities had high probability of remaining stable in PrEP group, but had a higher probability of transitioning to L. iners-dominant CT2 communities in non-PrEP group. L. iners-dominant communities were more likely to transition to communities associated with bacterial vaginosis (BV), irrespective of PrEP or antibiotic use. As expected, BV-linked CTs had a higher probability of transitioning to L. iners than L. crispatus dominant CTs and this shift was not associated with PrEP use.

Prospective study of oral pre-exposure prophylaxis initiation and adherence among young women in KwaZulu-Natal, South Africa.

INTRODUCTION: Oral tenofovir disoproxil fumarate/emtricitabine pre-exposure prophylaxis (PrEP), introduced into South Africa (SA) in 2016, has increasingly become part of HIV prevention standard of care. Given the urgent need for increased HIV prevention efforts for young women in SA, we conducted an implementation study to explore oral PrEP initiation and adherence, and the impact of oral PrEP on HIV incidence in this group. METHODS: This prospective cohort study (CAPRISA 082) was conducted at two sites (urban and rural) in KwaZulu-Natal, between March 2016 and February 2018. HIV-negative, sexually active women, aged 18-30 years, were enrolled and followed for approximately 10 months. Oral PrEP was offered as part of a comprehensive HIV prevention package. Adherence to oral PrEP was measured using pill counts and tenofovir-diphosphate (TFV-DP) levels. Characteristics of oral PrEP initiators versus non-initiators were compared using risk ratios. HIV incidence rates were measured using Poisson regression. RESULTS: Of 425 women enrolled, 262 (62%) initiated oral PrEP. Uptake was significantly higher at the rural site compared to the urban site (78% [n = 203/259] vs. 36% [n = 59/166], respectively, p-value<0.001). Approximately 25% and 50% had stopped using oral PrEP by 3 and 12 months post-initiation, respectively. Median pill count adherence was 90% (interquartile range: 81-97%); however, TFV-DP was only detected in 13% of samples tested, that is 56/431 samples from 97 (37%) participants who initiated oral PrEP. In total, 11 women seroconverted yielding an HIV incidence rate of 2.81 per 100 person-years (95% confidence interval: 1.40-5.03). Nine of 11 seroconverters had initiated oral PrEP; however, all showed drug levels equivalent to taking one to zero tablets per week. Among women who initiated oral PrEP, >50% had discontinued using oral PrEP by study end, with side effects, such as diarrhoea, nausea, headaches and rash, being the most frequent reason for discontinuation. CONCLUSIONS: Despite moderate oral PrEP initiation and high pill count adherence, adherence as measured by TFV-DP levels was low and early discontinuation was high. The overall HIV incidence rate was high underscoring the critical need to address barriers to oral PrEP initiation, adherence and continued use, as well as expanding HIV prevention options for young women.

Omicron infection enhances Delta antibody immunity in vaccinated persons.

The extent to which Omicron infection1-9, with or without previous vaccination, elicits protection against the previously dominant Delta (B.1.617.2) variant is unclear. Here we measured the neutralization capacity against variants of severe acute respiratory syndrome coronavirus 2 in 39 individuals in South Africa infected with the Omicron sublineage BA.1 starting at a median of 6 (interquartile range 3-9) days post symptom onset and continuing until last follow-up sample available, a median of 23 (interquartile range 19-27) days post symptoms to allow BA.1-elicited neutralizing immunity time to develop. Fifteen participants were vaccinated with Pfizer's BNT162b2 or Johnson & Johnson's Ad26.CoV2.S and had BA.1 breakthrough infections, and 24 were unvaccinated. BA.1 neutralization increased from a geometric mean 50% focus reduction neutralization test titre of 42 at enrolment to 575 at the last follow-up time point (13.6-fold) in vaccinated participants and from 46 to 272 (6.0-fold) in unvaccinated participants. Delta virus neutralization also increased, from 192 to 1,091 (5.7-fold) in vaccinated participants and from 28 to 91 (3.0-fold) in unvaccinated participants. At the last time point, unvaccinated individuals infected with BA.1 had low absolute levels of neutralization for the non-BA.1 viruses and 2.2-fold lower BA.1 neutralization, 12.0-fold lower Delta neutralization, 9.6-fold lower Beta variant neutralization, 17.9-fold lower ancestral virus neutralization and 4.8-fold lower Omicron sublineage BA.2 neutralization relative to vaccinated individuals infected with BA.1. These results indicate that hybrid immunity formed by vaccination and Omicron BA.1 infection should be protective against Delta and other variants. By contrast, infection with Omicron BA.1 alone offers limited cross-protection despite moderate enhancement.

Advancing HIV prevention using tenofovir-based pre-exposure prophylaxis.

Tenofovir-based pre-exposure prophylaxis (PrEP) revolutionized the global HIV prevention landscape. Prior to the proof-of concept trial in 2010, which demonstrated that tenofovir (TFV) could prevent sexual transmission of HIV, prevention options were largely limited to behavior change, condoms, and circumcision. Several subsequent studies evaluating oral tenofovir disoproxil fumarate (TDF) or the TDF/emtricitabine (FTC) combination as PrEP for HIV prevention provided evidence for regulatory approval and inclusion in national and international guidelines. By 2021, 1.5 million people had initiated oral tenofovir-based PrEP, contributing to declines in HIV incidence in some regions. Here we reflect on how oral tenofovir-based PrEP became an important component of combination HIV prevention programs across the globe.

Age-Restriction of a Validated Risk Scoring Tool Better Predicts HIV Acquisition in South African Women: CAPRISA 004.

We examined the predictive ability of the VOICE risk screening tool among adolescent girls and young women at heightened HIV risk in urban and peri-urban Kwa-Zulu-Natal, South Africa. Using participant data from CAPRISA 004's control arm (N = 444), we applied the initial VOICE risk screening score (IRS), a modified risk score (MRS) based on predictive and non-predictive variables in our data, and age-restricted (AIRS and AMRS, respectively). We estimated incidence rates, 95% confidence bounds, sensitivity, specificity, negative and positive predictive values and area under the curve (AUC). The sample's HIV incidence rate was 9.1/100 Person-Years [95% CI 6.9-11.7], resulting from 60 seroconversions (60/660.7 Person-Years). The IRS' ≥ 8 cutpoint produced moderate discrimination [AUC = 0.66 (0.54-0.74), sensitivity = 63%, specificity = 57%]. Restricting to age < 25 years improved the score's predictive ability (AIRS: AUC = 0.69, AMRS: AUC = 0.70), owing mainly to male partner having other partners and HSV-2. The risk tool predicted HIV acquisition at a higher cutpoint in this sample than in the initial VOICE analysis. After age-stratification, fewer variables were needed for maintaining score's predictiveness. In this high incidence setting, risk screening may still improve the efficiency or effectiveness of prevention counseling services. However, PrEP should be offered to all prevention-seeking individuals, regardless of risk ascertainment.

Kidney function in tenofovir disoproxil fumarate-based oral pre-exposure prophylaxis users: a systematic review and meta-analysis of published literature and a multi-country meta-analysis of individual participant data.

BACKGROUND: Previous WHO guidance on tenofovir disoproxil fumarate-based oral pre-exposure prophylaxis (PrEP) suggests measuring creatinine levels at PrEP initiation and regularly afterwards, which might represent barriers to PrEP implementation and uptake. We aimed to systematically review published literature on kidney toxicity among tenofovir disoproxil fumarate-based oral PrEP users and conducted an individual participant data meta-analysis (IPDMA) on kidney function among PrEP users in a global implementation project dataset. METHODS: In this systematic review and meta-analysis we searched PubMed up to June 30, 2021, for randomised controlled trials (RCTs) or cohort studies that reported on graded kidney-related adverse events among oral PrEP users (tenofovir disoproxil fumarate-based PrEP alone or in combination with emtricitabine or lamivudine). We extracted summary data and conducted meta-analyses with random-effects models to estimate relative risks of grade 1 and higher and grade 2 and higher kidney-related adverse events, measured by elevated serum creatinine or decline in estimated creatinine clearance or estimated glomerular filtration rate. The IPDMA included (largely unpublished) individual participant data from 17 PrEP implementation projects and two RCTs. Estimated baseline creatinine clearance and creatinine clearance change after initiation were described by age, gender, and comorbidities. We used random-effects regressions to estimate the risk in decline of creatinine clearance to less than 60 mL/min. FINDINGS: We identified 62 unique records and included 17 articles reporting on 11 RCTs with 13 523 participants in meta-analyses. PrEP use was associated with increased risk of grade 1 and higher kidney adverse events (pooled odds ratio [OR] 1·49, 95% CI 1·22-1·81; I2=25%) and grade 2 and higher events (OR 1·75, 0·68-4·49; I2=0%), although the grade 2 and higher association was not statistically significant and events were rare (13 out of 6764 in the intervention group vs six out of 6782 in the control group). The IPDMA included 18 676 individuals from 15 countries (1453 [7·8%] from RCTs) and 79 (0·42%) had a baseline estimated creatinine clearance of less than 60 mL/min (increasing proportions with increasing age). Longitudinal analyses included 14 368 PrEP users and 349 (2·43%) individuals had a decline to less than 60 mL/min creatinine clearance, with higher risks associated with increasing age and baseline creatinine clearance of 60·00-89·99 mL/min (adjusted hazard ratio [aHR] 8·49, 95% CI 6·44-11·20) and less than 60 mL/min (aHR 20·83, 12·83-33·82). INTERPRETATION: RCTs suggest that risks of kidney-related adverse events among tenofovir disoproxil fumarate-based oral PrEP users are increased but generally mild and small. Our global PrEP user analysis found varying risks by age and baseline creatinine clearance. Kidney function screening and monitoring might focus on older individuals, those with baseline creatinine clearance of less than 90 mL/min, and those with kidney-related comorbidities. Less frequent or optional screening among younger individuals without kidney-related comorbidities may reduce barriers to PrEP implementation and use. FUNDING: Unitaid, Bill & Melinda Gates Foundation, WHO.

Genital Immune Cell Activation and Tenofovir Gel Efficacy: A Case-Control Study.

Genital inflammation (GI) undermines topical human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) efficacy through unknown mechanisms. Here, associations between activated endocervical CD4 + T-cell numbers and higher deoxyadenosine triphosphate (dATP) concentrations suggest that competition for intracellular metabolites within HIV target cells may reduce the efficacy of antiretroviral-based PrEP in women with GI.